Journal: Acta Pharmaceutica Sinica. B

Article Title: USP20 as a super-enhancer-regulated gene drives T-ALL progression via HIF1A deubiquitination

doi: 10.1016/j.apsb.2025.07.003

Figure Lengend Snippet: USP20 promotes the progression of T-ALL by deubiquitinating modified HIF1A. (A) Using mass spectrometry and software analysis, 16 potential target proteins of USP20 were identified. The top 10 genes were sorted based on unique peptides, coverage (%), and intensity, with HIF1A leading the list. (B) Western blotting detected USP20, Flag, and HIF1A proteins after immunoprecipitation with the Flag antibody was applied to Jurkat and J.gamma1 cells overexpressing USP20. (C) The amounts of USP20, HIF1A, and β -actin proteins in Jurkat and J.gamma1 cells that stably expressed sh-NC, sh- USP20 #2, and sh- USP20 #3 were measured via WB analysis. (D) In Jurkat and J.gamma1 cells that stably expressed sh-NC, sh- USP20 #2, and sh- USP20 #3, the mRNA levels of USP20 and HIF1A were measured using qRT-PCR ( n = 3). Data are mean ± SD, non-significant (ns), ∗ P < 0.05, ∗∗ P < 0.01, ∗∗∗ P < 0.001, and ∗∗∗∗ P < 0.0001. (E) Twenty micromolar MG-132 was incubated for 6 h with sh-NC, sh- USP20 #2, and sh- USP20 #3 stably expressed in Jurkat and J.gamma1 cells. The protein levels of USP20, HIF1A, and β -actin were measured via Western blotting. (F) For the specified periods, 100 μg/mL CHX was applied to Jurkat and J.gamma1 cells that either overexpressed USP20 (Flag-USP20) or stably expressed the empty vector (Flag). Afterward, USP20, HIF1A, and β -actin protein levels were assessed using Western blotting. (G) HIF1A was immunoprecipitated from Jurkat and J.gamma1 cells stably expressing empty vector (Flag) or USP20 overexpression (Flag- USP20 ), respectively, followed by Western blotting of the precipitated proteins with the antibody specifically for k48-linked ubiquitin (Ub-k48).

Article Snippet: The following antibodies were used: USP20 (Proteintech, 17491-1-AP, Wuhan, China), β -actin (Proteintech, 66009-1-Ig, Wuhan, China), PARP (CST, 9542S, Danvers, USA), cleaved caspase-3 (CST, 9661S, Danvers, USA), cleaved caspase-8 (CST, 9746S, Danvers, USA); c-Myc (CST, 9402S, Danvers, USA); Flag (Sigma, F1804, St. Louis, MO, USA), HIF1A (Santa Cruz Biotechnology, sc-3515, Dallas, TX, USA), HA-Tag (CST, 3724S, Danvers, USA), IgG (Abcam, ab172730, Cambridge, MA, USA), β -Catenin (CST, 9582S, Danvers, USA), GFP (Proteintech, 66002-1-Ig, Wuhan, China), GAPDH (Abcam, ab128915, Cambridge, MA, USA), LDB1 (Abcam, ab96799, Cambridge, MA, USA), BRD4 (E2A7X) Rabbit mAb (CST, 13440, Danvers, USA).

Techniques: Modification, Mass Spectrometry, Software, Western Blot, Immunoprecipitation, Stable Transfection, Quantitative RT-PCR, Incubation, Plasmid Preparation, Expressing, Over Expression, Ubiquitin Proteomics

Journal: Acta Pharmaceutica Sinica. B

Article Title: USP20 as a super-enhancer-regulated gene drives T-ALL progression via HIF1A deubiquitination

doi: 10.1016/j.apsb.2025.07.003

Figure Lengend Snippet: USP20 regulates gene transcription through HIF1A. (A) A heatmap produced by CUT&Tag research revealed that USP20 and HIF1A were occupied in the J.gamma1 cell line. (B) Distribution of HIF1A and USP20 peaks within a ±1000 bp window of USP20 binding sites. (C) CUT&Tag IGV graphs displaying the H3K27ac signal, ATAC signal, and gene locus signal for USP20 and HIF1A in the T-ALL cell line (J.gamma1), highlighting potential target genes, such as SLC2A1 , PER1 , CIC , and MAZ .

Article Snippet: The following antibodies were used: USP20 (Proteintech, 17491-1-AP, Wuhan, China), β -actin (Proteintech, 66009-1-Ig, Wuhan, China), PARP (CST, 9542S, Danvers, USA), cleaved caspase-3 (CST, 9661S, Danvers, USA), cleaved caspase-8 (CST, 9746S, Danvers, USA); c-Myc (CST, 9402S, Danvers, USA); Flag (Sigma, F1804, St. Louis, MO, USA), HIF1A (Santa Cruz Biotechnology, sc-3515, Dallas, TX, USA), HA-Tag (CST, 3724S, Danvers, USA), IgG (Abcam, ab172730, Cambridge, MA, USA), β -Catenin (CST, 9582S, Danvers, USA), GFP (Proteintech, 66002-1-Ig, Wuhan, China), GAPDH (Abcam, ab128915, Cambridge, MA, USA), LDB1 (Abcam, ab96799, Cambridge, MA, USA), BRD4 (E2A7X) Rabbit mAb (CST, 13440, Danvers, USA).

Techniques: Produced, Binding Assay

Journal: Cell Death & Disease

Article Title: YIPF2 promotes chemotherapeutic agent-mediated apoptosis via enhancing TNFRSF10B recycling to plasma membrane in non-small cell lung cancer cells

doi: 10.1038/s41419-020-2436-x

Figure Lengend Snippet: a , b A549 ( a ) and H1792 ( b ) cells were treated with PEM at 5.0 μM for the indicated times (0, 6, 12, 24, 36 and 48 h). Cell lysates were analyzed by Western blotting with antibodies against YIPF2, TNFRSF10B and ACTB. c A549 cells were treated with doxorubicin (DOX) at various concentrations (0–2.0 μM) for 18 h. Cell lysates were analyzed by Western blotting with antibodies against YIPF2, TNFRSF10B and ACTB. d Overexpression of YIPF2 in H1792 and H1299 cells in the presence or absence of PEM at 5.0 μM for 36 h. Cell lysates were analyzed by Western blotting with antibodies against YIPF2, TNFRSF10B and ACTB. e Knockdown of YIPF2 expression by YIPF2–1 siRNA in A549 and H1792 cells in the presence or absence of PEM at 5.0 μM for 36 h. Cell lysates were analyzed by Western blotting with antibodies against YIPF2, TNFRSF10B and ACTB. f Overexpression of YIPF2 in H1299 cells (left) or Knockdown of YIPF2 expression by YIPF2–1 siRNA in H1792 cells (right) in the presence or absence of PEM at 5.0 μM for 36 h. Cell lysates were analyzed by Western blotting with antibodies against YIPF2, TNFRSF10A and ACTB. g Three A549 cell lines (Ctrl, YIPF2, YIPF2 + siTNFRSF10B) in the presence or absence of PEM at 5.0 μM for 36 h. Cell lysates were analyzed by Western blotting with antibodies against YIPF2, TNFRSF10B, CASP8, CASP3 and ACTB. h Three H1299 cell lines (Ctrl, siYIPF2–1, siYIPF2–1 + TNFRSF10B (short isoform)) in the presence or absence of PEM at 5.0 μM for 36 h. Cell lysates were analyzed by Western blotting with antibodies against YIPF2, TNFRSF10B, CASP8, CASP3 and ACTB.

Article Snippet: The primary antibodies used in western blot assays and Immunoprecipitation were as follows: anti-YIPF2 (Cat. no. HPA019902; Sigma-Aldrich), ACTB (Cat. no. A1978; Sigma-Aldrich), CASP8 (Cat. no. 9746S; CST), CASP3 (Cat. no. NB100-56708; Novus Biologicals), PARP1 (Cat. no. 9542S; CST), TNFRSF10B (Cat. no. 2019; ProSci Incorporated), RAB8 (Cat. no. 6975S; CST), Flag (Cat. no. F7425, F1804; Sigma-Aldrich) and GFP (Cat. no. G1544, Sigma-Aldrich; Cat. no. sc-9996, Santa Cruz).

Techniques: Western Blot, Over Expression, Expressing

Journal: Cell Death & Disease

Article Title: YIPF2 promotes chemotherapeutic agent-mediated apoptosis via enhancing TNFRSF10B recycling to plasma membrane in non-small cell lung cancer cells

doi: 10.1038/s41419-020-2436-x

Figure Lengend Snippet: a Overexpression of YIPF2 in A549 and H1792 cells. The surface expression of TNFRSF10B was confirmed by flow cytometry analyses. b Knockdown of YIPF2 expression by YIPF2–1 and YIPF2–2 siRNA in A549 cells. The surface expression of TNFRSF10B was confirmed by flow cytometry analyses. c Relative RT-qPCR analyses of YIPF2 and TNFRSF10B mRNA levels after YIPF2 overexpression in H1792 (left) and H1299 (right) cells ( n = 3). d Relative RT-qPCR analyses of YIPF2 and TNFRSF10B mRNA levels after YIPF2 knocking down in H1792 (left) and H1299 (right) cells ( n = 3). e Left: Overexpression of YIPF2 in H1299 cells in the presence or absence of cycloheximide (CHX) at 10 μg/ml for the indicated times (0, 4, 8 and 12 h). Cell lysates were analyzed by Western blotting with antibodies against YIPF2, TNFRSF10B and ACTB. Right: The band intensity of TNFRSF10B was quantified by ImageJ software and plotted. This experiment was repeated three times independently with similar results. f Left: Knockdown of YIPF2 expression by YIPF2–1 siRNA in A549 cells in the presence or absence of cycloheximide (CHX) at 10 μg/ml for the indicated times (0, 4, 8 and 12 h). Cell lysates were analyzed by Western blotting with antibodies against YIPF2, TNFRSF10B and ACTB. Right: The band intensity of TNFRSF10B was quantified by ImageJ software and plotted. This experiment was repeated three times independently with similar results. (mean ± SEM, n = 3 independent experiments; NS, not significant; * P < 0.05, ** P < 0.01 and *** P < 0.001; P -values in c , e and f were obtained using two-tailed Student’s t -tests, P -values in d were obtained using one-way ANOVA followed by Bonferroni’s post-hoc test).

Article Snippet: The primary antibodies used in western blot assays and Immunoprecipitation were as follows: anti-YIPF2 (Cat. no. HPA019902; Sigma-Aldrich), ACTB (Cat. no. A1978; Sigma-Aldrich), CASP8 (Cat. no. 9746S; CST), CASP3 (Cat. no. NB100-56708; Novus Biologicals), PARP1 (Cat. no. 9542S; CST), TNFRSF10B (Cat. no. 2019; ProSci Incorporated), RAB8 (Cat. no. 6975S; CST), Flag (Cat. no. F7425, F1804; Sigma-Aldrich) and GFP (Cat. no. G1544, Sigma-Aldrich; Cat. no. sc-9996, Santa Cruz).

Techniques: Over Expression, Expressing, Flow Cytometry, Quantitative RT-PCR, Western Blot, Software, Two Tailed Test

Journal: Cell Death & Disease

Article Title: YIPF2 promotes chemotherapeutic agent-mediated apoptosis via enhancing TNFRSF10B recycling to plasma membrane in non-small cell lung cancer cells

doi: 10.1038/s41419-020-2436-x

Figure Lengend Snippet: a , b Knockdown of RAB8 expression by RAB8 siRNA in H1792 ( a ) and A549 ( b ) cells in the presence or absence of PEM at 5.0 μM for 36 h. The surface expression of TNFRSF10B was confirmed by flow cytometry analyses. c Overexpression of RAB8 in H1299 cells in the presence or absence of PEM at 5.0 μM for 36 h. Cell lysates were analyzed by Western blotting with antibodies against RAB8, TNFRSF10B, CASP8, CASP3, PARP1 and ACTB. d Knockdown of RAB8 expression by RAB8 siRNA in H1792 cells in the presence or absence of PEM at 5.0 μM for 36 h. Cell lysates were analyzed by Western blotting with antibodies against RAB8, TNFRSF10B, CASP8, CASP3, PARP1 and ACTB.

Article Snippet: The primary antibodies used in western blot assays and Immunoprecipitation were as follows: anti-YIPF2 (Cat. no. HPA019902; Sigma-Aldrich), ACTB (Cat. no. A1978; Sigma-Aldrich), CASP8 (Cat. no. 9746S; CST), CASP3 (Cat. no. NB100-56708; Novus Biologicals), PARP1 (Cat. no. 9542S; CST), TNFRSF10B (Cat. no. 2019; ProSci Incorporated), RAB8 (Cat. no. 6975S; CST), Flag (Cat. no. F7425, F1804; Sigma-Aldrich) and GFP (Cat. no. G1544, Sigma-Aldrich; Cat. no. sc-9996, Santa Cruz).

Techniques: Expressing, Flow Cytometry, Over Expression, Western Blot

Journal: Cell Death & Disease

Article Title: YIPF2 promotes chemotherapeutic agent-mediated apoptosis via enhancing TNFRSF10B recycling to plasma membrane in non-small cell lung cancer cells

doi: 10.1038/s41419-020-2436-x

Figure Lengend Snippet: a H1299 cells were transfected with pcDNA3.1 or pcDNA3.1-Flag-TNFRSF10B (short isoform) plasmids. Then the co-IP assays were carried out with Flag antibody and the co-eluted proteins were detected by western blot assays with Flag and YIPF2 antibodies. b , c H1792 cells were transfected with pEGFP-N1 or pEGFP-N1-YIPF2 plasmids. Then the co-IP assays were carried out with GFP antibody and the co-eluted proteins were detected by western blot assays with GFP, TNFRSF10B ( b ) and RAB8 ( c ) antibodies. d H1792 cells were transfected with pEGFP-N1 or pEGFP-N1-RAB8 plasmids. Then the co-IP assays were carried out with GFP antibody and the co-eluted proteins were detected by western blot assays with GFP, and TNFRSF10B antibodies. e Cell lysates were prepared from H1792 cells transiently co-expressing pEGFP-N1-RAB8 and pcDNA3.1-YIPF2. The co-IP assays were carried out with GFP antibody and the co-eluted proteins were detected by western blot assays with YIPF2, GFP and TNFRSF10B antibodies. f Schematic illustration showing that YIPF2 promotes PEM-mediated apoptosis via enhancing TNFRSF10B recycling to plasma membrane in NSCLC cells.

Article Snippet: The primary antibodies used in western blot assays and Immunoprecipitation were as follows: anti-YIPF2 (Cat. no. HPA019902; Sigma-Aldrich), ACTB (Cat. no. A1978; Sigma-Aldrich), CASP8 (Cat. no. 9746S; CST), CASP3 (Cat. no. NB100-56708; Novus Biologicals), PARP1 (Cat. no. 9542S; CST), TNFRSF10B (Cat. no. 2019; ProSci Incorporated), RAB8 (Cat. no. 6975S; CST), Flag (Cat. no. F7425, F1804; Sigma-Aldrich) and GFP (Cat. no. G1544, Sigma-Aldrich; Cat. no. sc-9996, Santa Cruz).

Techniques: Transfection, Co-Immunoprecipitation Assay, Western Blot, Expressing

Journal: Cell Death & Disease

Article Title: YIPF2 promotes chemotherapeutic agent-mediated apoptosis via enhancing TNFRSF10B recycling to plasma membrane in non-small cell lung cancer cells

doi: 10.1038/s41419-020-2436-x

Figure Lengend Snippet: a Box plots of YIPF2 mRNA levels determined from two Oncomine datasets, namely TCGA Lung 2 and Weiss Lung (** P < 0.01 and *** P < 0.001; P- values were obtained using two-tailed Student’s t -tests). b Box plots of TNFRSF10B mRNA levels determined from two Oncomine datasets, namely TCGA Lung 2 and Bhattacharjee Lung. c Kaplan-Meier plots of the first-progression survival (FPS, upper) and post-progression survival (PPS, lower) of chemotherapy-treated patients stratified by YIPF2 expression. The data were acquired from the Kaplan-Meier plotter database ( P -values were obtained using the log-rank test). d Kaplan-Meier plots of the first-progression survival (FPS, upper) and post-progression survival (PPS, lower) of chemotherapy-treated patients stratified by TNFRSF10B expression. The data were acquired from the Kaplan-Meier plotter database ( P -values were obtained using the log-rank test). e Scatter plots showing the correlation of YIPF2 expression with TNFRSF10B expression in lung cancer cells in two GEO datasets (upper: GDS1688 which contains 29 lung cancer cell lines; lower: GDS3627 which contains 58 NSCLC cell lines). The r value was calculated via Spearman’s rank correlation coefficient analysis.

Article Snippet: The primary antibodies used in western blot assays and Immunoprecipitation were as follows: anti-YIPF2 (Cat. no. HPA019902; Sigma-Aldrich), ACTB (Cat. no. A1978; Sigma-Aldrich), CASP8 (Cat. no. 9746S; CST), CASP3 (Cat. no. NB100-56708; Novus Biologicals), PARP1 (Cat. no. 9542S; CST), TNFRSF10B (Cat. no. 2019; ProSci Incorporated), RAB8 (Cat. no. 6975S; CST), Flag (Cat. no. F7425, F1804; Sigma-Aldrich) and GFP (Cat. no. G1544, Sigma-Aldrich; Cat. no. sc-9996, Santa Cruz).

Techniques: Two Tailed Test, Expressing