Journal: Advanced science (Weinheim, Baden-Wurttemberg, Germany)

Article Title: EZH2 Inhibition Enhances PD-L1 Protein Stability Through USP22-Mediated Deubiquitination in Colorectal Cancer.

doi: 10.1002/advs.202308045

Figure Lengend Snippet: Figure 1. EZH2 inhibition enhance the expression of PD-L1 in colorectal cancer. A) Gene set enrichment analysis in HCT116 cells treated with Taz 40 μM comparing to DMSO control. HCT116 cells were treated with 40 ng mL−1 IFN-𝛾for an additional 24 h. Immune-related gene sets enriched in Taz-treated tumor cells are labeled in red. B) GSEA analysis highlighting six Taz-induced immune relate enriched gene sets. C–F) Representative qRT-PCR analysis of PD-L1 mRNA level in various colon cancer cell lines, C)HCT116, D)LS180, E)SW480, and F)MC38 cells were treated with Taz for 48 h and 40 ng mL−1

Article Snippet: This was repeated five more times using the following antibodies: PD-L1 (# 64988 Cell Signaling Technology), Tri-MethylHistone H3 (Lys27) (#9733, Cell Signaling Technology), CD8 (#98941, Cell Signaling Technology), USP22 (ab195289, Abcam), GZMB (ab289888, Abcam), perforin (ab16074, Abcam), CD86 (ER1906-01, HUABIO), CD206 (#24595, Cell Signaling Technology), F4/80 (#70076, Cell Signaling Technology), PD-1(#84651, Cell Signaling Technology), TIM-3 (#83882, Cell Signaling Technology).

Techniques: Inhibition, Expressing, Control, Labeling, Quantitative RT-PCR

Journal: Advanced science (Weinheim, Baden-Wurttemberg, Germany)

Article Title: EZH2 Inhibition Enhances PD-L1 Protein Stability Through USP22-Mediated Deubiquitination in Colorectal Cancer.

doi: 10.1002/advs.202308045

Figure Lengend Snippet: Figure 2. Inhibition of EZH2 enhances the stability of PD-L1 protein and attenuates K48-mediated ubiquitination. A) Representative immunoblot and B) quantification analysis of endogenous PD-L1 degradation in HCT116 treated with Taz or DMSO control, 20 μg mL−1 cycloheximide (CHX) was incubated indicated time. Results are presented as mean ± SEM, two-tailed unpaired t-test. C) GSEA analysis highlighting Taz-induced enriched gene sets of KEGG_UBIQUTIN_MEDIATED_PROREOLYSIS. D) Representative immunoblot analysis and quantification of the effect of Taz on PD-L1 expression in the

Article Snippet: This was repeated five more times using the following antibodies: PD-L1 (# 64988 Cell Signaling Technology), Tri-MethylHistone H3 (Lys27) (#9733, Cell Signaling Technology), CD8 (#98941, Cell Signaling Technology), USP22 (ab195289, Abcam), GZMB (ab289888, Abcam), perforin (ab16074, Abcam), CD86 (ER1906-01, HUABIO), CD206 (#24595, Cell Signaling Technology), F4/80 (#70076, Cell Signaling Technology), PD-1(#84651, Cell Signaling Technology), TIM-3 (#83882, Cell Signaling Technology).

Techniques: Inhibition, Ubiquitin Proteomics, Western Blot, Control, Incubation, Two Tailed Test, Expressing

Journal: Advanced science (Weinheim, Baden-Wurttemberg, Germany)

Article Title: EZH2 Inhibition Enhances PD-L1 Protein Stability Through USP22-Mediated Deubiquitination in Colorectal Cancer.

doi: 10.1002/advs.202308045

Figure Lengend Snippet: Figure 3. USP22 is a key deubiquitinase (DUB) involved in the regulation of PD-L1 stability by EZH2. A) Representative immunoblot analysis and B) qPCR of USP22 in various colon cancer cell lines. Results are presented as mean ± SEM, two-tailed unpaired t-test. C) Immunoblot analysis of the USP22 and PD-L1 protein expression in HCT116 cell after treatment with USP22 shRNAs or scrambled shRNA. D) Immunoblot analysis of the PD-L1 protein expression after ectopic Myc-USP22 overexpression in HCT116 cells. E) Representative immunoblot analysis of the PD-L1 protein expression

Article Snippet: This was repeated five more times using the following antibodies: PD-L1 (# 64988 Cell Signaling Technology), Tri-MethylHistone H3 (Lys27) (#9733, Cell Signaling Technology), CD8 (#98941, Cell Signaling Technology), USP22 (ab195289, Abcam), GZMB (ab289888, Abcam), perforin (ab16074, Abcam), CD86 (ER1906-01, HUABIO), CD206 (#24595, Cell Signaling Technology), F4/80 (#70076, Cell Signaling Technology), PD-1(#84651, Cell Signaling Technology), TIM-3 (#83882, Cell Signaling Technology).

Techniques: Western Blot, Two Tailed Test, Expressing, shRNA, Over Expression

Journal: Advanced science (Weinheim, Baden-Wurttemberg, Germany)

Article Title: EZH2 Inhibition Enhances PD-L1 Protein Stability Through USP22-Mediated Deubiquitination in Colorectal Cancer.

doi: 10.1002/advs.202308045

Figure Lengend Snippet: Figure 4. EZH2 enhances the protein stability of PD-L1 through the deubiquitination effect mediated by USP22 A) Representative immunoblot and B) quantification analysis of endogenous PD-L1 degradation in HCT116 treated with shUSP22 or scrambled shRNA, 20 μg mL−1 cycloheximide (CHX) was incubated indicated time. C) HCT116-shUSP22 cells were cocultured with or without activated T-cells at a ratio of 1:2 for 72 h and subjected to crystal violet staining. D) Representative immunoblot and E) quantification analysis of endogenous PD-L1 degradation in HCT116 treated with ectopic Myc-USP22

Article Snippet: This was repeated five more times using the following antibodies: PD-L1 (# 64988 Cell Signaling Technology), Tri-MethylHistone H3 (Lys27) (#9733, Cell Signaling Technology), CD8 (#98941, Cell Signaling Technology), USP22 (ab195289, Abcam), GZMB (ab289888, Abcam), perforin (ab16074, Abcam), CD86 (ER1906-01, HUABIO), CD206 (#24595, Cell Signaling Technology), F4/80 (#70076, Cell Signaling Technology), PD-1(#84651, Cell Signaling Technology), TIM-3 (#83882, Cell Signaling Technology).

Techniques: Western Blot, shRNA, Incubation, Staining

Journal: Advanced science (Weinheim, Baden-Wurttemberg, Germany)

Article Title: EZH2 Inhibition Enhances PD-L1 Protein Stability Through USP22-Mediated Deubiquitination in Colorectal Cancer.

doi: 10.1002/advs.202308045

Figure Lengend Snippet: Figure 5. Taz enhances the sensitivity of tumors to immunotherapy in vivo. A) Working model of engraftment and treatment of the subcutaneous transplantation model of MC38. B) Representative immunoblot analysis of the effects of USP22 knockdown on PD-L1 expression in MC38 cell after treatment with USP22 shRNAs or scrambled shRNA. C) Overview of tumors, D) tumor volume, and E) weights of tumor bulk. F) PD-L1 H-scores in EZH2−or EZH2+ cells of subcutaneous MC38 transplant tumors in control or Taz groups. G) Representative immunofluorescence staining of EZH2 and PD-L1 in MC38 transplant subcutaneous tumor of control or Taz groups. Scale bar, 200 μm. ns., not significant; p > 0.05, * p < 0.05, ** p < 0.01, ***

Article Snippet: This was repeated five more times using the following antibodies: PD-L1 (# 64988 Cell Signaling Technology), Tri-MethylHistone H3 (Lys27) (#9733, Cell Signaling Technology), CD8 (#98941, Cell Signaling Technology), USP22 (ab195289, Abcam), GZMB (ab289888, Abcam), perforin (ab16074, Abcam), CD86 (ER1906-01, HUABIO), CD206 (#24595, Cell Signaling Technology), F4/80 (#70076, Cell Signaling Technology), PD-1(#84651, Cell Signaling Technology), TIM-3 (#83882, Cell Signaling Technology).

Techniques: In Vivo, Transplantation Assay, Western Blot, Knockdown, Expressing, shRNA, Control, Staining

Journal: Advanced science (Weinheim, Baden-Wurttemberg, Germany)

Article Title: EZH2 Inhibition Enhances PD-L1 Protein Stability Through USP22-Mediated Deubiquitination in Colorectal Cancer.

doi: 10.1002/advs.202308045

Figure Lengend Snippet: Figure 6. Inhibition of EZH2 and USP22 affects the tumor microenvironment. A) Representative immunofluorescence staining of H3K27me3, PD-L1, and USP22 in MC38-WT and B) MC38-shUSP22 transplant subcutaneous tumor of control or Taz groups. Scale bar, 100 μm. C) Average fluorescence intensity of PD-L1 and D) USP22 in MC38-WT tumor microenvironment of different groups. E) Average fluorescence intensity of PD-L1 and F) USP22 in MC38-shUSP22 tumor microenvironment of different groups. G) The proportion of late-stage exhausted TIM-3+ cells among CD8+ T-cells in MC38-WT

Article Snippet: This was repeated five more times using the following antibodies: PD-L1 (# 64988 Cell Signaling Technology), Tri-MethylHistone H3 (Lys27) (#9733, Cell Signaling Technology), CD8 (#98941, Cell Signaling Technology), USP22 (ab195289, Abcam), GZMB (ab289888, Abcam), perforin (ab16074, Abcam), CD86 (ER1906-01, HUABIO), CD206 (#24595, Cell Signaling Technology), F4/80 (#70076, Cell Signaling Technology), PD-1(#84651, Cell Signaling Technology), TIM-3 (#83882, Cell Signaling Technology).

Techniques: Inhibition, Staining, Control