Fig 6A . A diagram showing two groups of ER - resident proteins : those that become macro - ER - phagy cargos ( in green ) , and those that do not ( in blue or grey ) . Like overexpressed Snc1 - PEM or Snq2 , the ER - resident membrane proteins Sec61 ( translocon subunit ) and Hmg1 ( sterol biogenesis ) are transported to the vacuole via macro - ER - phagy . In contrast , the ER - to - Golgi exit regulators Sec12 and Sec13 and the ER lumen chaperone Kar2 are not co - transported to the lysosome through macro - ER - phagy . Four or six different strains were used for this analysis : WT ( YPT1 ) , pep4 ∆ , and pep4 prb1 ∆ , ypt1 - 1 , ypt1 - 1pep4 ∆ , and ypt1 - 1pep4 ∆ prb1 ∆ . In each strain , one ER - resident protein was tagged at its C - terminus ( except for Kar2 ) . ( The pep4 ∆ strains were not used in all experiments because they do not show the full defect ) . The level of ER - resident proteins was determined by immunoblot analysis in cells that either do not ( B - D ) or do overexpress GFP - Snc1 - PEM ( F - H ) . B - C . Sec61 - 3xHA ( B ) and Sec13 - 3x HA ( C ) expressing cells ( 2 independent un - transformed colonies ) were tested by immunoblot analysis ( using anti - HA antibodies ) . Shown from top to bottom : strain genotype , HA - tagged protein , G6PDH ( loading control ) , quantification of HA - tagged protein expressed as average fold of WT ( p - value = 0 . 025 for Sec61 ) . D . Summary of immunoblot analyses of ER - resident proteins level in cells that do not overexpress GFP - Snc1 - PEM : WT ( PEP4PRB1 ) vs pep4 ∆ prb1 ∆ ( left ) , ypt1 - 1PEP4PRB1 vs ypt1 - 1pep4 ∆ prb1 ∆ ( right ) . Immunoblots and quantification are shown in Fig 6B - 6C and S6A - S6C Fig . In YPT1 cells , the levels of Sec61 and Hmg1 ( green ) are slightly increased ( 14 and 38 % , respectively ) in strains defective in vacuolar proteolysis ; the levels of Sec12 , Sec13 and Kar2 are not increased ( blue and grey ) . The levels of Sec61 and Hmg1 ( green ) are increased by 2 - 2 . 5 fold in ypt1 - 1 mutant cells when compared to WT cells , regardless if they are defective in vacuolar proteolysis or not . In contrast , the levels of Sec12 , Sec13 and Kar2 are only slightly increased ( blue and grey ) . E . The level of the ER - resident protein Sec61 is similar whether GFP - Snc1 - PEM is overexpressed or not . Wild - type cells were transformed with a 2μ plasmid , either empty or for overexpression of GFP - Snc1 - GEM ( 2 independent transformants ) . Shown from top to bottom : plasmid , Sec61 , G6PDH ( loading control ) , GFP - Snc1 - PEM , and quantification of Sec61 expressed as average fold of WT with empty plasmid . F - G . Sec61 - 3xHA ( F ) and Sec13 - 3xHA ( G ) expressing cells were transformed with a 2μ plasmid for overexpression of GFP - Snc1 - PEM . Cell lysates were tested by immunoblot analysis ( using ant - HA and anti - GFP antibodies ) . Shown from top to bottom : strain genotype , the specific ER - resident protein tested , quantification of the ER - resident protein bands expressed as average fold of WT , GFP - Snc1 - PEM , quantification of the GFP - Snc1 - PEM bands expressed as average fold of WT , and G6PDH ( loading control ) . H . Summary of immunoblot analysis of ER - resident proteins level in cells overexpressing GFP - Snc1 - PEM : WT ( PEP4PRB1 ) vs pep4 ∆ prb1 ∆ ( left ) , ypt1 - 1PEP4PRB1 vs ypt1 - 1pep4 ∆ prb1 ∆ ( right ) . Immunoblots and quantification are shown in Fig 6F and 6G and S6F - S6H Fig . In YPT1 cells , like Snc1 - PEM ( black ) , the levels of Hmg1 and Sec61 ( green ) are increased > 10 fold in strains defective in vacuolar proteolysis . In contrast , the levels of Sec12 , Sec13 and Kar2 are not changed ( blue and grey ) . Like Snc1 - PEM ( black ) , the protein levels of Hmg1 and Sec61 ( green ) are increased ~ 15 fold in ypt1 - 1 mutant cells when compared to WT cells , regardless if they are defective in vacuolar proteolysis or not . In contrast , the levels of Sec12 , Sec13 and Kar2 are only slightly increased ( blue and grey ) . + / - and error bars represent STDEV . Results in this figure represent at least two independent experiments .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Fig 6A. A diagram showing two groups of ER-resident proteins: those that become macro-ER-phagy cargos (in green), and those that do not (in blue or grey). Like overexpressed Snc1-PEM or Snq2, the ER-resident membrane proteins Sec61 (translocon subunit) and Hmg1 (sterol biogenesis) are transported to the vacuole via macro-ER-phagy. In contrast, the ER-to-Golgi exit regulators Sec12 and Sec13 and the ER lumen chaperone Kar2 are not co-transported to the lysosome through macro-ER-phagy. Four or six different strains were used for this analysis: WT (YPT1), pep4∆, and pep4 prb1∆, ypt1-1, ypt1-1pep4∆, and ypt1-1pep4∆ prb1∆. In each strain, one ER-resident protein was tagged at its C-terminus (except for Kar2). (The pep4∆ strains were not used in all experiments because they do not show the full defect). The level of ER-resident proteins was determined by immunoblot analysis in cells that either do not (B-D) or do overexpress GFP-Snc1-PEM (F-H). B-C. Sec61-3xHA (B) and Sec13-3x HA (C) expressing cells (2 independent un-transformed colonies) were tested by immunoblot analysis (using anti-HA antibodies). Shown from top to bottom: strain genotype, HA-tagged protein, G6PDH (loading control), quantification of HA-tagged protein expressed as average fold of WT (p-value = 0.025 for Sec61).D. Summary of immunoblot analyses of ER-resident proteins level in cells that do not overexpress GFP-Snc1-PEM: WT (PEP4PRB1) vs pep4∆ prb1∆ (left), ypt1-1PEP4PRB1 vs ypt1-1pep4∆ prb1∆ (right). Immunoblots and quantification are shown in Fig 6B-6C and S6A-S6C Fig. In YPT1 cells, the levels of Sec61 and Hmg1 (green) are slightly increased (14 and 38%, respectively) in strains defective in vacuolar proteolysis; the levels of Sec12, Sec13 and Kar2 are not increased (blue and grey). The levels of Sec61 and Hmg1 (green) are increased by 2-2.5 fold in ypt1-1 mutant cells when compared to WT cells, regardless if they are defective in vacuolar proteolysis or not. In contrast, the levels of Sec12, Sec13 and Kar2 are only slightly increased (blue and grey).E. The level of the ER-resident protein Sec61 is similar whether GFP-Snc1-PEM is overexpressed or not. Wild-type cells were transformed with a 2μ plasmid, either empty or for overexpression of GFP-Snc1-GEM (2 independent transformants). Shown from top to bottom: plasmid, Sec61, G6PDH (loading control), GFP-Snc1-PEM, and quantification of Sec61 expressed as average fold of WT with empty plasmid.F-G. Sec61-3xHA (F) and Sec13-3xHA (G) expressing cells were transformed with a 2μ plasmid for overexpression of GFP-Snc1-PEM. Cell lysates were tested by immunoblot analysis (using ant-HA and anti-GFP antibodies). Shown from top to bottom: strain genotype, the specific ER-resident protein tested, quantification of the ER-resident protein bands expressed as average fold of WT, GFP-Snc1-PEM, quantification of the GFP-Snc1-PEM bands expressed as average fold of WT, and G6PDH (loading control).H. Summary of immunoblot analysis of ER-resident proteins level in cells overexpressing GFP-Snc1-PEM: WT (PEP4PRB1) vs pep4∆ prb1∆ (left), ypt1-1PEP4PRB1 vs ypt1-1pep4∆ prb1∆ (right). Immunoblots and quantification are shown in Fig 6F and 6G and S6F-S6H Fig. In YPT1 cells, like Snc1-PEM (black), the levels of Hmg1 and Sec61 (green) are increased >10 fold in strains defective in vacuolar proteolysis. In contrast, the levels of Sec12, Sec13 and Kar2 are not changed (blue and grey). Like Snc1-PEM (black), the protein levels of Hmg1 and Sec61 (green) are increased ~15 fold in ypt1-1 mutant cells when compared to WT cells, regardless if they are defective in vacuolar proteolysis or not. In contrast, the levels of Sec12, Sec13 and Kar2 are only slightly increased (blue and grey). +/- and error bars represent STDEV. Results in this figure represent at least two independent experiments.


Fig 7A . In cells defective in vacuolar proteolysis ( pep4 ∆ prb1 ∆ ) , Hmg1 co - localizes with 70 % of intra - cellular Snc1 - PEM , but Sec13 does not ( at least 36 cells were analyzed for each strain ) . No co - localization was observed for resident ER proteins and overexpressed Snc1 - PEM in WT cells ( PEP4PRB1 ) . Hmg1 ( left ) and Sec13 ( right ) were tagged with mCherry at their C - terminus in PEP4PRB1 ( WT ) and pepb4 ∆ prb1 ∆ mutant cells . Cells were transformed with a 2μ plasmid for overexpression of GFP - Snc1 - PEM and analyzed by live - cell microscopy . Shown from top to bottom : DIC , GFP , mCherry , merge ( yellow ) , % cells with intra - cellular Snc1 - PEM , and % co - localization of the ER protein with intra - cellular Snc1 - PEM . B . The ER - resident protein Hmg1 is delivered to the vacuole with GFP - Snc1 - PEM . The vacuole of PEP4PRB1 ( WT ) and pepb4 ∆ prb1 ∆ mutant cells expressing Hmg1 - mCherry and overexpressing GFP - Snc1 - PEM ( see Panel A , left ) was stained with CMAC - Arg ( blue ) , and the cells were analyzed by live - cell microscopy . In WT cells ( top ) , there is no intracellular Snc1 - PEM and the red - labeled ER is distinguished from the blue vacuole . In contrast , in ~ 75 % pep4 ∆ prb1 ∆ mutant cells ( bottom ) , which are defective in vacuolar proteolysis , Hmg1 ( red ) co - localizes with Snc1 - PEM ( green ) in the vacuole ( blue ) . Shown from left to right : DIC , GFP , mCherry , blue , merge ( white shows merge of the three colors ) , % cells with intracellular Snc1 - PEM , % cells in which Hmg1 with Snc1 - PEM co - localize , and % co - localization of the two proteins with the vacuolar dye ( 38 cells were analyzed for each strain ) . C . > 80 % of ypt1 - 1 mutant cells , regardless if they are PEP4PRB1 or pep4 ∆ prb1 ∆ , accumulate aberrant GFP - Snc1 - PEM structures . Hmg1 co - localizes with > 70 % of these structures , whereas Sec13 co - localizes with only ~ 15 % of these structures ( 36 - 43 cells were analyzed for each strain ) . The experiment was done as described for Panel A . D . Summary of microscopy analysis of ER - resident proteins co - localization with GFP - Snc1 - PEM in cells overexpressing GFP - Snc1 - PEM : WT ( PEP4PRB1 ) vs pep4Δ prb1Δ ( left ) , and ypt1 - 1PEP4PRB1 vs ypt1 - 1pep4Δ prb1Δ ( right ) . Microscopy and quantification are shown in Fig 7A and 7C and S7 Fig . In YPT1 cells , whereas > 60 % of Hmg1 and Sec61 ( green ) co - localize with Snc1 - PEM in strains defective in vacuolar proteolysis , Sec12 , Sec13 and Kar2 are not changed significantly ( blue and grey ) . Hmg1 and Sec61 ( green ) co - localize with Snc1 - PEM in > 70 % of ypt1 - 1 and > 80 % of ypt1 - 1pep4Δ prb1Δ mutant cells , whereas Sec13 and Kar2 ( blue and grey ) do so in < 15 % of the cells . The level of Sec12 co - localization with Snc1 - PEM is intermediate , ~ 38 % in ypt1 - 1 and ypt1 - 1pep4Δ prb1Δ . Error bars represent STDEV . Results in panels A and B represent at least two independent experiments .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Fig 7A. In cells defective in vacuolar proteolysis (pep4∆ prb1∆), Hmg1 co-localizes with 70% of intra-cellular Snc1-PEM, but Sec13 does not (at least 36 cells were analyzed for each strain). No co-localization was observed for resident ER proteins and overexpressed Snc1-PEM in WT cells (PEP4PRB1). Hmg1 (left) and Sec13 (right) were tagged with mCherry at their C-terminus in PEP4PRB1 (WT) and pepb4∆ prb1∆ mutant cells. Cells were transformed with a 2μ plasmid for overexpression of GFP-Snc1-PEM and analyzed by live-cell microscopy. Shown from top to bottom: DIC, GFP, mCherry, merge (yellow), % cells with intra-cellular Snc1-PEM, and % co-localization of the ER protein with intra-cellular Snc1-PEM.B. The ER-resident protein Hmg1 is delivered to the vacuole with GFP-Snc1-PEM. The vacuole of PEP4PRB1 (WT) and pepb4∆ prb1∆ mutant cells expressing Hmg1-mCherry and overexpressing GFP-Snc1-PEM (see Panel A, left) was stained with CMAC-Arg (blue), and the cells were analyzed by live-cell microscopy. In WT cells (top), there is no intracellular Snc1-PEM and the red-labeled ER is distinguished from the blue vacuole. In contrast, in ~75% pep4∆ prb1∆ mutant cells (bottom), which are defective in vacuolar proteolysis, Hmg1 (red) co-localizes with Snc1-PEM (green) in the vacuole (blue). Shown from left to right: DIC, GFP, mCherry, blue, merge (white shows merge of the three colors), % cells with intracellular Snc1-PEM, % cells in which Hmg1 with Snc1-PEM co-localize, and % co-localization of the two proteins with the vacuolar dye (38 cells were analyzed for each strain).C. >80% of ypt1-1 mutant cells, regardless if they are PEP4PRB1 or pep4∆ prb1∆, accumulate aberrant GFP-Snc1-PEM structures. Hmg1 co-localizes with >70% of these structures, whereas Sec13 co-localizes with only ~15% of these structures (36-43 cells were analyzed for each strain). The experiment was done as described for Panel A.D. Summary of microscopy analysis of ER-resident proteins co-localization with GFP-Snc1-PEM in cells overexpressing GFP-Snc1-PEM: WT (PEP4PRB1) vs pep4Δ prb1Δ (left), and ypt1-1PEP4PRB1 vs ypt1-1pep4Δ prb1Δ (right). Microscopy and quantification are shown in Fig 7A and 7C and S7 Fig. In YPT1 cells, whereas >60% of Hmg1 and Sec61 (green) co-localize with Snc1-PEM in strains defective in vacuolar proteolysis, Sec12, Sec13 and Kar2 are not changed significantly (blue and grey). Hmg1 and Sec61 (green) co-localize with Snc1-PEM in >70% of ypt1-1 and >80% of ypt1-1pep4Δ prb1Δ mutant cells, whereas Sec13 and Kar2 (blue and grey) do so in <15% of the cells. The level of Sec12 co-localization with Snc1-PEM is intermediate, ~38% in ypt1-1 and ypt1-1pep4Δ prb1Δ. Error bars represent STDEV. Results in panels A and B represent at least two independent experiments.


Figure 2A . Top : DDX42 KO and CTRL KO U87 - MG / CD4 / CXCR4 / Cas9 / Firefly cells were generated using 3 sgRNAs and 4 non - targeting sgRNAs , respectively ( for CTRL , the average of data obtained with 4 cell populations is shown ) . Cells were treated or not with IFN 24 h prior to infection with HIV - 1 Renilla ( NL4 - 3 / Nef - IRES - Renilla ) . Relative luminescence results for IFN - treated and - untreated conditions are shown . Two - way ANOVA on log - transformed data with Sidak ' s test . Bottom : Immunoblot analysis of DDX42 levels is shown for 1 CTRL and DDX42 - depleted populations ; Actin served as a loading control . C . DDX42 silencing efficiency measured by RT - qPCR ( top ) and immunoblot ( bottom ) in parallel samples from B . G . Primary CD4 + T cells were electroporated with Cas9 - sgRNA RNPs using 2 non - targeting sgRNAs ( sgCTRL1 and 2 ) and 5 sgRNAs targeting DDX42 . Top : Cells were then infected with HIV - 1 Renilla ( NL4 - 3 / Nef - IRES - Renilla ) and relative infection efficiencies obtained with cells from 3 donors are shown . Two - way ANOVA on log - transformed data with Dunnett ' s test . Bottom : DDX42 protein levels were determined by immunoblot , Actin served as a loading control . A representative immunoblot is shown .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 2A. Top: DDX42 KO and CTRL KO U87-MG/CD4/CXCR4/Cas9/Firefly cells were generated using 3 sgRNAs and 4 non-targeting sgRNAs, respectively (for CTRL, the average of data obtained with 4 cell populations is shown). Cells were treated or not with IFN 24 h prior to infection with HIV-1 Renilla (NL4-3/Nef-IRES-Renilla). Relative luminescence results for IFN-treated and -untreated conditions are shown. Two-way ANOVA on log-transformed data with Sidak's test. Bottom: Immunoblot analysis of DDX42 levels is shown for 1 CTRL and DDX42-depleted populations; Actin served as a loading control.C. DDX42 silencing efficiency measured by RT-qPCR (top) and immunoblot (bottom) in parallel samples from B.G. Primary CD4+ T cells were electroporated with Cas9-sgRNA RNPs using 2 non-targeting sgRNAs (sgCTRL1 and 2) and 5 sgRNAs targeting DDX42. Top: Cells were then infected with HIV-1 Renilla (NL4-3/Nef-IRES-Renilla) and relative infection efficiencies obtained with cells from 3 donors are shown. Two-way ANOVA on log-transformed data with Dunnett's test. Bottom: DDX42 protein levels were determined by immunoblot, Actin served as a loading control. A representative immunoblot is shown.


Figure 3C . Heat - annealed ODN / RNA 1 - 294 complex was incubated with HIV - 1 RT and increasing concentrations of recombinant DDX42 . Reverse transcription was initiated by addition of the four dNTPs . Extension was for 1 , 5 , 20 or 60 min and samples were analysed by PAGE 8 % ( P / T : primer / template ; SSDNA : Strong - Stop DNA ) . A representative autoradiograph is shown . D . Quantification of 3 biological replicates performed as in C . Two - way ANOVA on log - transformed data with Dunnett ' s test . E . PLAs were performed in MDMs infected with HIV - 1 or not ( N . I . CTRL ) , using anti - Capsid and anti - DDX42 antibodies ( nuclei stained with Hoechst ) . Images were acquired using a LSM880 Airyscan microscope . Left : representative images , scale - bar : 10 μm . Right : Average punctae quantified per cell in 3 biological replicates done on MDMs from different donors with mean ± SD ( n > 65 cells per condition ) . K . HEK293T were co - transfected with pRPS - GFP and a Flag - Firefly - ( negative control ) or Flag - DDX42 - coding plasmid , followed by Flag immunoprecipitation and immunoblot analysis . A representative immunoblot is shown .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 3C. Heat-annealed ODN/RNA 1-294 complex was incubated with HIV-1 RT and increasing concentrations of recombinant DDX42. Reverse transcription was initiated by addition of the four dNTPs. Extension was for 1, 5, 20 or 60 min and samples were analysed by PAGE 8% (P/T: primer/template; SSDNA: Strong-Stop DNA). A representative autoradiograph is shown.D. Quantification of 3 biological replicates performed as in C. Two-way ANOVA on log-transformed data with Dunnett's test.E. PLAs were performed in MDMs infected with HIV-1 or not (N.I. CTRL), using anti-Capsid and anti-DDX42 antibodies (nuclei stained with Hoechst). Images were acquired using a LSM880 Airyscan microscope. Left: representative images, scale-bar: 10 μm. Right: Average punctae quantified per cell in 3 biological replicates done on MDMs from different donors with mean ± SD (n>65 cells per condition).K. HEK293T were co-transfected with pRPS-GFP and a Flag-Firefly- (negative control) or Flag-DDX42-coding plasmid, followed by Flag immunoprecipitation and immunoblot analysis. A representative immunoblot is shown.


Figure 5 ( A ) Left , A549 - ACE2 cells were infected or not with SARS - CoV - 2 for 24 h prior to PLA using mouse anti - dsRNA ( J2 ) and rabbit anti - DDX42 antibodies , followed by additional immunofluorescence ( IF ) staining with anti - mouse Alexa Fluor 546 antibody ( PLA in green , IF in magenta ) . Representative Z - stack projection images are shown ; scale bar : 15 μm . Right , Average punctae were quantified in 3 biological replicates with mean ± SD ( n > 75 cells per condition ) . Left , A549 - ACE2 cells were infected or not with SARS - CoV - 2 for 24 h prior to PLA rabbit anti - DDX42 antibodies , followed by additional immunofluorescence ( IF ) staining with anti - mouse Alexa Fluor 546 antibody ( PLA in green , IF in magenta ) . Representative Z - stack projection images are shown ; scale bar : 15 μm . Right , Average punctae were quantified in 3 biological replicates with mean ± SD ( n > 75 cells per condition ) . using an anti - N antibodyF . The following biotinylated RNAs were used to pull - down recombinant DDX42 poly ( I : C ) , CHIKV G4 , TRF2 G4 and a mutated TRF2 G4 sequence ( Mut . TRF2 G4 ) , and DDX42 was revealed by an immunoblot .

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Figure 5(A) Left, A549-ACE2 cells were infected or not with SARS-CoV-2 for 24 h prior to PLA using mouse anti-dsRNA (J2) and rabbit anti-DDX42 antibodies, followed by additional immunofluorescence (IF) staining with anti-mouse Alexa Fluor 546 antibody (PLA in green, IF in magenta). Representative Z-stack projection images are shown; scale bar: 15 μm. Right, Average punctae were quantified in 3 biological replicates with mean ± SD (n>75 cells per condition).Left, A549-ACE2 cells were infected or not with SARS-CoV-2 for 24 h prior to PLA rabbit anti-DDX42 antibodies, followed by additional immunofluorescence (IF) staining with anti-mouse Alexa Fluor 546 antibody (PLA in green, IF in magenta). Representative Z-stack projection images are shown; scale bar: 15 μm. Right, Average punctae were quantified in 3 biological replicates with mean ± SD (n>75 cells per condition). using an anti-N antibodyF. The following biotinylated RNAs were used to pull-down recombinant DDX42 poly(I:C), CHIKV G4, TRF2 G4 and a mutated TRF2 G4 sequence (Mut. TRF2 G4), and DDX42 was revealed by an immunoblot.


Figure 1U2OS cells were transfected with siRNA , and subjected to immunofluorescence staining with DAPI , anti - human centromere autoantibody ( ACA ) , and antibodies for Bub1 and TOP2A . Example images are shown ( A ) . The immunofluorescence intensity ratios of centromeric TOP2A / ACA ( B ) , centromeric TOP2A / arm TOP2A ( C ) , and arm TOP2A / DNA ( D ) , were determined on around 600 ( B and C ) or 400 ( D ) chromosomes in 20 cells . ns , no statistical significance . Data information : Means and error bars representing S . D . are shown unpaired t test ) . Scale bars , 10 μm . U2OS cells were transfected with siRNA , and subjected to immunofluorescence staining with DAPI , and antibodies for Bub1 and TOP2A . Lysates of nocodazole - arrested mitotic cells were immunoblotted with the indicated antibodies ( E ) . ns , no statistical significance . U2OS cells were transfected with siRNA , and subjected to immunofluorescence staining . Example images are shown ( F ) . U2OS cells were transfected with siRNA , and subjected to immunofluorescence staining . The immunofluorescence intensity ratio of centromeric TOP2A / arm TOP2A was determined on around 500 chromosomes in 15 cells ( G ) . Data information : Means and error bars representing S . D . are shown Scale bars , 10 μm . U2OS cells treated with STLC and MG132 for 0 . 5 hr were subjected to further treatment with Reversine , AZ3146 or dimethyl sulfoxide ( DMSO , vehicle control ) for 1 hr in the continued presence of STLC and MG132 . Example images of the immunofluorescence staining are shown ( H ) . U2OS cells treated with STLC and MG132 for 0 . 5 hr were subjected to further treatment with Reversine , AZ3146 or dimethyl sulfoxide ( DMSO , vehicle control ) for 1 hr in the continued presence of STLC and MG132 . The immunofluorescence intensity ratios of centromeric Bub1 / ACA ( I ) were determined on around 300 chromosomes in 15 cells . Data information : Means and error bars representing S . D . are shown Scale bars , 10 μm . U2OS cells treated with STLC and MG132 for 0 . 5 hr were subjected to further treatment with Reversine , AZ3146 or dimethyl sulfoxide ( DMSO , vehicle control ) for 1 hr in the continued presence of STLC and MG132 . The immunofluorescence intensity ratios of centromeric TOP2A / ACA ( J ) , and centromeric TOP2A / arm TOP2A ( K ) were determined on around 300 chromosomes in 15 cells . Data information : Means and error bars representing S . D . are shown Scale bars , 10 μm . L U2OS cells stably expressing CB - GFP or CB - Bub1 - K - GFP were treated as in ( H ) . Example images of the immunofluorescence staining are shown .

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Figure 1U2OS cells were transfected with siRNA, and subjected to immunofluorescence staining with DAPI, anti-human centromere autoantibody (ACA), and antibodies for Bub1 and TOP2A. Example images are shown (A). The immunofluorescence intensity ratios of centromeric TOP2A/ACA (B), centromeric TOP2A/arm TOP2A (C), and arm TOP2A/DNA (D), were determined on around 600 (B and C) or 400 (D) chromosomes in 20 cells. ns, no statistical significance. Data information: Means and error bars representing S.D. are shown unpaired t test). Scale bars, 10 μm.U2OS cells were transfected with siRNA, and subjected to immunofluorescence staining with DAPI, and antibodies for Bub1 and TOP2A. Lysates of nocodazole-arrested mitotic cells were immunoblotted with the indicated antibodies (E). ns, no statistical significance.U2OS cells were transfected with siRNA, and subjected to immunofluorescence staining. Example images are shown (F).U2OS cells were transfected with siRNA, and subjected to immunofluorescence staining. The immunofluorescence intensity ratio of centromeric TOP2A/arm TOP2A was determined on around 500 chromosomes in 15 cells (G). Data information: Means and error bars representing S.D. are shown Scale bars, 10 μm.U2OS cells treated with STLC and MG132 for 0.5 hr were subjected to further treatment with Reversine, AZ3146 or dimethyl sulfoxide (DMSO, vehicle control) for 1 hr in the continued presence of STLC and MG132. Example images of the immunofluorescence staining are shown (H).U2OS cells treated with STLC and MG132 for 0.5 hr were subjected to further treatment with Reversine, AZ3146 or dimethyl sulfoxide (DMSO, vehicle control) for 1 hr in the continued presence of STLC and MG132. The immunofluorescence intensity ratios of centromeric Bub1/ACA (I) were determined on around 300 chromosomes in 15 cells. Data information: Means and error bars representing S.D. are shown Scale bars, 10 μm.U2OS cells treated with STLC and MG132 for 0.5 hr were subjected to further treatment with Reversine, AZ3146 or dimethyl sulfoxide (DMSO, vehicle control) for 1 hr in the continued presence of STLC and MG132. The immunofluorescence intensity ratios of centromeric TOP2A/ACA (J), and centromeric TOP2A/arm TOP2A (K) were determined on around 300 chromosomes in 15 cells. Data information: Means and error bars representing S.D. are shown Scale bars, 10 μm.L U2OS cells stably expressing CB-GFP or CB-Bub1-K-GFP were treated as in (H). Example images of the immunofluorescence staining are shown.


Figure 2U2OS cells stably expressing CB - GFP , CB - Bub1 - K - GFP or CB - Bub1 - K - D946N - GFP were transfected with siRNA , and subjected to immunofluorescence staining with DAPI and the antibodies for H2ApT120 and TOP2A ( A ) . Data information : Scale bars , 10 μm . U2OS cells stably expressing CB - GFP , CB - Bub1 - K - GFP or CB - Bub1 - K - D946N - GFP were transfected with siRNA Lysates of nocodazole - arrested mitotic cells were immunoblotted ( B ) . * represents non - specific bands . U2OS cells were treated with DMSO or BAY 1816032 at the indicated concentrations for 1 hr , and subjected to immunofluorescence staining . Example images are shown ( C ) . Data information : Scale bars , 10 μm . U2OS cells were treated with DMSO or BAY 1816032 at the indicated concentrations for 1 hr , and subjected to immunofluorescence staining . The immunofluorescence intensity ratios of centromeric H2ApT120 / ACA ( D ) , centromeric TOP2A / ACA ( E ) , and centromeric TOP2A / arm TOP2A ( F ) were determined on around 450 chromosomes in 15 cells . Means and error bars representing S . D . are shown ( D - F ; unpaired t test ) .

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Figure 2U2OS cells stably expressing CB-GFP, CB-Bub1-K-GFP or CB-Bub1-K-D946N-GFP were transfected with siRNA, and subjected to immunofluorescence staining with DAPI and the antibodies for H2ApT120 and TOP2A (A). Data information: Scale bars, 10 μm.U2OS cells stably expressing CB-GFP, CB-Bub1-K-GFP or CB-Bub1-K-D946N-GFP were transfected with siRNA Lysates of nocodazole-arrested mitotic cells were immunoblotted (B). * represents non-specific bands.U2OS cells were treated with DMSO or BAY 1816032 at the indicated concentrations for 1 hr, and subjected to immunofluorescence staining. Example images are shown (C). Data information: Scale bars, 10 μm.U2OS cells were treated with DMSO or BAY 1816032 at the indicated concentrations for 1 hr, and subjected to immunofluorescence staining. The immunofluorescence intensity ratios of centromeric H2ApT120/ACA (D), centromeric TOP2A/ACA (E), and centromeric TOP2A/arm TOP2A (F) were determined on around 450 chromosomes in 15 cells. Means and error bars representing S.D. are shown (D-F; unpaired t test).


Figure 3A U2OS cells were subjected to immunofluorescence staining . Example images of mitotic cells at various stages are shown . Data information : Scale bars , 10 μm . B U2OS cells stably expressing CB - GFP , CB - Bub1 - K - GFP or CB - Bub1 - K - D946N - GFP were subjected to immunofluorescence staining . Example images of anaphase cells are shown . Data information : Scale bars , 10 μm . C U2OS cells stably expressing CB - GFP , CB - Bub1 - K - GFP or CB - Bub1 - K - D946N - GFP were subjected to immunofluorescence staining with DAPI , and antibodies for TOP2A and H2ApT120 or H3pS10 . Example images of interphase cells are shown . The cells in the top line , which is H3pS10 - positive , are in early prophase / late G2 . Data information : Scale bars , 10 μm . D U2OS - LacO cells transiently expressing EGFP - LacI , EGFP - LacI - Bub1 - K ( WT or D946N ) were subjected to immunofluorescence staining . Arrows point to the transgene loci . Data information : Scale bars , 10 μm . E , F MBP - TOP2A ( 429 - 1531 ) - 10xHis was subjected to pulldown by GST , GST - H2A , or GST - H2ApS120 , followed by immunoblotting with antibodies for MBP , GST or H2ApT120 ( E ) . The relative pulldown efficiency was determined ( F ) . Means and error bars representing S . D . from three independent experiments are shown ( unpaired t - test ) .

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Figure 3A U2OS cells were subjected to immunofluorescence staining. Example images of mitotic cells at various stages are shown. Data information: Scale bars, 10 μm.B U2OS cells stably expressing CB-GFP, CB-Bub1-K-GFP or CB-Bub1-K-D946N-GFP were subjected to immunofluorescence staining. Example images of anaphase cells are shown. Data information: Scale bars, 10 μm.C U2OS cells stably expressing CB-GFP, CB-Bub1-K-GFP or CB-Bub1-K-D946N-GFP were subjected to immunofluorescence staining with DAPI, and antibodies for TOP2A and H2ApT120 or H3pS10. Example images of interphase cells are shown. The cells in the top line, which is H3pS10-positive, are in early prophase/late G2. Data information: Scale bars, 10 μm.D U2OS-LacO cells transiently expressing EGFP-LacI, EGFP-LacI-Bub1-K (WT or D946N) were subjected to immunofluorescence staining. Arrows point to the transgene loci. Data information: Scale bars, 10 μm.E, F MBP-TOP2A (429-1531)-10xHis was subjected to pulldown by GST, GST-H2A, or GST-H2ApS120, followed by immunoblotting with antibodies for MBP, GST or H2ApT120 (E). The relative pulldown efficiency was determined (F). Means and error bars representing S.D. from three independent experiments are shown (unpaired t-test).


Figure 4A , B HeLa cells and the Sgo1 - K492A mutant cells were immunostained with the indicated antibodies . Example images are shown ( A ) . The immunofluorescence intensity ratio of centromeric INCENP / ACA was determined on around 250 chromosomes in 10 cells ( B ) . Data information : Means and error bars representing S . D . are shown ; unpaired t - test ) . Scale bars , 10 μm . C , D HeLa and the Sgo1 - K492A cells were exposed to nocodazole for 4 hr . Mitotic cells were cytospun onto coverslips and fixed for immunostaining . Example images are shown ( C ) . The immunofluorescence intensity ratio of centromeric TOP2A / arm TOP2A was determined on around 150 chromosomes in 10 cells ( D ) . Data information : Means and error bars representing S . D . are shown unpaired t - test ) . Scale bars , 10 μm . E , F HeLa and the Sgo1 - K492A cells were transfected with siRNA , and immunostained with the indicated antibodies . Example images are shown ( E ) . The immunofluorescence intensity ratio of centromeric Aurora B / ACA was determined on around 250 chromosomes in 10 cells ( F ) . Data information : Means and error bars representing S . D . are shown unpaired t - test ) . Scale bars , 10 μm . G , H HeLa and the Sgo1 - K492A cells were transfected with siRNA , and treated with nocodazole for 5 hr . Mitotic cells were cytospun onto coverslips and fixed for immunostaining . Example images are shown ( G ) . The immunofluorescence intensity ratio of centromeric TOP2A / arm TOP2A was determined on around 280 chromosomes in 10 cells ( H ) . Data information : Means and error bars representing S . D . are shown unpaired t - test ) . Scale bars , 10 μm .

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Figure 4A, B HeLa cells and the Sgo1-K492A mutant cells were immunostained with the indicated antibodies. Example images are shown (A). The immunofluorescence intensity ratio of centromeric INCENP/ACA was determined on around 250 chromosomes in 10 cells (B). Data information: Means and error bars representing S.D. are shown ; unpaired t-test). Scale bars, 10 μm.C, D HeLa and the Sgo1-K492A cells were exposed to nocodazole for 4 hr. Mitotic cells were cytospun onto coverslips and fixed for immunostaining. Example images are shown (C). The immunofluorescence intensity ratio of centromeric TOP2A/arm TOP2A was determined on around 150 chromosomes in 10 cells (D). Data information: Means and error bars representing S.D. are shown unpaired t-test). Scale bars, 10 μm.E, F HeLa and the Sgo1-K492A cells were transfected with siRNA, and immunostained with the indicated antibodies. Example images are shown (E). The immunofluorescence intensity ratio of centromeric Aurora B/ACA was determined on around 250 chromosomes in 10 cells (F). Data information: Means and error bars representing S.D. are shown unpaired t-test). Scale bars, 10 μm.G, H HeLa and the Sgo1-K492A cells were transfected with siRNA, and treated with nocodazole for 5 hr. Mitotic cells were cytospun onto coverslips and fixed for immunostaining. Example images are shown (G). The immunofluorescence intensity ratio of centromeric TOP2A/arm TOP2A was determined on around 280 chromosomes in 10 cells (H). Data information: Means and error bars representing S.D. are shown unpaired t-test). Scale bars, 10 μm.


Figure 5B HeLa cells were transfected with siRNA and the plasmids encoding the indicated proteins , and then exposed to nocodazole for 5 hr . Mitotic cells were cytospun onto coverslips and subjected to immunofluorescence staining with DAPI , ACA and anti - GFP antibodies . Example images are shown . Scale bars , 10 μm . HeLa cells were transfected and treated Example images are shown ( C ) . Scale bars , 10 μm . HeLa cells were transfected and treated The immunofluorescence intensity ratios of centromeric EGFP - TOP2A / ACA ( D ) , centromeric EGFP - TOP2A / arm EGFP - TOP2A ( E ) , and arm EGFP - TOP2A / DNA ( F ) , were determined on around 160 chromosomes in 8 cells . Data information : Means and error bars representing S . D . are shown unpaired t - test ) . HeLa cells transiently expressing EGFP - TOP2A ( 970 - 1531 ) or EGFP - TOP2A ( 970 - 1531 ) - Δ ( 1015 - 1032 ) were treated and immunostained Example images are shown ( G ) . Scale bars , 10 μm . HeLa cells transiently expressing EGFP - TOP2A ( 970 - 1531 ) or EGFP - TOP2A ( 970 - 1531 ) - Δ ( 1015 - 1032 ) were treated and immunostained The immunofluorescence intensity ratio of centromeric EGFP - TOP2A / arm EGFP - TOP2A was determined on around 250 chromosomes in 10 cells ( H ) . Data information : Means and error bars representing S . D . are shown unpaired t - test ) . U2OS - LacO cells transiently expressing Myc - LacI - Bub1 and EGFP - TOP2A ( 970 - 1531 ) were subjected to immunofluorescence staining with DAPI and antibodies for the Myc - tag and GFP . Example images are shown ( I ) . Scale bars , 10 μm . U2OS - LacO cells transiently expressing Myc - LacI - Bub1 and EGFP - TOP2A ( 970 - 1531 ) were subjected to immunofluorescence staining with DAPI and antibodies for the Myc - tag and GFP . The relative enrichment of EGFP - TOP2A at the LacO repeats was quantified in 130 cells ( J ) . Data information : Means and error bars representing S . D . are shown unpaired t - test ) . U2OS - LacO cells transiently expressing Myc - LacI - Bub1 and EGFP - TOP2A ( 970 - 1531 ) were subjected to immunofluorescence staining with DAPI and antibodies for the Myc - tag and GFP . Cell lysates were immunoblotted ( K ) .

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Figure 5B HeLa cells were transfected with siRNA and the plasmids encoding the indicated proteins, and then exposed to nocodazole for 5 hr. Mitotic cells were cytospun onto coverslips and subjected to immunofluorescence staining with DAPI, ACA and anti-GFP antibodies. Example images are shown. Scale bars, 10 μm.HeLa cells were transfected and treated Example images are shown (C). Scale bars, 10 μm.HeLa cells were transfected and treated The immunofluorescence intensity ratios of centromeric EGFP-TOP2A/ACA (D), centromeric EGFP-TOP2A/arm EGFP-TOP2A (E), and arm EGFP-TOP2A/DNA (F), were determined on around 160 chromosomes in 8 cells. Data information: Means and error bars representing S.D. are shown unpaired t-test).HeLa cells transiently expressing EGFP-TOP2A (970-1531) or EGFP-TOP2A (970-1531)-Δ(1015-1032) were treated and immunostained Example images are shown (G). Scale bars, 10 μm.HeLa cells transiently expressing EGFP-TOP2A (970-1531) or EGFP-TOP2A (970-1531)-Δ(1015-1032) were treated and immunostained The immunofluorescence intensity ratio of centromeric EGFP-TOP2A/arm EGFP-TOP2A was determined on around 250 chromosomes in 10 cells (H). Data information: Means and error bars representing S.D. are shown unpaired t-test).U2OS-LacO cells transiently expressing Myc-LacI-Bub1 and EGFP-TOP2A (970-1531) were subjected to immunofluorescence staining with DAPI and antibodies for the Myc-tag and GFP. Example images are shown (I). Scale bars, 10 μm.U2OS-LacO cells transiently expressing Myc-LacI-Bub1 and EGFP-TOP2A (970-1531) were subjected to immunofluorescence staining with DAPI and antibodies for the Myc-tag and GFP. The relative enrichment of EGFP-TOP2A at the LacO repeats was quantified in 130 cells (J). Data information: Means and error bars representing S.D. are shown unpaired t-test).U2OS-LacO cells transiently expressing Myc-LacI-Bub1 and EGFP-TOP2A (970-1531) were subjected to immunofluorescence staining with DAPI and antibodies for the Myc-tag and GFP. Cell lysates were immunoblotted (K).


Figure 6A HeLa cells were transfected with siRNA and plasmids encoding the indicated proteins , and then exposed to nocodazole for 3 hr . Mitotic cells were cytospun onto coverslips and subjected to immunofluorescence staining with DAPI , ACA and anti - GFP antibodies . Example images are shown . Scale bars , 10 μm . U2OS - LacO cells transiently expressing Myc - LacI - Bub1 and the indicated EGFP - TOP2A proteins were subjected to immunofluorescence staining with DAPI and antibodies for the Myc - tag and H2ApT120 . Example images are shown ( B ) . The relative enrichment of EGFP signal at the LacO repeats was quantified in 30 cells ( C ) . Arrows point to the transgene loci . Data information : Means and error bars representing S . D . are shown ; unpaired t test ) . Scale bars , 10 μm . U2OS - LacO cells transiently expressing Myc - LacI - Bub1 and the indicated EGFP - TOP2A proteins Cell lysates were immunoblotted ( D ) . E , F HeLa cells transfected with siRNA and plasmids encoding the indicated proteins were treated and immunostained Example images are shown ( E ) . The immunofluorescence intensity ratio of centromeric EGFP - TOP2A / arm EGFP - TOP2A was determined on around 300 chromosomes in 15 cells ( F ) . Means and error bars representing S . D . are shown unpaired t test ) . Scale bars , 10 μm . G , H HeLa cells transfected with siRNA and plasmids encoding the indicated proteins were treated and immunostained Example images are shown ( G ) . The immunofluorescence intensity ratio of centromeric TOP2A - Myc - 6xHis / arm TOP2A - Myc - 6xHis was determined on around 200 chromosomes in 10 cells ( H ) . Data information : Means and error bars representing S . D . are shown unpaired t test ) . Scale bars , 10 μm . U2OS cells stably expressing the indicated CENP - B fusion proteins were transfected with the plasmid encoding TOP2A - Myc - 6xHis or TOP2A - ΔChT - Myc - 6xHis , and then subjected to immunofluorescence staining with DAPI and antibodies for the Myc - tag and H2ApT120 . Example images are shown ( I ) . Scale bars , 10 μm . U2OS cells stably expressing the indicated CENP - B fusion proteins were transfected with the plasmid encoding TOP2A - Myc - 6xHis or TOP2A - ΔChT - Myc - 6xHis , and then subjected to immunofluorescence staining with DAPI and antibodies for the Myc - tag and H2ApT120 . The relative enrichment of anti - Myc staining signal at the centromere region versus that in the nearby nuclear region was quantified on around 200 chromosomes in 10 cells ( J ) . Data information : Means and error bars representing S . D . are shown unpaired t test ) . U2OS cells stably expressing the indicated CENP - B fusion proteins were transfected with the plasmid encoding TOP2A - Myc - 6xHis or TOP2A - ΔChT - Myc - 6xHis , and then subjected to immunofluorescence staining with DAPI and antibodies for the Myc - tag and H2ApT120 . Cell lysates were immunoblotted ( K ) . L MBP - TOP2A ( 429 - 1531 ) - 10xHis and MBP - TOP2A ( 429 - 1531 ) - ΔChT - 10xHis were subjected to pulldown by GST , GST - H2A , or GST - H2ApS120 , followed by immunoblotting with antibodies for MBP or GST .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 6A HeLa cells were transfected with siRNA and plasmids encoding the indicated proteins, and then exposed to nocodazole for 3 hr. Mitotic cells were cytospun onto coverslips and subjected to immunofluorescence staining with DAPI, ACA and anti-GFP antibodies. Example images are shown. Scale bars, 10 μm.U2OS-LacO cells transiently expressing Myc-LacI-Bub1 and the indicated EGFP-TOP2A proteins were subjected to immunofluorescence staining with DAPI and antibodies for the Myc-tag and H2ApT120. Example images are shown (B). The relative enrichment of EGFP signal at the LacO repeats was quantified in 30 cells (C). Arrows point to the transgene loci. Data information: Means and error bars representing S.D. are shown ; unpaired t test). Scale bars, 10 μm.U2OS-LacO cells transiently expressing Myc-LacI-Bub1 and the indicated EGFP-TOP2A proteins Cell lysates were immunoblotted (D).E, F HeLa cells transfected with siRNA and plasmids encoding the indicated proteins were treated and immunostained Example images are shown (E). The immunofluorescence intensity ratio of centromeric EGFP-TOP2A/arm EGFP-TOP2A was determined on around 300 chromosomes in 15 cells (F). Means and error bars representing S.D. are shown unpaired t test). Scale bars, 10 μm.G, H HeLa cells transfected with siRNA and plasmids encoding the indicated proteins were treated and immunostained Example images are shown (G). The immunofluorescence intensity ratio of centromeric TOP2A-Myc-6xHis/arm TOP2A-Myc-6xHis was determined on around 200 chromosomes in 10 cells (H). Data information: Means and error bars representing S.D. are shown unpaired t test). Scale bars, 10 μm.U2OS cells stably expressing the indicated CENP-B fusion proteins were transfected with the plasmid encoding TOP2A-Myc-6xHis or TOP2A-ΔChT-Myc-6xHis, and then subjected to immunofluorescence staining with DAPI and antibodies for the Myc-tag and H2ApT120. Example images are shown (I). Scale bars, 10 μm.U2OS cells stably expressing the indicated CENP-B fusion proteins were transfected with the plasmid encoding TOP2A-Myc-6xHis or TOP2A-ΔChT-Myc-6xHis, and then subjected to immunofluorescence staining with DAPI and antibodies for the Myc-tag and H2ApT120. The relative enrichment of anti-Myc staining signal at the centromere region versus that in the nearby nuclear region was quantified on around 200 chromosomes in 10 cells (J). Data information: Means and error bars representing S.D. are shown unpaired t test).U2OS cells stably expressing the indicated CENP-B fusion proteins were transfected with the plasmid encoding TOP2A-Myc-6xHis or TOP2A-ΔChT-Myc-6xHis, and then subjected to immunofluorescence staining with DAPI and antibodies for the Myc-tag and H2ApT120. Cell lysates were immunoblotted (K).L MBP-TOP2A (429-1531)-10xHis and MBP-TOP2A (429-1531)-ΔChT-10xHis were subjected to pulldown by GST, GST-H2A, or GST-H2ApS120, followed by immunoblotting with antibodies for MBP or GST.


Figure 7HeLa cells with or without transient expression of H2B - GFP or EGFP - TOP2A were exposed to nocodazole for 3 hr . Mitotic cells were cytospun onto coverslips and subjected to immunofluorescence staining with DAPI and antibodies for GFP , Sgo1 and ACA . Example images are shown ( A ) . The immunofluorescence intensity ratio of centromeric Sgo1 / ACA was determined on 400 chromosomes in 20 cells ( B ) . The means and S . D . s from three independent experiments with the Sgo1 / ACA ratio determined on around total 1200 chromosomes in 60 cells are shown ( C ) . Data information : Means and error bars representing S . D . are shown unpaired t test ) . Scale bars , 10 μm . HeLa cells with or without transient expression of H2B - GFP or EGFP - TOP2A were exposed to nocodazole for 3 hr . Mitotic cells were cytospun onto coverslips and Lysates of asynchronous cells were immunoblotted ( D ) . * represents non - specific bands . U2OS - LacO cells transiently expressing Myc - LacI - Bub1 , with or without EGFP - TOP2A ( 970 - 1531 ) , were subjected to immunofluorescence staining with DAPI and antibodies for H2ApT120 and Sgo1 . Example images are shown ( E ) . Scale bars , 10 μm . U2OS - LacO cells transiently expressing Myc - LacI - Bub1 , with or without EGFP - TOP2A ( 970 - 1531 ) , were subjected to immunofluorescence staining with DAPI and antibodies for H2ApT120 and Sgo1 . The relative enrichment of EGFP - TOP2A ( F ) and Sgo1 ( G ) at the LacO repeats was quantified in 120 cells . Data information : Means and error bars representing S . D . are shown unpaired t test ) . U2OS - LacO cells transiently expressing Myc - LacI - Bub1 , with or without EGFP - TOP2A ( 970 - 1531 ) Cell lysates were immunoblotted ( H ) . U2OS - LacO cells transiently expressing EGFP - LacI - Bub1 - K together with VSV - Sgo1 or a control vector were subjected to immunofluorescence staining with DAPI and antibodies for TOP2A and VSV - tag . Example images are shown ( I ) . The relative enrichment of TOP2A ( J ) and VSV - Sgo1 ( K ) at the LacO repeats was quantified in 50 cells . Data information : Means and error bars representing S . D . are shown unpaired t test ) . Scale bars , 10 μm . U2OS - LacO cells transiently expressing EGFP - LacI - Bub1 - K together with VSV - Sgo1 or a control vector Cell lysates were immunoblotted ( L ) . * represents non - specific bands .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 7HeLa cells with or without transient expression of H2B-GFP or EGFP-TOP2A were exposed to nocodazole for 3 hr. Mitotic cells were cytospun onto coverslips and subjected to immunofluorescence staining with DAPI and antibodies for GFP, Sgo1 and ACA. Example images are shown (A). The immunofluorescence intensity ratio of centromeric Sgo1/ACA was determined on 400 chromosomes in 20 cells (B). The means and S.D.s from three independent experiments with the Sgo1/ACA ratio determined on around total 1200 chromosomes in 60 cells are shown (C). Data information: Means and error bars representing S.D. are shown unpaired t test). Scale bars, 10 μm.HeLa cells with or without transient expression of H2B-GFP or EGFP-TOP2A were exposed to nocodazole for 3 hr. Mitotic cells were cytospun onto coverslips and Lysates of asynchronous cells were immunoblotted (D). * represents non-specific bands.U2OS-LacO cells transiently expressing Myc-LacI-Bub1, with or without EGFP-TOP2A (970-1531), were subjected to immunofluorescence staining with DAPI and antibodies for H2ApT120 and Sgo1. Example images are shown (E). Scale bars, 10 μm.U2OS-LacO cells transiently expressing Myc-LacI-Bub1, with or without EGFP-TOP2A (970-1531), were subjected to immunofluorescence staining with DAPI and antibodies for H2ApT120 and Sgo1. The relative enrichment of EGFP-TOP2A (F) and Sgo1 (G) at the LacO repeats was quantified in 120 cells. Data information: Means and error bars representing S.D. are shown unpaired t test).U2OS-LacO cells transiently expressing Myc-LacI-Bub1, with or without EGFP-TOP2A (970-1531) Cell lysates were immunoblotted (H).U2OS-LacO cells transiently expressing EGFP-LacI-Bub1-K together with VSV-Sgo1 or a control vector were subjected to immunofluorescence staining with DAPI and antibodies for TOP2A and VSV-tag. Example images are shown (I). The relative enrichment of TOP2A (J) and VSV-Sgo1 (K) at the LacO repeats was quantified in 50 cells. Data information: Means and error bars representing S.D. are shown unpaired t test). Scale bars, 10 μm.U2OS-LacO cells transiently expressing EGFP-LacI-Bub1-K together with VSV-Sgo1 or a control vector Cell lysates were immunoblotted (L). * represents non-specific bands.


Figure 8HeLa cells were transfected with siRNA and subjected to immunofluorescence staining with DAPI , ACA and the anti - PICH antibody . Example images of anaphase cells are shown ( A ) . The percentage of PICH - positive cells was determined in at least 339 anaphase cells in each condition . Means and S . D . s from three independent experiments are shown ( B ) . Data information : Means and error bars representing S . D . are shown ; unpaired t test ) . Scale bars , 10 μm . HeLa cells were transfected with siRNA and Lysates of asynchronously growing cells were immunoblotted ( C ) . HeLa cells were released from single thymidine block , and after 7 hr , BAY 1816032 or DMSO was added for 5 hr . Cells were then fixed and subjected to immunofluorescence staining Example images of anaphase cells are shown ( D ) . Scale bars , 10 μm . E HeLa cells were released from single thymidine block , and after 7 hr , BAY 1816032 or DMSO was added for 5 hr . Cells were then fixed and subjected to immunofluorescence staining The percentage of anaphases with PICH was determined ( E ) . HeLa cells were arrested in a prometaphase - like state with nocodazole treatment for 5hr , and then BAY 1816032 or DMSO was added for 1 hr in the continued presence of nocodazole . Then , nocodazole was removed by washing into fresh medium containing BAY 1816032 or DMSO . After 1 hr , cells were fixed and subjected to immunofluorescence staining . Example images are shown ( F ) . The percentage of anaphases with PICH was determined ( G ) . Arrows point to the UFBs . Scale bars , 10 μm . HeLa and the Sgo1 - K492A cells transfected with the indicated siRNA were synchronized and treated with BAY 1816032 or DMSO and then immunostained with the indicated antibodies . Example images of anaphase cells are shown ( H ) . The percentage of anaphases with PICH was determined in at least 256 cells in each condition . Means and S . D . s from three independent experiments are shown ( I ) . Example images of prometaphase cells immunostained with the indicated antibodies are shown ( J ) . Data information : Means and error bars representing S . D . are shown unpaired t test ) . Scale bars , 10 μm . HeLa and the Sgo1 - K492A cells transfected with the indicated siRNA were synchronized and treated with BAY 1816032 or DMSO Lysates of nocodazole - arrested mitotic cells were immunoblotted ( K ) .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 8HeLa cells were transfected with siRNA and subjected to immunofluorescence staining with DAPI, ACA and the anti-PICH antibody. Example images of anaphase cells are shown (A). The percentage of PICH-positive cells was determined in at least 339 anaphase cells in each condition. Means and S.D.s from three independent experiments are shown (B). Data information: Means and error bars representing S.D. are shown ; unpaired t test). Scale bars, 10 μm.HeLa cells were transfected with siRNA and Lysates of asynchronously growing cells were immunoblotted (C).HeLa cells were released from single thymidine block, and after 7 hr, BAY 1816032 or DMSO was added for 5 hr. Cells were then fixed and subjected to immunofluorescence staining Example images of anaphase cells are shown (D). Scale bars, 10 μm.E HeLa cells were released from single thymidine block, and after 7 hr, BAY 1816032 or DMSO was added for 5 hr. Cells were then fixed and subjected to immunofluorescence staining The percentage of anaphases with PICH was determined (E).HeLa cells were arrested in a prometaphase-like state with nocodazole treatment for 5hr, and then BAY 1816032 or DMSO was added for 1 hr in the continued presence of nocodazole. Then, nocodazole was removed by washing into fresh medium containing BAY 1816032 or DMSO. After 1 hr, cells were fixed and subjected to immunofluorescence staining. Example images are shown (F). The percentage of anaphases with PICH was determined (G). Arrows point to the UFBs. Scale bars, 10 μm.HeLa and the Sgo1-K492A cells transfected with the indicated siRNA were synchronized and treated with BAY 1816032 or DMSO and then immunostained with the indicated antibodies. Example images of anaphase cells are shown (H). The percentage of anaphases with PICH was determined in at least 256 cells in each condition. Means and S.D.s from three independent experiments are shown (I). Example images of prometaphase cells immunostained with the indicated antibodies are shown (J). Data information: Means and error bars representing S.D. are shown unpaired t test). Scale bars, 10 μm.HeLa and the Sgo1-K492A cells transfected with the indicated siRNA were synchronized and treated with BAY 1816032 or DMSO Lysates of nocodazole-arrested mitotic cells were immunoblotted (K).


Figure 9A HeLa cells were transfected with plasmids encoding CB - GFP or CB - TOP2A - GFP . Lysates of asynchronously growing cells were immunoblotted with the indicated antibodies . HeLa cells transfected with siRNA and plasmids encoding the indicated proteins were released from single thymidine block . At 12 hr post - release , cells were fixed and subjected to immunofluorescence staining with DAPI and the antibodies for PICH , GFP and CENP - C . Example images of anaphase cells are shown ( B ) . The percentage of anaphases with PICH was determined in at least 152 cells in each condition . Means and S . D . s from three independent experiments are shown ( C ) . Arrows point to the UFBs . Data information : Means and error bars representing S . D . are shown unpaired t test ) . Scale bars , 10 μm . HeLa cells transfected with siRNA and plasmids encoding the indicated proteins were released from single thymidine block . At 12 hr post - release , cells Lysates of nocodazole - arrested mitotic cells were immunoblotted ( D ) . * represents non - specific bands . E , F HeLa cells transiently expressing CB - GFP or CB - TOP2A - GFP were released from single thymidine block , and after 7 hr , BAY 1816032 or DMSO was added for 5 hr . Cells were then fixed and subjected to immunofluorescence staining as in ( B ) . Example images of anaphase cells are shown ( E ) . The percentage of anaphases with PICH was determined in at least 142 cells in each condition . Means and S . D . s from three independent experiments are shown ( F ) . Arrows point to the UFBs . Data information : Means and error bars representing S . D . are shown ; unpaired t test ) . Scale bars , 10 μm . HeLa cells transfected with siRNA and plasmids encoding EGFP - TOP2A ( WT or ΔChT ) were synchronized and then subjected to immunofluorescence staining with DAPI , ACA and the antibodies for PICH and GFP . Example images of anaphase cells are shown ( G ) . Arrows point to the UFBs . Scale bars , 10 μm . HeLa cells transfected with siRNA and plasmids encoding EGFP - TOP2A ( WT or ΔChT ) were synchronized and then subjected to immunofluorescence staining with DAPI , ACA and the antibodies for PICH and GFP . The percentage of anaphases with PICH was determined in at least 174 cells in each condition . Means and S . D . s from three independent experiments are shown ( H ) . Data information : Means and error bars representing S . D . are shown unpaired t test ) . HeLa cells transfected with siRNA and plasmids encoding EGFP - TOP2A ( WT or ΔChT ) were synchronized and then Lysates of asynchronous cells were immunoblotted ( I ) .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 9A HeLa cells were transfected with plasmids encoding CB-GFP or CB-TOP2A-GFP. Lysates of asynchronously growing cells were immunoblotted with the indicated antibodies.HeLa cells transfected with siRNA and plasmids encoding the indicated proteins were released from single thymidine block. At 12 hr post-release, cells were fixed and subjected to immunofluorescence staining with DAPI and the antibodies for PICH, GFP and CENP-C. Example images of anaphase cells are shown (B). The percentage of anaphases with PICH was determined in at least 152 cells in each condition. Means and S.D.s from three independent experiments are shown (C). Arrows point to the UFBs. Data information: Means and error bars representing S.D. are shown unpaired t test). Scale bars, 10 μm.HeLa cells transfected with siRNA and plasmids encoding the indicated proteins were released from single thymidine block. At 12 hr post-release, cells Lysates of nocodazole-arrested mitotic cells were immunoblotted (D). * represents non-specific bands.E, F HeLa cells transiently expressing CB-GFP or CB-TOP2A-GFP were released from single thymidine block, and after 7 hr, BAY 1816032 or DMSO was added for 5 hr. Cells were then fixed and subjected to immunofluorescence staining as in (B). Example images of anaphase cells are shown (E). The percentage of anaphases with PICH was determined in at least 142 cells in each condition. Means and S.D.s from three independent experiments are shown (F). Arrows point to the UFBs. Data information: Means and error bars representing S.D. are shown ; unpaired t test). Scale bars, 10 μm.HeLa cells transfected with siRNA and plasmids encoding EGFP-TOP2A (WT or ΔChT) were synchronized and then subjected to immunofluorescence staining with DAPI, ACA and the antibodies for PICH and GFP. Example images of anaphase cells are shown (G). Arrows point to the UFBs. Scale bars, 10 μm.HeLa cells transfected with siRNA and plasmids encoding EGFP-TOP2A (WT or ΔChT) were synchronized and then subjected to immunofluorescence staining with DAPI, ACA and the antibodies for PICH and GFP. The percentage of anaphases with PICH was determined in at least 174 cells in each condition. Means and S.D.s from three independent experiments are shown (H). Data information: Means and error bars representing S.D. are shown unpaired t test).HeLa cells transfected with siRNA and plasmids encoding EGFP-TOP2A (WT or ΔChT) were synchronized and then Lysates of asynchronous cells were immunoblotted (I).


Figure 1 ( A ) H1299 cells were transfected with Myc - β - catenin , CK1α and MDMX mutants . Phosphorylation of β - catenin S45 was determined by western blot . Co - expression of MDMX blocked the increase of pS45 level . MDMXSG ( W200S / W201G ) and MDMX - 1 - 300 were defective for CK1α binding . ( B ) GST - β - catenin - 1 - 200 was incubated with purified FLAG - CK1α , FLAG - MDMX and ATP at indicated ratios . Phosphorylation of β - catenin S45 was determined by western blot . MDMXS289A contains mutation of CK1α phosphorylation site . ( C ) Kinetic characterization of CK1α inhibition by MDMX . Reaction velocity as a function of β - catenin peptide substrate and increasing MDMX concentrations at 100 µM ATP and 2 nM CK1α . Data were fitted to the Michaelis - Menten equation . The resulting parameter values are shown in the table . ( D ) Reaction velocity as a function of ATP and increasing MDMX concentrations at 800 µM peptide substrate and 2 nM CK1α . Data were fitted to the Michaelis - Menten equation .

O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 1(A) H1299 cells were transfected with Myc-β-catenin, CK1α and MDMX mutants. Phosphorylation of β-catenin S45 was determined by western blot. Co-expression of MDMX blocked the increase of pS45 level. MDMXSG (W200S/W201G) and MDMX-1-300 were defective for CK1α binding.(B) GST-β-catenin-1-200 was incubated with purified FLAG-CK1α, FLAG-MDMX and ATP at indicated ratios. Phosphorylation of β-catenin S45 was determined by western blot. MDMXS289A contains mutation of CK1α phosphorylation site.(C) Kinetic characterization of CK1α inhibition by MDMX. Reaction velocity as a function of β-catenin peptide substrate and increasing MDMX concentrations at 100 µM ATP and 2 nM CK1α. Data were fitted to the Michaelis-Menten equation. The resulting parameter values are shown in the table.(D) Reaction velocity as a function of ATP and increasing MDMX concentrations at 800 µM peptide substrate and 2 nM CK1α. Data were fitted to the Michaelis-Menten equation.


Figure 2 ( A , B ) H1299 cells and human foreskin fibroblasts HFF were stably infected with lentivirus expressing MDMX and analyzed by western blot . ( C ) H1299 cells were transiently transfected with CK1α plasmid and analyzed for β - catenin phosphorylation by western blot . ( D ) Cytoplasmic and nuclear fractions of H1299 cells stably expressing MDMX were analyzed for indicated markers . ( E ) Extract of H1299 expressing MDMX at indicated amounts were analyzed for β - catenin S45 kinase activity in a titration assay . ( F ) Extracts of H1299 cells separately transfected with MDMX or CK1α were mixed in the presence of CK1α inhibitors . In vitro formation of MDMX - CK1α complex was detected by MDMX IP followed by CK1α western blot .

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Figure 2(A, B) H1299 cells and human foreskin fibroblasts HFF were stably infected with lentivirus expressing MDMX and analyzed by western blot.(C) H1299 cells were transiently transfected with CK1α plasmid and analyzed for β-catenin phosphorylation by western blot.(D) Cytoplasmic and nuclear fractions of H1299 cells stably expressing MDMX were analyzed for indicated markers.(E) Extract of H1299 expressing MDMX at indicated amounts were analyzed for β-catenin S45 kinase activity in a titration assay.(F) Extracts of H1299 cells separately transfected with MDMX or CK1α were mixed in the presence of CK1α inhibitors. In vitro formation of MDMX-CK1α complex was detected by MDMX IP followed by CK1α western blot.


Figure 3 ( A , B , C , D ) Cells were treated with MDMX siRNA for 48 hrs and analyzed for β - catenin pS45 level by western blot . ( E ) H1299 and 293T cells were treated with indicated siRNA for 48 hrs . The activity of β - catenin S45 kinase in cell extracts was determined using an ELISA assay . The results are average of 3 experiments ( mean + / - SD ) . * p < 0 . 05 ( Student ' s t test ) .

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Figure 3(A, B, C, D) Cells were treated with MDMX siRNA for 48 hrs and analyzed for β-catenin pS45 level by western blot.(E) H1299 and 293T cells were treated with indicated siRNA for 48 hrs. The activity of β-catenin S45 kinase in cell extracts was determined using an ELISA assay. The results are average of 3 experiments (mean +/- SD). *p<0.05 (Student's t test).


Figure 4 ( B GST - β - catenin - 1 - 200 was incubated with ATP , CK1α and GST - MDMX mutants at indicated ratios . The level of pS45 was determined by western blot . C , D ) GST - β - catenin - 1 - 200 was incubated with ATP , CK1α and GST - MDMX mutants at indicated ratios . The level of pS45 was determined by western blot .

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Figure 4(B GST-β-catenin-1-200 was incubated with ATP, CK1α and GST-MDMX mutants at indicated ratios. The level of pS45 was determined by western blot.C, D) GST-β-catenin-1-200 was incubated with ATP, CK1α and GST-MDMX mutants at indicated ratios. The level of pS45 was determined by western blot.


Figure 5 ( B ) Purified FLAG - CK1α was incubated with ATP , GST - MDMX , GST - β - catenin - 1 - 200 , pDI peptide ( LTFEHYWAQLTS , 5 µM ) , and pDI3A peptide ( LTAEHYAAQATS , 5 µM ) . β - catenin pS45 level was determined by western blot . ( C ) H1299 cells were cotransfected with MDMX , CK1α and p53 mutants . MDMX - CK1α binding was determined by IP - western blot . ( D ) Endogenous p53 in A549 ( wt ) and Panc1 ( R273H ) cells were depleted by CRISPR / Cas9 knockout . β - catenin pS45 level was determined by western blot . ( E ) The β - catenin S45 kinase activity in A549 and Panc1 cells was

O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O

Figure 5(B) Purified FLAG-CK1α was incubated with ATP, GST-MDMX, GST-β-catenin-1-200, pDI peptide (LTFEHYWAQLTS, 5 µM), and pDI3A peptide (LTAEHYAAQATS, 5 µM). β-catenin pS45 level was determined by western blot.(C) H1299 cells were cotransfected with MDMX, CK1α and p53 mutants. MDMX-CK1α binding was determined by IP-western blot.(D) Endogenous p53 in A549 (wt) and Panc1 (R273H) cells were depleted by CRISPR/Cas9 knockout. β-catenin pS45 level was determined by western blot.(E) The β-catenin S45 kinase activity in A549 and Panc1 cells was determined


Figure 6 ( A ) H1299 cells were transfected with MDMX and CK1α mutants . MDMX - CK1α binding was determined by IP - western blot . ( B ) GST - β - catenin - 1 - 200 was incubated with ATP , CK1α and GST - MDMX . The level of pS45 was determined by western blot . ( C ) GST - β - catenin - 1 - 200 was incubated with identical amounts of purified FLAG - CK1α mutants and ATP . The level of pS45 was determined by western blot . ( D ) . ELISA plates coated with GST - β - catenin - 1 - 200 was incubated with purified FLAG - CK1α mutants , ATP , and MDMX . The level of pS45 was determined by ELISA assay . The results are average of 3 experiments ( mean + / - SD ) .

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Figure 6(A) H1299 cells were transfected with MDMX and CK1α mutants. MDMX-CK1α binding was determined by IP-western blot.(B) GST-β-catenin-1-200 was incubated with ATP, CK1α and GST-MDMX. The level of pS45 was determined by western blot.(C) GST-β-catenin-1-200 was incubated with identical amounts of purified FLAG-CK1α mutants and ATP. The level of pS45 was determined by western blot.(D). ELISA plates coated with GST-β-catenin-1-200 was incubated with purified FLAG-CK1α mutants, ATP, and MDMX. The level of pS45 was determined by ELISA assay. The results are average of 3 experiments (mean +/- SD).


Figure 7 ( A ) MEF cell lines derived from 3 p53 - / - ; MDMXSG / SG and 3 p53 - / - ; MDMXwt / wt embryos were analyzed for β - catenin pS45 level by western blot . ( B ) CK1α activity in MEF cell extracts was determined using ELISA kinase activity assay . The results are average of 3 experiments ( mean + / - SD ) . * p < 0 . 01 ( Student ' s t test ) . ( C ) Volcano plot of differential gene expression between p53 - / - ; MDMXSG / SG and p53 - / - ; MDMXwt / wt MEF lines . ( D ) Multiple Wnt signaling genes were down regulated in p53 - / - ; MDMXSG / SG MEF cell lines . ( E ) RT - PCR confirmation of Wnt pathway genes down - regulated in p53 - / - ; MDMXSG / SG MEF ( average of 3 cell lines , mean + / - SD ) and thymus from 1 month old mice ( average of 3 mice , mean + / - SD ) . * * p < 0 . 01 , * p < 0 . 05 ( Student ' s t test ) . ( F ) p53 - / - ; MDMXSG / SG and p53 - / - ; MDMXwt / wt cells were treated with conditioned medium containing Wnt3a . Axin2 mRNA level was determined by RT - PCR ( average of 3 experiments , mean + / - SD ) . * p < 0 . 05 ( Student ' s t test ) .

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Figure 7(A) MEF cell lines derived from 3 p53-/-;MDMXSG/SG and 3 p53-/-;MDMXwt/wt embryos were analyzed for β-catenin pS45 level by western blot.(B) CK1α activity in MEF cell extracts was determined using ELISA kinase activity assay. The results are average of 3 experiments (mean +/- SD). *p<0.01 (Student's t test).(C) Volcano plot of differential gene expression between p53-/-;MDMXSG/SG and p53-/-;MDMXwt/wt MEF lines.(D) Multiple Wnt signaling genes were down regulated in p53-/-;MDMXSG/SG MEF cell lines.(E) RT-PCR confirmation of Wnt pathway genes down-regulated in p53-/-;MDMXSG/SG MEF (average of 3 cell lines, mean +/- SD) and thymus from 1 month old mice (average of 3 mice, mean +/- SD). **p<0.01, *p<0.05 (Student's t test).(F) p53-/-;MDMXSG/SG and p53-/-;MDMXwt/wt cells were treated with conditioned medium containing Wnt3a. Axin2 mRNA level was determined by RT-PCR (average of 3 experiments, mean +/- SD). *p<0.05 (Student's t test).


Figure 8 ( A ) Identical starting number of cells from the MEF lines were serially passaged following identical schedules . The differences in cell number were determined after 3 and 5 passages . The results are average of 3 experiments ( mean + / - SD ) . ( B ) Identical number of cells from the MEF lines were plated at low density and cultured for 14 days . Colony formation efficiency was determined by crystal violate staining . ( C ) Survival curves of p53 - / - ; MDMXSG / SG and p53 - / - ; MDMXwt / wt mice . Thymic lymphomas were the major cause of mortality in both genotypes .

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Figure 8(A) Identical starting number of cells from the MEF lines were serially passaged following identical schedules. The differences in cell number were determined after 3 and 5 passages. The results are average of 3 experiments (mean +/- SD).(B) Identical number of cells from the MEF lines were plated at low density and cultured for 14 days. Colony formation efficiency was determined by crystal violate staining.(C) Survival curves of p53-/-;MDMXSG/SG and p53-/-;MDMXwt/wt mice. Thymic lymphomas were the major cause of mortality in both genotypes.


Figure 1 Time - lapse microscopy of sporozoites , 10 min after intradermal inoculation into mice with CD31 - labeled vascular endothelia . Maximum intensity projection over 240 sec shows trajectories of moving sporozoites ( green ) and blood vessels ( magenta ) . The light green structures in the first two panels are autofluorescent hair follicles . Scale bar , 50 μm .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 1 Time-lapse microscopy of sporozoites, 10 min after intradermal inoculation into mice with CD31-labeled vascular endothelia. Maximum intensity projection over 240 sec shows trajectories of moving sporozoites (green) and blood vessels (magenta). The light green structures in the first two panels are autofluorescent hair follicles. Scale bar, 50 μm.


Figure 2 ( A ) P . berghei sporozoite trajectories obtained with manual ( left ) and automated ( right ) tracking methods . ( B ) Image processing and automated tracking method . Shown are images illustrating each step of automated tracking : The raw data image shows the first frame of time - lapse microscopy of P . berghei sporozoites ( red ) after intradermal inoculation , followed by the same image after background subtraction and thresholding using Fiji software . Spot detection was run using ICY software and the final image shows the overlay with tracks generated using the spot tracking plugin of ICY software . Scale bars , 50 μm . ( C ) Apparent speed of sporozoites 5 to 120 min after intradermal inoculation , obtained with manual and automated tracking methods . Data is displayed in box and whisker plots with whiskers showing 10 to 90 percentiles and values below and above the whiskers shown individually . Horizontal line indicates the median speed . No statistically significant differences between the corresponding timepoints of manual and automated tracking methods were found ( Kruskal - Wallis test ) . ( D ) Displacement of sporozoites 5 to 120 min after intradermal inoculation , obtained with manual and automated tracking methods . To normalize between tracks of different duration , displacement is displayed as final displacement per 30 seconds interval . Data is displayed in box and whisker plots with whiskers showing 10 to 90 percentiles and values below and above the whiskers shown individually . Horizontal line indicates the median . The only statistically significant difference in sporozoite displacement between the manual and automated tracking methods was at 120 minutes ( Kruskal - Wallis test , * * * * p < 0 . 0001 ) . For panels C & D , a varying number of videos were processed for each time point : C : 5 min ( 3 videos / 37 manual tracks / 203 automated tracks ) , 10 min ( 6 videos / 112 manual tracks / 292 automated tracks ) , 20 min ( 4 videos / 95 manual tracks / 184 automated tracks ) , 30 min ( 6 videos / 63 manual tracks / 235 automated tracks ) , 60 min ( 7 videos / 77 manual tracks / 162 automated tracks ) , and 120 min ( 6 videos / 44 manual tracks / 151 automated tracks ) . D : 5 min ( 3 videos / 61 manual tracks / 203 automated tracks ) , 10 min ( 6 videos / 74 manual tracks / 292 automated tracks ) , 20 min ( 4 videos / 47 manual tracks / 184 automated tracks ) , 30 min ( 6 videos / 53 manual tracks / 235 automated tracks ) , 60 min ( 7 videos / 65 manual tracks / 162 automated tracks ) , and 120 min ( 6 videos / 48 manual tracks / 151 automated tracks ) .

O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 2(A) P. berghei sporozoite trajectories obtained with manual (left) and automated (right) tracking methods. (B) Image processing and automated tracking method. Shown are images illustrating each step of automated tracking: The raw data image shows the first frame of time-lapse microscopy of P. berghei sporozoites (red) after intradermal inoculation, followed by the same image after background subtraction and thresholding using Fiji software. Spot detection was run using ICY software and the final image shows the overlay with tracks generated using the spot tracking plugin of ICY software. Scale bars, 50 μm. (C) Apparent speed of sporozoites 5 to 120 min after intradermal inoculation, obtained with manual and automated tracking methods. Data is displayed in box and whisker plots with whiskers showing 10 to 90 percentiles and values below and above the whiskers shown individually. Horizontal line indicates the median speed. No statistically significant differences between the corresponding timepoints of manual and automated tracking methods were found (Kruskal-Wallis test). (D) Displacement of sporozoites 5 to 120 min after intradermal inoculation, obtained with manual and automated tracking methods. To normalize between tracks of different duration, displacement is displayed as final displacement per 30 seconds interval. Data is displayed in box and whisker plots with whiskers showing 10 to 90 percentiles and values below and above the whiskers shown individually. Horizontal line indicates the median. The only statistically significant difference in sporozoite displacement between the manual and automated tracking methods was at 120 minutes (Kruskal-Wallis test, ****p<0.0001). For panels C&D, a varying number of videos were processed for each time point: C: 5 min (3 videos/37 manual tracks/203 automated tracks), 10 min (6 videos/112 manual tracks/292 automated tracks), 20 min (4 videos/95 manual tracks/184 automated tracks), 30 min (6 videos/63 manual tracks/235 automated tracks), 60 min (7 videos/77 manual tracks/162 automated tracks), and 120 min (6 videos/44 manual tracks/151 automated tracks). D: 5 min (3 videos/61 manual tracks/203 automated tracks), 10 min (6 videos/74 manual tracks/292 automated tracks), 20 min (4 videos/47 manual tracks/184 automated tracks), 30 min (6 videos/53 manual tracks/235 automated tracks), 60 min (7 videos/65 manual tracks/162 automated tracks), and 120 min (6 videos/48 manual tracks/151 automated tracks).


Figure 3 ( A - C ) Tracks generated through automated tracking were plotted to a common origin to visualize parasite dispersal P . berghei ( A ) , P . yoelii ( B ) and P . falciparum ( C ) at 5 , 10 , 30 and 60 min after intradermal inoculation . For each timepoint , the tracks are representative of the average displacement of the entire sporozoite population . Track numbers used for each timepoint were : 5 min , 40 tracks ; 10 min , 32 tracks ; 30 min , 25 tracks ; 60 min , 21 tracks . ( D - F ) Displacement of P . berghei ( D ) , P . yoelii ( E ) and P . falciparum ( F ) sporozoites 5 - 120 min after inoculation in box and whisker plots with whiskers displaying the 10 - 90 percentiles and values below and above the whiskers shown individually . Horizontal lines show median displacement at each time point . Percentage values in boxes indicate the fraction of tracks displacing over 75 micrometers at each time point . Within a given species , the difference in sporozoite displacement between 5 min and 120 min is statistically significant ( Kruskal - Wallis test : P . berghei p < 0 . 0001 , P . yoelii p < 0 . 0001 , P . falciparum p < 0 . 01 ) . Comparisons between species showed no statistically significant difference in displacements for any given time point , with the exception of the 10 min time point in which there was a significant difference between P . berghei and P . falciparum ( Kruskal - Wallis test , * p < 0 . 05 ) . For panels ( D - F ) , a varying number of videos were processed for each time point after inoculation : ( D ) Same dataset as used for Figure 2D : 5 min ( 3 videos / 203 tracks ) , 10 min ( 6 videos / 292 tracks ) , 20 min ( 4 videos / 184 tracks ) , 30 min ( 6 videos / 235 tracks ) , 60 min ( 7 videos / 162 tracks ) and 120 min ( 6 videos / 151 tracks ) . ( E ) 5 min ( 7 videos / 714 tracks ) , 10 min ( 16 videos / 1514 tracks ) , 20 min ( 4 videos / 415 tracks ) , 30 min ( 4 videos / 319 tracks ) , 60 min ( 4 videos / 255 tracks ) , and 120 min ( 4 videos / 180 tracks ) . ( F ) 5 min ( 6 videos / 389 tracks ) , 10 min ( 6 videos / 464 tracks ) , 20 min ( 3 videos / 184 tracks ) , 30 min ( 2 videos / 195 tracks ) , 60 min ( 2 videos / 239 tracks ) , and 120 min ( 2 videos / 103 tracks ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 3  (A-C) Tracks generated through automated tracking were plotted to a common origin to visualize parasite dispersal P. berghei (A), P. yoelii (B) and P. falciparum (C) at 5, 10, 30 and 60 min after intradermal inoculation. For each timepoint, the tracks are representative of the average displacement of the entire sporozoite population. Track numbers used for each timepoint were: 5 min, 40 tracks; 10 min, 32 tracks; 30 min, 25 tracks; 60 min, 21 tracks. (D-F) Displacement of P. berghei (D), P. yoelii (E) and P. falciparum (F) sporozoites 5-120 min after inoculation in box and whisker plots with whiskers displaying the 10-90 percentiles and values below and above the whiskers shown individually. Horizontal lines show median displacement at each time point. Percentage values in boxes indicate the fraction of tracks displacing over 75 micrometers at each time point. Within a given species, the difference in sporozoite displacement between 5 min and 120 min is statistically significant (Kruskal-Wallis test: P. berghei p<0.0001, P. yoelii p<0.0001, P. falciparum p<0.01). Comparisons between species showed no statistically significant difference in displacements for any given time point, with the exception of the 10 min time point in which there was a significant difference between P. berghei and P. falciparum (Kruskal-Wallis test, *p<0.05). For panels (D-F), a varying number of videos were processed for each time point after inoculation: (D) Same dataset as used for Figure 2D: 5 min (3 videos/203 tracks), 10 min (6 videos/292 tracks), 20 min (4 videos/184 tracks), 30 min (6 videos/235 tracks), 60 min (7 videos/162 tracks) and 120 min (6 videos/151 tracks). (E) 5 min (7 videos/714 tracks), 10 min (16 videos/1514 tracks), 20 min (4 videos/415 tracks), 30 min (4 videos/319 tracks), 60 min (4 videos/255 tracks), and 120 min (4 videos/180 tracks). (F) 5 min (6 videos/389 tracks), 10 min (6 videos/464 tracks), 20 min (3 videos/184 tracks), 30 min (2 videos/195 tracks), 60 min (2 videos/239 tracks), and 120 min (2 videos/103 tracks).