EMBO/SourceData huggingface dataset:
Figure 6(A) Using flow cytometry, binding to Expi293F cells expressing S of BA.2 omicron was measured for monomeric sACE2-8his proteins. (B) Binding of antibodies and dimeric sACE22-IgG1 proteins to Expi293F cells expressing BA.2 omicron S measured by flow cytometry.(C) BA.2 omicron pseudovirus was incubated with sACE22-IgG1 (grey) or sACE22.v2.4-IgG1 (blue) for 1 h and added to HeLa-hACE2-11 cells. Infection was measured after 48 h.(D-E) Binding of monomeric sACE2-8his proteins (D) and dimeric IgG1 proteins (E) to Expi293F cells expressing BA.4/BA.5 omicron S as measured by flow cytometry.
Figure 1.(A and B) ARPE-19 cells were transfected with siRNA duplexes to raptor, RagA+B, p18, or nontarget. 60 h after transfection, cells were infected with adenovirus expressing either TFEB-FLAG-WT (A) or TFEB-FLAG-S211A (B). 12 h later, cells were fixed, permeabilized with 0.2% Triton X-100, and double stained with antibodies against FLAG (used to detect TFEB) and mTOR. Bars, 10 µm. (C) Quantification of A. (D) Quantification of B. Values are means ± SD of three independent experiments. ***, P < 0.001.
Figure 2.(A) ARPE-19 cells were nucleofected with the indicated Rag-expressing plasmids. 6 h after nucleofection, cells were infected with adenovirus expressing TFEB-FLAG-WT. 12 h later, cells were lysed and subjected to immunoprecipitation with the anti-FLAG antibody. The immunoprecipitates were analyzed by immunoblotting with antibodies against Rag proteins and FLAG (used to detect TFEB-WT).(B) ARPE-19 cells were nucleofected with the indicated Rag- and TFEB-GFP-expressing plasmids. 18 h later, cells were lysed and subjected to immunoprecipitation with the anti-HA antibody (used to immunoprecipitate Rag proteins). The immunoprecipitates were analyzed by immunoblotting with antibodies against GST and GFP (used to detect Rag proteins and TFEB-WT, respectively).(C) ARPE-19 coexpressing TFEB and the indicated Rag-expressing plasmids were lysed and subjected to immunoprecipitation with the anti-FLAG antibody. The immunoprecipitates were analyzed by immunoblotting with antibodies against GST and FLAG (used to detect Rag proteins and TFEB-WT, respectively).(D) Immunoblotting analysis of coimmunoprecipitated TFEB-FLAG-S211A with Rag heterodimers. IP, immunoprecipitation.
Figure 3.(A) The indicated cell lines were incubated in medium containing DMSO or 250 nM Torin-1 for 1 h. Cells were then lysed and subjected to immunoblotting with antibodies against TFEB (used to detect endogenous TFEB) and actin.(B) HeLa cells were treated as indicated in A, and nuclei and membrane plus cytosol fractions were obtained by low speed centrifugation. Proteins from different fractions were subjected to immunoblotting with antibodies against TFEB (used to detect endogenous TFEB), Lamp1, or Histone H3.(C) Immunofluorescence confocal microscopy showing nuclear and lysosomal localization of endogenous TFEB upon treatment of HeLa cells with Torin-1 as indicated in A.(D) HeLa cells were starved in serum- and amino acid-free medium (starvation) for 3 h and analyzed by immunoblotting with antibodies against TFEB.(E) HeLa cells were incubated in normal medium (control) and serum- and amino acid-free medium (Starvation) for 4 h or starved for 4 h followed by restimulation with amino acids (starvation + amino acids) for 30 min and analyzed by immunofluorescence with antibodies against TFEB (endogenous TFEB is shown in green) and Lamp1 (red). The region within the dotted box is magnified in the insets.(F) HeLa cells were nucleofected with the indicated Rag-expressing plasmids. 18 h later, cells were lysed, and RagB/D heterodimers were pulled down using glutathione-Sepharose beads. Proteins bound to the beads were analyzed by immunoblotting with antibodies against TFEB and GST (used to detect endogenous TFEB and Rag proteins, respectively). Bars, 10 µm.
Figure 4.(A) ARPE-19 cells were infected with adenovirus expressing TFEB-FLAG-WT. 16 h later, cells were incubated with 250 nM Torin-1 for 1 h or starved in serum- and amino acid-free medium for 3 h. Cells were then fixed, permeabilized with 0.2% saponin, and double stained with antibodies against TFEB (used to detect recombinant TFEB) and Lamp1.(B) ARPE-19 cells were infected with adenovirus expressing TFEB-FLAG-S211A. 12 h later, cells were starved in serum- and amino acid-free medium for 4 h (starvation) or kept in complete medium (control). Cells were then fixed, permeabilized with 0.2% Triton X-100, and stained with antibodies against FLAG (used to detect TFEB-S211A).(C) HeLa cells were incubated with 250 nM Torin-1 for 1 h or starved in serum- and amino acid-free medium for 4 h with the addition of Torin-1 during the last hour of starvation. Cells were then washed, fixed, permeabilized with 0.2% Triton X-100, and double stained with antibodies against TFEB (used to detect endogenous TFEB) and Lamp1. Regions within the dotted boxes are magnified in the insets.
Figure 5.(A and B) ARPE-19 cells were transfected with either active or inactive RagB/D heterodimers and infected with adenovirus expressing TFEB-FLAG-WT (A) or TFEB-FLAG-S211A (B). After 12 h, cells were double stained with antibodies against FLAG and GST (used to detect TFEB or Rag proteins, respectively). (C) Quantification of TFEB-WT nuclear localization from A. (D) Quantification of TFEB-S211A lysosomal localization from B.(E and F) ARPE-19 cells were cotransfected with plasmids expressing TFEB-GFP together with inactive (E) or active Rag heterodimers (F). Cells were then incubated with 250 nM Torin-1 for 1 h (E) or starved in serum- and amino acid-free medium for 3 h (F). Anti-GST antibodies were used to detect Rag proteins. Arrows point to cells that do not express Rag heterodimers.
Figure 6.(A) Summary of the nuclear and lysosomal distribution of several TFEB amino acid and deletion mutants in ARPE-19 cells treated with either DMSO or Torin-1.(B) ARPE-19 cells were nucleofected with the indicated Rag- and TFEB-expressing plasmids. After 12 h, cell lysates were immunoprecipitated with the anti-FLAG antibody and analyzed by immunoblotting with antibodies against FLAG and GST (used to detect TFEB and Rag proteins, respectively).(C) FLAG-tagged TFEB-WT or TFEB deletion mutants were immunoprecipitated with the anti-FLAG antibody and analyzed by immunoblotting with antibodies against FLAG, 14-3-3 binding motif, or 14-3-3.(D) Immunofluorescence confocal microscopy showing the subcellular distribution of TFEB-WT and TFEB-S3A/R4A mutant upon incubation with DMSO (vehicle) or 250 nM Torin-1 for 1 h. Cells were fixed, permeabilized with 0.2% Triton X-100, and stained with antibodies against FLAG (used to detect TFEB).(E) ARPE-19 cells expressing either TFEB-S211A or TFEB-S211A-Δ30 were double stained with antibodies against TFEB and Lamp1. Regions within the dotted boxes are magnified in the insets.
Figure 7.(A) ARPE-19 cells coexpressing active RagB/D heterodimer and the indicated TFEB plasmids were fixed, permeabilized with 0.2% saponin, and stained with antibodies against GST (used to detect Rag proteins). Regions within the dotted boxes are magnified in the insets. Bars, 10 µm.(B) ARPE-19 cells were cotransfected with active RagB/D heterodimers and the indicated TFEB constructs. After 18 h, cell lysates were immunoprecipitated with the anti-HA antibody (used to immunoprecipitate Rag proteins) and immunoblotted with antibodies against GFP and GST (used to detect TFEB-GFP and Rag proteins, respectively). The white line indicates that intervening lanes have been spliced out.(C) Immunofluorescence confocal microscopy showing the subcellular distribution of TFEB-(1-30)-GFP in ARPE-19 cells coexpressing active RagB/D heterodimers (antibodies against GST were used to detect Rags). Bar, 4 µm.(D) Relative RT-PCR analysis of the mRNA expression of autophagy (ATG9B and UVRAG) and lysosomal (MCOLN1) genes in ARPE-19 cells infected with the indicated adenovirus for 48 h. The values are expressed as a ratio to RNA from cells infected with control adenovirus (Ad.-Null). Values are means ± SD of two independent experiments. ***, P < 0.001; **, P < 0.01.(E) Cells were infected with adenovirus as in D and analyzed by immunoblotting with the indicated antibodies. IP, immunoprecipitation.
Figure 8.(B-D) ARPE-19 cells were infected with adenovirus expressing MITF1-FLAG. 16 h later, cells were incubated with DMSO or 250 nM Torin-1 for 1 h and analyzed by immunoblotting (C) or immunofluorescence (B and D) with anti-FLAG antibodies. The region within the dotted box is magnified in the insets. Bars: (B and D, main images) 10 µm; (D, insets) 5 µm.(E) ARPE-19 cells were incubated with 250 nM Torin-1 for 1 h. Cells were then lysed and analyzed by immunoblotting with antibodies against MITF.(F) HEK-293T cells were incubated in medium containing DMSO or Torin-1 for 1 h. Cells were lysed, and nuclei and membrane plus cytosol fractions were obtained by low speed centrifugation. Proteins from the different fractions were subjected to immunoblotting with antibodies against MITF, Lamp1, and Histone H3.(G) ARPE-19 cells expressing active (RagBGTP/RagDGDP) or inactive (RagBGDP/RagDGTP) Rag heterodimers were immunoprecipitated with the anti-FLAG antibody and immunoblotted with antibodies against FLAG and GST (used to detect MITF and Rag proteins, respectively).(H) HEK-293T cells expressing active (RagBGTP/RagDGDP) or inactive (RagBGDP/RagDGTP) Rag heterodimers were pulled down using glutathione-Sepharose beads and immunoblotted with antibodies against GST and MITF (used to detect Rag proteins and endogenous MITF, respectively). IP, immunoprecipitation.
Figure 9.(A and B) ARPE-19 cells were nucleofected with the indicated Rag- and MITF-expressing plasmids. 12 h later, cells were fixed, permeabilized with 0.2% Triton X-100, and double stained with antibodies against FLAG and GST (used to detect MITF and Rag proteins, respectively). Bars, 10 µm.
Figure 1  (C) Electrophoretic mobility-shift assay (EMSA) with biotin-labeled RORA consensus, rs2978980T or rs2978980G probes and HGC-27 or MGC80-3 nuclear extracts. HGC-27 or MGC80-3 left panel: EMSA with RORA consensus or rs2978980T probes. Lane 1-8: from the first lane at the left side to the right side. Lanes 4 and 8, probe only; lanes 2 and 6, probe and nuclear extracts; lanes 1, 3, 5 and 7, probe and nuclear extracts plus 100× unlabeled rs2978980T (lanes 1 and 5) or RORA consensus probes (lanes 3 and 7). HGC-27 or MGC80-3 right panel: EMSA with rs2978980T or rs2978980G probes. Lanes 1 and 6, probe only; lanes 2 and 7, probe and nuclear extracts; lanes 3-5 and 8-10, probe and nuclear extracts plus 100× unlabeled rs2978980T (lanes 5 and 8), rs2978980G (lanes 3 and 10) or RORA consensus probes (lanes 4 and 9).   (F) Suppression of RORA using siRNAs (siRORA-1 and siRORA-2) could markedly down-regulate lncPSCA expression in MGC80-3 cells but not in HGC-27 cells. In detail, MGC80-3 and HGC-27 cells were transfected with siRORA-1 and siRORA-2, lncPSCA and RORA expression levels were measured by RT-qPCR and Western Blot.
Figure 2 (E) lncPSCA-knockout significantly promoted growth of MGC80-3 (n = 8) or HGC-27 (n = 7) xenografts compared with control xenografts after 27 or 18 days.  (F) HE and Ki67 staining of MGC80-3 or HGC-27 xenografts. Scale bar = 100 μm.
Figure 3 (B) lncPSCA inhibits invasion abilities of MGC80-3 and HGC-27 cells. Cells on the lower surface of the chamber were stained by crystal violet. Scale bar = 100 μm.  (C) In MGC80-3 and HGC-27 cells, expression changes of different markers (β-catenin, ZO-1, SNAIL and VIMENTIN) of epithelial-to-mesenchymal transition were examined after overexpression or knockout of lncPSCA.
Figure 4 (F) Gastric cancer cells after lncPSCA-knockout or stably overexpressing lncPSCA and control cells were treated with cycloheximide (CHX) or vehicle for the indicated periods of time. DDX5 levels were analyzed by Western blot.  (I) Knockdown of DDX5 with siRNA (siD-1 and siD-2) substantially reduced the proliferation of gastric cancer cells. Data show one representative example of three biological replicates. (J) Silencing of DDX5 with shRNA (shD) significantly reduced the proliferation of gastric cancer cells. Data show one representative example of three biological replicates. (
Figure 5 (E) RNA Pol II ChIPseq and RNAseq signals of PMAIP1, CDKN1A, SESN2 and SESN3 in HGC-27 cells. Red bars represent the RNA Pol II ChIPseq signal. Orange or blue bars represent the RNAseq signals of HGC-27 cells overexpressing lncPSCA (lncP) or with silenced DDX5 (shD), respectively.  (F) Validation of candidate downstream genes of the lncPSCA-DDX5 axis identified through the integrated analyses. qRT-PCR was performed for PMAIP1, CDKN1A and SESN2 genes upon overexpression of lncPSCA (lncP), silencing of DDX5 (shD) or lncPSCA konckout. Error bars indicate the SEM. Data show one representative example of three biological replicates.
Figure 1A. (Top) USP44 transcript levels across CD4+ T cell populations. Suspension of the lymph node and spleen cells of 6-8 week old female C57BL/6 mice (n= 5 experiments) were obtained, and naïve (CD62Lhigh/CD25-) CD4+ T cells and nTregs (CD4+/CD25high) were obtained by FACS. The indicated effector T cell populations were generated by activating 1x10^6 naïve CD4+ T precursors with anti-CD3 and anti-CD28 antibodies (1 and 4 ug/ml, respectively) for 4 days under the indicated in vitro skewing conditions (described in the Methods section). After harvesting RNA by Trizol reagent and generating cDNA, the Usp44 mRNA levels expressed by the resulting Teff and iTreg, as well as freshly isolated nTreg cells were determined by qRT-PCR. (Bottom) Suspension of lymph node and spleen cells of 6-8 week old female C57BL/6 mice were obtained, and naïve (CD62LhighCD25-) CD4+ T cells and nTregs (CD4+/CD25high) were sorted by FACS. iTregs were generated by activating 1x10^6 naïve CD4+ T cells with anti-CD3 and anti-CD28 antibodies (1 and 4 ug/ml, respectively) for 4 days in the presence of IL-2 (100u/ml) and TGF-β (5ng/ml) before the cells were harvested for SDS-PAGE. Panels A and B depict the mean mRNA expression normalized to the housekeeping gene GAPDH.B. Naïve murine CD4+ T cells were obtained and differentiated into the iTreg lineage as in A. Expression levels of Usp44 and Foxp3 mRNA were assessed daily over time by qRT-PCR.C. Expression of USP44 and FOXP3 protein by T cells during in vitro differentiation was also measured. After 1-4 days of iTreg skewing, cell lysates were resolved by SDS PAGE following by immunoblotting with the indicated antibodies.D. Murine iTregs generated in vitro as above were activated in the presence of recombinant TNFα, IL-6, or IL-1β (each at a 10 ng/ml concentration), or LPS (1µg/ml) for 24 hours. Treated Tregs were then harvested and USP44 protein levels were determined as above.
Figure 2A. HEK293T cells were transfected with MYC-FOXP3 and FLAG-USP44 encoding expression constructs using Polyethylenimine. 48hrs post-transfection, cells were harvested, lysed, and anti-FLAG or anti-MYC antibody coated beads were used to immunoprecipitate the given labeled protein along with its binding partner. Co-IP'ed proteins were subjected to SDS PAGE followed by immunoblot analysis. Antibodies recognizing FLAG or MYC tags were used to probe for USP44 and FOXP3, respectively.B. Endogenous co-IP of USP44 and FOXP3 in murine iTregs. iTregs were generated as in Fig. 1 from naïve CD4+ T cells FACS isolated from pooled suspensions of the lymph node and spleen cells of wild type C57BL/6 mice (n=2-3/experiment). iTregs were lysed and key proteins were immunoprecipitated using either anti-USP44 (right panel) or anti-FOXP3 (left panel) antibody. Proteins pulled-down in this experiment were then resolved and analyzed by immunoblot using anti-FOXP3 or anti-USP44 antibodies.C. Endogenous co-IP of USP44 and FOXP3 in murine nTregs. nTregs (CD4+CD25high) isolated by FACS were activated by anti-CD3 and anti-CD28 (1 and 4 ug/ml, respectively) overnight in the presence of IL-2 (100 U/ml). The cells were lysed and proteins were immunoprecipitated using either anti-Foxp3 (left panel) or anti-Usp44 (right panel). Proteins pulled down in this experiment were then resolved and identified with the indicated antibodies.D. Naïve murine CD4+ T cells were isolated by FACS from lymph node and spleen cell suspension of USP44fl/fl CD4Cre+ mice and that of their wild type littermates (USP44fl/fl CD4Cre- mice; n=2-3/group/experiment). iTreg cells were generated from these mice as described for Fig. 1 before incubation on a microscope slide pre-coated with poly-L lysine for 1h. Adhered cells were then fixed by PFA for 0.5 followed by blocking with 1% BSA for 1h, then incubation with the specified antibodies. Representative confocal microscopy images (40X) were visualized for endogenous USP44 (red) and FOXP3 Baxter et al(). DAPI was used to visualize cell nuclei (blue); scale bar 50μm.
Figure 3A. HEK293T cells were transfected with expression constructs encoding MYC-FOXP3, His-Ubiquitin, and either a wild type or catalytically inactive (C282S, "cs") version of USP44 tagged with FLAG. Transfected cells were treated with 20 μM MG132 for 4 hrs and subsequently harvested for cell lysis. Pull-down of His-labeled proteins using Ni-NTA beads allowed recovery of ubiquitinated FOXP3 species that were then visualized by immunobot probing with antibodies specific for MYC.B. Effect of USP44 knock down on FOXP3 ubiquitination. Two distinct shRNA lentiviral constructs each containing a G418 resistance cassette were delivered into HEK293T cells. After selected for 7d, cells were transfected with MYC-FOXP3 and His-Ubiquitin, then treated with 20 μM MG132 for 4 hrs before harvest and lysis. Ubiquitinated FOXP3 proteins were visualized as in A.C. Levels of polyubiquitinated FOXP3 in the presence or absence of USP44. As in Fig 2b, murine iTregs were generated from naïve CD4+ precursors isolated from wild type and mice globally deficient in USP44-/- mice (n=3/group/experiment). Ubiquitinated proteins were extracted from iTreg lysates with anti-ubiquitin antibodies (anti-Ubi) prior to resolution by SDS PAGE and immunoblot analysis, probing for FOXP3.D, E. HEK293T cells were transfected with plasmids encoding MYC-FOXP3, FLAG-USP44 (either wild type USP44, "wt" or the C282S mutant). These cells were subsequently treated with cycloheximide (CHX; 5μg/ml) for the indicated time points before harvested and cell lysis. FOXP3 levels and the relative turnover rate of this factor was determined by immunoblotting analysis with anti-MYC antibodies.F, USP44 knock down in human Tregs. Naïve CD4+ T cells (CD4+CD25-CD45RA+) were isolated from the peripheral blood of healthy donors by FACS, and iTreg were generated after 7 days of in vitro skewing conditions. As in Fig. 3b, shRNA constructs targeting USP44 were delivered by lentivirus. Here human iTregs received either shCK (control), or one of two different shUSP44 constructs. Endogenous FOXP3 and USP44 protein levels were visualized by immunoblot (F)G. USP44 knock down in human Tregs. Naïve CD4+ T cells (CD4+CD25-CD45RA+) were isolated from the peripheral blood of healthy donors by FACS, and iTreg were generated after 7 days of in vitro skewing conditions. As in Fig. 3b, shRNA constructs targeting USP44 were delivered by lentivirus. Here human iTregs received either shCK (control), or one of two different shUSP44 constructs. FOXP3 expression was assessed by intracellular staining followed by flow cytometry (G).H. Murine naïve CD4 T cells (CD62L+/CD25-/CD4+) from WT and USP44-/- mice (n=2-3/group/experiment) were FACS purified and activated in vitro for four days by CD3/CD28 cross-linking antibodies (1ug and 4ug/ml, respectively) in the presence of 100 U/ml IL-2 and the indicated dose of TGF-β. Down-regulation of FOXP3 was observed in USP44-/- mice by intracellular immunostaining and flow cytometry.
Figure 4A. Reciprocal Co-IP of USP44 and USP7. HEK293T cells were transfected with constructs encoding MYC-USP44 and FLAG-USP7. These cells were lysed and pertinent factors were immunoprecipitated using either anti-FLAG or anti-MYC. Pulled-down proteins were observed by immunoblot analysis using either anti-MYC or anti-FLAG antibodies.B. Effect of USP44 on USP7 levels. HEK293T cells were transfected with MYC-FOXP3. Cells also received combinations of FLAG-USP7 and varying doses of pLVX-USP44 encoding plasmids. FOXP3 was immunoprecipitated using anti-MYC, and immunoblots were analyzed using anti-FLAG, anti-USP44 or anti-MYC antibodies.C. Effect of USP44 and USP7 coexpression on FOXP3 ubiquitination. HEK293T cells were transfected with plasmids encoding MYC-FOXP3, either FLAG-USP44 or FLAG-USP7 or both, His-ubiquitin. Cells were treated with 20 μM MG132 for 4 hrs before being harvested, lysed, and incubated on Ni-NTA beads to pull down ubiquitinated FOXP3, which was visualized by immunoblotting using antibodies specific for MYC.D. (left panel) iTreg were polarized from human naïve CD4+ T cells over 7 days (n=3 donors /experiment) receiving control (shCK), or shRNA knockdown constructs targeting USP44 (shUSP44-1, -2), or USP7 (shUSP7-1, -2), or both shRNA constructs. Freshly isolated nTregs were treated with either control or shRNA constructs. After transduction with lentivirus for 48 hours, cells were harvested and run SDS-PAGE gels. Protein levels were visualized by immunoblotting using antibodies specific for FOXP3, USP44, USP7 and GAPDH. Shown are representative findings from 3 independent experiments (biological replicates).
Figure 5A. Jurkat T cells were transfected with the indicated combinations and relative doses of expression constructs encoding MYC-FOXP3, FLAG-USP44 along with an IL-2 promoter-driven firefly luciferase reporter. After 8 hours stimulation with PMA/Inomycin, cells were lysed and luciferase activity, which was observed and normalized to renilla luciferase activity.B. Tregs (CD4+/CD25High) were recovered from the lymph nodes and spleens of wild type C57BL/6 (WT) and USP44-/- mice by FACS purification. Cells were activated with anti-CD3/CD28 agonist antibodies overnight, and RNA was isolated, and cDNA was generated from these samples for the measurement of several signature Treg-associated transcripts by qRT-PCR. The relative expression level of these by WT and KO-derived Tregs is shown.C, D. Analysis of the in vitro suppressive function of Tregs isolated from WT mice or age- and sex-matched USP44-/- mice (n=3/group/experiment). Tregs were mixed with CFSE-labeled responder naïve T cells from CD45.1+ C57BL/6 mice (n=3/experiment) for three days. CFSE dilution in CD45.1+ T cells was observed by flow cytometry and the percent suppression of responder cell proliferation by the co-cultured Tregs was determined.