EMBO/soda-nel huggingface dataset:
UBQLN4 recognizes mislocalized transmembrane domain proteins and targets these to proteasomal degradation (2016)
Figure 3

Figure 3. ΔSS mutant form of IL-2Rα as a model for mislocalized proteins.

(A) Schematic representation of the IL-2Rα proteins used in this study. WT: wild-type, ΔSS: signal sequence (SS)-deleted mutant, and ΔSSΔTM: SS and transmembrane domain (TM)-deleted mutant.

(B) IL-2Rα ΔSS mislocalized to the cytosolic fraction. HeLa cells, expressing Flag-tagged IL-2Rα WT or ΔSS, were fractionated to the cytosolic (Cyt.) and membrane (Mem.) fractions. Tubulin was used as a cytoplasmic marker, while calnexin was used for the ER membrane fraction.

(C) IL-2Rα ΔSS was not glycosylated. Flag-tagged IL-2Rα WT or ΔSS proteins was expressed in HeLa cells, and immunoprecipitated with an anti-Flag antibody. The precipitates were incubated with (+) or without (-) 10 unit of the deglycosylation enzyme PNGase F for 2 h, and subjected to western blot analysis with an anti-Flag antibody. Glycosylated and non-glycosylated signals are indicated.

(D) The glycosylated form of IL-2Rα WT is a stable protein, while mislocalized IL-2Rα ΔSS is degraded rapidly via its TMD. A series of IL-2Rα deletion proteins were expressed in HeLa cells and then chased with 20 μg/mL CHX for the indicated periods. Actin was used as a loading control. The right panel is a quantified graph of the left blot signals represent mean ± SD calculated from 4 independent experiments (n=4).

(E) IL-2Rα ΔSS is degraded in a proteasome-dependent manner. HeLa cells expressing Flag-tagged IL-2Rα ΔSS were treated with 20 μg/mL CHX as well as 0.1% DMSO (DM.), 10 μM MG-132 (MG.), 2 μM Bortezomib (Bor.), 10 μM Leupeptin (Leu.), 0.1 μM Bafilomycin A1 (Baf.), or 10 μg/ml Pepstatin A (pep. A)/E64d for the indicated periods. The right panel indicates that the quantified data of the left blot signals represent mean ± SD calculated from 3 independent experiments (n=3).

(F) The Flag-tagged IL-2Rα ΔSS mutant and T7-tagged ubiquitin were co-expressed in HeLa cells and the cells were treated with (+) or without (-) 10 μM MG-132. After 4 h, Flag-precipitates were blotted with anti-T7 and anti-Flag antibodies.

(G) HeLa cells expressing Flag-tagged IL-2Rα WT,ΔSS, and ΔSSΔTM proteins were treated with (+) or without (-) 10 μM MG-132. At 4 h after MG-132 treatment, the cells were lysed and analyzed by immunoblotting using the indicated antibodies. Note that a higher migrating form of non-glycosylated (and thus defective) IL-2Rα WT (indicated by the arrowhead) is detected strongly in the presence of MG-132.
UBQLN4 recognizes mislocalized transmembrane domain proteins and targets these to proteasomal degradation (2016)
Figure 4

Figure 4. UBQLN4 is essential for the degradation of mislocalized IL-2Rα ΔSS.

(A, B) IL-2Rα ΔSS is stabilized in UBQLN4-knockdown cells. HeLa cells were transfected with three distinct siRNA duplexes for UBQLN4 (UBQLN4 siRNA#1~#3) or control siRNA. At 48 h after siRNA transfection, Flag tagged-IL-2Rα ΔSS was expressed in the cells. At 24 h after IL-2Rα ΔSS transfection, the cells were chased with 20 μg/mL CHX and harvested at the indicated time after CHX addition. Anti-Flag signals in the control or UBQLN4 siRNA-treated cells (siRNA#1~#3) were quantified at the indicated time points. The data represent mean ± SEM calculated from 4 independent experiments (n=4). The UBQLN4 signal is marked by an arrowhead, while the band just below it (indicated by an asterisk) is a non-specific band, since this signal was never affected by any of the UBQLN4 siRNAs. Actin was used as a loading control.

(C) Polyubiquitin modification of IL-2Rα ΔSS was not diminished, but rather strengthened, in UBQLN4 knockdown cells. At 48 h after transfection of HeLa cells with siRNA for UBQLN4 or BAG6, Flag-tagged IL-2Rα ΔSS and T7-tagged ubiquitin were expressed with (+) or without (-) 10 mM MG-132. From these cells, Flag-tagged IL-2Rα ΔSS was affinity-purified using Hot lysis analysis, and Flag-precipitates were blotted with an anti-T7 antibody to detect polyubiquitination of IL-2Rα ΔSS.
UBQLN4 recognizes mislocalized transmembrane domain proteins and targets these to proteasomal degradation (2016)
Figure 5

Figure 5. UBQLN4 recognizes misassembled IL-2Rα through its unembedded TMD. (A) A series of Flag-tagged truncated mutants of IL-2Rα substrates were expressed in HeLa cells with T7-tagged UBQLN4 and treated with (+) or without (-) 10 µM MG-132 for 4 h. Flag-IL-2Rα substrates were immunoprecipitated and their co-precipitation with UBQLN4 was analyzed.

(B) T7-UBQLN4 was immunoprecipitated, and its interactions with a series of IL-2Rα proteins were detected.
UBQLN4 recognizes mislocalized transmembrane domain proteins and targets these to proteasomal degradation (2016)
Figure 6

Figure 6. The STI-II region of UBQLN4 determines its specificity to misassembled IL-2Rα

(A) Schematic representation of the UBQLN1, UBQLN4, and STI-II mutant proteins used in this study.

(B) UBQLN4 dominantly co-precipitates IL-2Rα ΔSS over UBQLN1. Precipitates of Flag-tagged UBQLN1 or UBQLN4 from HeLa cell lysates were probed with IL-2Rα ΔSS.

(C) The complete amino acid sequences of human UBQLN1 and UBQLN4 were analyzed using the disorder/order structure prediction program PONDR-FIT. The STI-II region of UBQLN4 is indicated by the red box, and that of UBQLN1 is boxed blue. Note that the disorder tendency of the UBL, STI-I, and UBA regions is nearly identical between UBQLN1 and UBQLN4 (indicated by black and green boxes), while that of the STI-II region is quite distinct, and the STI-II of UBQLN4 shows a high probability of an ordered structure. The numbers denote the corresponding amino acid positions in these proteins.

(D) Amino acid sequence alignments of the STI-II regions of UBQLN1 and UBQLN4. UBQLN4-specific residues are shown as red characters. HS: Homo sapiens (Mammalia), MM: Mus musculus (Mammalia), GG: Gallus gallus (Aves), PS: Pelodiscus sinensis (Reptilia), and XL: Xenopus laevis (Amphibia).

(E) Substitution of the STI-II sequence of UBQLN4 with that of UBQLN1 (designated as the 4-STI-II-1 mutant protein) abolished its high affinity to the IL-2Rα ΔSS client protein.

(F) Substitution of the STI-II sequence of UBQLN1 with that of UBQLN4 (designated as the 1-STI-II-4) did not enable UBQLN1 to recognize IL-2Rα ΔSS.
UBQLN4 recognizes mislocalized transmembrane domain proteins and targets these to proteasomal degradation (2016)
Figure 7

Figure 7. STI-II region of UBQLN4 is essential for its binding to client proteins.

Schematic representation of the UBQLN4 deletion mutant proteins used in this study.

(B, C and D) The wild-type (WT) form of Flag-tagged UBQLN4 and its truncated derivatives were expressed in HeLa cells with T7-tagged IL-2Rα ΔSS (B and C) and T7-tagged ubiquitin (D). At 4 h after the addition of 10 μM MG-132, UBQLN4 was immunoprecipitated with an anti-Flag antibody and the precipitates were blotted with an anti-T7 antibody.

(E) GST-tagged and purified UBQLN4 and its truncated derivatives (ΔUBA and ΔSTI-II) were incubated with K48-linked polyubiquitin chains. After in vitro GST pull-down, the precipitates were probed with an anti-polyubiquitin (FK2) antibody.
UBQLN4 recognizes mislocalized transmembrane domain proteins and targets these to proteasomal degradation (2016)
Figure 8

Figure 8. SRP deficiency enhances the interaction between UBQLN4 and its client proteins.

(A) UBQLN4 interacts with non-glycosylated (and thus defective) IL-2Ra WT in SRP54-knockdown cells. After treatment of HeLa cells with SRP54 siRNA (10 nM) for 48 h, Flag-tagged IL-2Rα WT and T7-tagged UBQLN4 proteins were expressed, and the cells were treated with (+) or without (-) 10 µM MG-132 at 4 h before harvesting. UBQLN4 was affinity-purified from cell extracts and probed with an anti-Flag antibody. Note that the levels of non-glycosylated IL-2Rα WT (indicated as Defective) increased, while the glycosylated form of this protein (indicated as Assembled) decreased in SRP54 knockdown cells.

(B, C) SRP54 knockdown stimulates polyubiquitinated proteins co-precipitation of UBQLN4. Flag-tagged UBQLN4 and T7-tagged ubiquitin were expressed in SRP54 siRNA-treated cells, and Flag precipitates were probed with an anti-T7 antibody to detect polyubiquitinated clients co-precipitation with UBQLN4 under no addition of MG-132 (B). Note that the interaction of UBQLN4 with polyubiquitinated substrates was further increased in SRP54 knockdown cells treated with MG-132 for 4 h before harvesting (C).
IMPDH inhibition activates TLR-VCAM1 pathway and suppresses the development of MLL-fusion leukemia (2022)
Figure 1

Figure 1. IMPDH inhibitors suppress the growth of MLL-fusion leukemia in vitro

(A) Schematic of de novo purine nucleotide synthesis.

(B and C) IC50 values for MPA-response curves using cell viability assay were shown. Human AML cell lines (with MLL-fusions: MV4;11, MOLM13, NOMO1 and THP1, without MLL-fusions: HL60, Kasumi-1, OCI-AML3 and U937), human cord blood (CB) CD34+ cells, and those expressing MLL-AF9 (CB-MLL-AF9#1, 2, 3) or MLL-ENL (CB#MLL-ENL-1, 2), and patient cells with or without MLL-fusions (AML#1, 2, 3 or AML#4, 5, 6) were treated with titrating doses of MPA (1-100 μM) for 72 hours in three technical replicates.

(D) Response curves of FF-10501-01 (1-100 μM) for 72 hours in three technical replicates.

(E) FCM plots of the cell cycle and apoptosis analyses in MLL-AF9 expressing CB cells and MV4;11 cells. Cells were incubated with/without 1 μM MPA and 100 mM Guanosine for 36 hours. Numbers indicate the frequency of S/G2/M-phase cells (left) and Annexin V+ cells (right).

(F and G) Immunophenotypes and Wright-Giemsa staining of MLL-AF9 expressing CB cells after four days of culture with/without 3.3 μM MPA and 100 μM Guanosine (Scale bar: 20 mm).

(H) MLL-ENL-expressing CB cells and MOLM13 cells were transduced with Cas9 together with non-targeting (NT) sgRNA or IMPDH2-targeting sgRNAs (IMPDH2-KO1 and KO2) co-expressing tRFP657. Changes in the frequency of tRFP657+ cells (sgRNA-transduced cells) in cell cultures are shown. Results are normalized to the frequency of tRFP657+ cells at day 4, set to 1.

(I) The tRFP657+ cells in (H) were sorted and subjected to western blotting. The depletion of IMPDH2 protein in MLL-ENL cells and MOLM13 cells was confirmed by western blotting.

Data information: All data are shown as mean ± SEM.
IMPDH inhibition activates TLR-VCAM1 pathway and suppresses the development of MLL-fusion leukemia (2022)
Figure 3

Figure 3. The therapeutic effect of IMPDH inhibition on AML is p53-p21 independent

(A) Immunoblotting of the cord blood cells and those expressing RUNX1-ETO or MLL-AF9. The cells were incubated with/without 1 μM MPA (M) and 100 μM Guanosine (G) for 24 h.

(B) CB-MLL-AF9#2 cells were transduced with a Non-targeting (NT) control and an shRNA targeting p21 (sh-p21). Cells were incubated with/without 1 μM MPA (M) and 100 μM guanosine (G) for 24 hours. Total cell lysates were analyzed by western blotting using the antibodies for p21 and Tubulin.

(C) CB-MLL-AF9#2 cells transduced with NT or sh-p21 shRNAs were incubated with MPA at the indicated concentration for 72 hours. Cell viability assays were performed using WST-1 in three technical replicates.

(D) CB-MLL-AF9#2 cells were transduced by vector control or p53DD (dominant-negative) and were incubated with/without 1 μM MPA (M) and 100 μM guanosine (G) for 24 h. Total cell lysates were analyzed by western blotting using the antibodies for p53 and Tubulin. Note the elevated levels of endogenous p53 protein (upper arrow) in p53DD (lower arrow)-expressing cells.

(E) Cells were incubated with MPA at the indicated concentration with/without 100 μM guanosine for 72 hours then measured with WST-1 in three technical replicates.

(F) Mouse bone marrow c-Kit+ cells derived from Trp53-/- mice were transduced with MLL-AF9-GFP and were transplanted into mice to generate p53-deficient (p53-/-) leukemia cells. GFP+ MLL-AF9 leukemia cells were collected from bone marrows of vehicle- or FF-10501-01-treated wild-type and p53-/- leukemic mice. Expression levels of p53 and Tubulin were assessed by western blotting 24 hours after the treatment.

(G) Experimental scheme used in (H). The p53-deficient MLL-AF9-bearing mice were treated with vehicle, FF-10501-01 or DS-5272 every other day from day 1.

(H) Kaplan-Meier survival curves of p53-/- MLL-AF9 leukemia mice treated with vehicle or FF-10501-01or DS-5272 (n = 6 per group). Statistical significance was evaluated by the log-rank test (left). Frequency of GFP+ leukemia cells in peripheral blood at day 19 (right, n = 6 per group). A two-tailed unpaired t-test was used for the comparison.

Data information: All data are shown as mean ± SEM.
IMPDH inhibition activates TLR-VCAM1 pathway and suppresses the development of MLL-fusion leukemia (2022)
Figure 5

Figure 5. IMPDH inhibition leads to overactivation of the TLR-TRAF6-NF-κB pathway

(A) Experimental scheme used in (B). Ba/F3 cells were transduced with NF-κB-GFP reporter and were then transduced with individual mouse TLR4, TLR5, or TLR7 to establish BaκB cells expressing each TLR. TLR2 is expressed in parent Ba/F3 cells.

(B) The TLR-expressing BaκB cells were stimulated with the corresponding TLR ligands (100 ng/mL Pam3CSK4 for TLR2, 100 ng/mL LPS for TLR4, 10 ng/mL Flagellin for TLR5, 100 ng/ml R848 for TLR7) with titrating doses of MPA (1 - 10 μM). GFP expression was assessed 24 hours after the stimulation.

(C) 293T cells were transfected with FLAG-tagged TRAF6 and HA-tagged K63-ubiquitin. After 24 hours, these cells were treated with 3 or 10 μM MPA (M-3 μM or M-10 μM) with/without 100 μM guanosine (G). IRAK1/4 inhibitor (IRAK1/4 i) was also used as a control. Whole-cell extracts were immunoprecipitated with anti-FLAG antibody, and ubiquitinated TRAF6 was detected with anti-HA antibody.

(D, E) Murine bone marrow-derived macrophages (BMDMs) were treated with vehicle, 10 μM MPA alone (D) or co-treated with vehicle, 10 μM MPA (M), 100 ng/mL Pam3CSK4 and 100 μM Guanosine (G) for 4 hours (E), as indicated. N=3 (technical replicates) per group. mRNA levels of TNF-α and IL-1β were measured by qPCR. Two-tailed unpaired t-tests were used for the comparison in (D). Ordinary one-way ANOVA was used for the comparison in (E).

(F, G) (F) THP1 cells and (G) MLL-AF9-expressing CB cells (CB-MLL-AF9#1) were pretreated with 10 μM MPA for 2 hours and then treated with 1000 ng/mL Pam3CSK4 for 0, 30, 60, and (120) minutes. Cells were lysed and subjected to SDS-PAGE. Levels of total IκBα, phosphate-p38, total p38, and GAPDH were evaluated by western blotting.

Data information: All data are shown as mean ± SEM.
The phagosomal solute transporter SLC15A4 promotes inflammasome activity via mTORC1 signaling and autophagy restraint in dendritic cells (2022)
Figure 1

Figure 1. SLC15A4 promotes inflammasome activity by inhibiting autophagy

(A-J). WT BMDCs transduced with retrovirus encoding SLC15A4-GFP (A, B), or lentiviruses encoding non-target (ctrl) or any of three SLC15A4-specific shRNAs (C, D) or WT or SLC15A4feeble BMDCs (E-J) were treated with LPS/OVA-TxR polystyrene beads (A) or STm-mcherry (B), unstimulated or infected with flagellin-expressing STm (C, E-J) or primed for 3 h with LPS and stimulated with alum or MSU (D). (A, B). Cells were analyzed by live-cell imaging 2 h after the pulse (A) or 1 h after infection (B). Representative images. Differential interference contrast (DIC) image shows cell shape and outline. Arrowheads, phagosomes; arrows, phagosomal tubules. Scale bars, 6 μm. (C-J). Cell supernatants collected 2 h (C, J) or 4 h (D) after treatment were assayed for IL-1β by ELISA. (C, D, J). Representative plots of three independent experiments. (E-I). Cell pellets collected at the indicated time points after STm infection were lysed, fractionated by SDS-PAGE and immunoblotted for caspase-1 (E, F), GSDMD or LC-3 (G-I). (E, G). Representative immunoblots showing pro-caspase-1 (pro-casp.-1) and cleaved p20 (casp.-1 p20) bands (E) or GSDMD full length (GSDMD FL), cleaved GSDMD N-terminal fragment (GSDMD N-ter), LC3-II and LC3-I bands in cell lysates (G). (F, H, I) Quantification of band intensities for caspase-1 p20 normalized to pro-caspase-1 and actin (E), GSDMD N-ter normalized to actin (G) and LC3-II normalized to LC3-I and actin (H) from three independent experiments are shown as fold change (F, H) or fold induction (I) relative to time 0.

Data information: Data represent mean ± SD. * p<0.05; **p<0.01; ***p<0.001. Two-tailed Student's t-test. See also Figs. EV1, EV2 and EV3.
The phagosomal solute transporter SLC15A4 promotes inflammasome activity via mTORC1 signaling and autophagy restraint in dendritic cells (2022)
Figure 3

Figure 3. SLC15A4feeble intestinal CD11c+ DCs show reduced IL-1β secretion and increased autophagy

Intestinal CD11c+ DCs were isolated from colons of WT and SLC15A4feeble mice on day 16 and cultured overnight. A. IL-1β was measured from culture supernatants by ELISA in three independent experiments. B. Cell pellets from three independent experiments were immunoblotted for LC3 and actin. Upper panel. Representative immunoblot. Lower panel. Quantification of band intensities for LC3-II normalized to LC3-I and actin are shown as fold induction relative to samples from untreated mice. Note that LC3-I signal from ex vivo samples is almost undetectable.

Data information: Mean is indicated. **p<0.01. Mann-Whitney non-parametric statistical test. See also Fig. EV2G.
The phagosomal solute transporter SLC15A4 promotes inflammasome activity via mTORC1 signaling and autophagy restraint in dendritic cells (2022)
Figure 4

Figure 4. SLC15A4 promotes inflammasome perinuclear positioning away from autophagic membranes

(A-G). WT or SLC15A4feeble BMDCs expressing ASC-GFP and mCherry-LC3 (A, B, D) or non-transduced (C) or SLC15A4feeble BMDCs transduced with SLC15A4-GFP (E, F, G) were infected with flagellin-expressing STm and fixed (A-F) or lysed (G) 1 h after infection. A. Cells were analyzed by fluorescence microscopy. Representative images showing ASC speck (green) relative to mCherry-LC3 (red) in two infected WT and SLC15A4feeble DCs each. B. Quantification of LC3 fluorescence per unit area in a radius of 1 μm surrounding the ASC speck in 20 cells per cell type in each of 3 independent experiments. C. Cells were stained for endogenous ASC and p62, labeled with DAPI and analyzed by fluorescence microscopy. Representative images showing ASC speck (red) relative to p62 (green) in two infected WT and SLC15A4feeble DCs each. Note p62 staining surrounding ASC specks in SLC15A4feeble DCs. D. Quantification of perinuclear (within a radius of 3 μm from the nucleus) ASC specks in at least 30 cells per cell type in each of 3 independent experiments. (E, F). Cells were stained for endogenous ASC, labeled with DAPI and analyzed by fluorescence microscopy. Representative images showing ASC speck (red) and SLC15A4-GFP (green) in SLC15A4feeble DCs together with non-transduced SLC15A4feeble DCs. Note the perinuclear positioning of ASC specks in the transduced cells (E). Quantification of perinuclear (within a radius of 3 μm from the nucleus) and non-perinuclear ASC specks in at least 30 cells per cell type in each of three independent experiments (F). G. Representative immunoblots showing pro-caspase-1 (pro-casp.-1) and cleaved p20 (casp.-1 p20), LC3-II and LC3-I, and actin bands. N, nucleus. Dotted white lines, cell outlines. Corresponding DIC images show nuclear position. Scale bar, 8 μm.

Data information: Data represent mean ± SD. **p<0.01. Two-tailed Student's t-test. See also Fig. EV4.
The phagosomal solute transporter SLC15A4 promotes inflammasome activity via mTORC1 signaling and autophagy restraint in dendritic cells (2022)
Figure 5

Figure 5. Constitutive mTORC1 signaling restores inflammasome activity and positioning in SLC15A4feeble DCs

(A-I). WT or SLC15A4feeble BMDCs transduced with constitutively active RagB (RagBQ99L) or metap2 (control) (A-E, H, I) and mcherry-LC3 (H, I), or non-target control, ATG5 or ATG7 shRNAs (F, G) were unstimulated or infected with flagellin-expressing STm. (A, B). Cell pellets collected 1 h after STm infection were lysed, fractionated by SDS-PAGE and immunoblotted for phospho (P) and total mTOR, ULK1 kinase, p70 kinase and S6, FLAG and actin. Representative immunoblots. Non-specific band right above FLAG-RagBQ99L is indicated with an asterisk (A). Quantification of band intensities for P-mTOR normalized to total mTOR, P-ULK1 normalized to total ULK1, P-p70 normalized to total p70 and P-S6 normalized to S6, in transduced cells from 3 independent experiments are shown as fold change relative to WT control (B). (C, D, F). Cell supernatants collected 1 h after infection were assayed for IL-1β by ELISA. (C, F). Representative plots of 3 independent experiments. D. IL-1β values of transduced SLC15A4feeble BMDCs from 3 independent experiments are shown as percent of WT DC values to represent rescue of the defective phenotype. (E, G). Cell pellets collected 1 h after STm infection were probed for caspase-1, GSDMD, actin (E, G), ATG5 and ATG 7 (G). Representative immunoblots showing pro-caspase-1 (pro-casp.-1) and cleaved p20 (casp.-1 p20), GSDMD full length (GSDMD FL) and cleaved GSDMD N-terminal fragment (GSDMD N-ter) bands. (H, I). Cells were stained for endogenous ASC, labeled with DAPI and analyzed by fluorescence microscopy. Representative images showing ASC speck (green) and LC3 (red) in SLC15A4feeble DCs (H). Quantification of perinuclear (within a radius of 3 μm from the nucleus) in at least 30 cells per cell type in each of three independent experiments (I). N, nucleus. Corresponding DIC images show nuclear position. Scale bar, 8 μm.

Data information: Data represent mean ± SD. **p<0.01; ***p<0.001. Two-tailed Student's t-test. See also Fig. EV5.

Expanded View Figure legends
A regulatory phosphorylation site on Mec1 controls chromatin occupancy of RNA polymerases during replication stress (2021)
Figure 2

Figure 2: Mec1 phosphorylation on Serine 1991 helps cells cope with replication stress
(A) The domain structure of Mec1, which is typical for PI3K-related protein kinases, including the N-terminal HEAT repeat domain, FAT domain, kinase domain and the C-terminal FAT domain. The Mec1 consensus SQ/TQ sites identified in silico are marked in black, while in vivo validated phosphorylation sites induced by genotoxic agents are in red.

(B) A 10-fold dilution series of cells from exponential SC cultures of the indicated strains were spotted on SC +/- the indicated dose of genotoxic agents.

(C) Asynchronous exponentially growing cells were treated with 0.2M HU (+HU), then HU was removed from the medium (-HU). SDS-PAGE of total protein extracts taken at the indicated times were used to detect the kinetic of Rad53 phosphorylation upshift (Rad53-P=**). Red and green asterisk indicate the time where mec1-S1991 phosphomutants show altered Rad53 recovery.

(D-F) A 5-fold dilution series of cells from exponential SC cultures of the indicated strains were spotted on SC +/- the indicated dose of HU.