Additional file 1 of PSMD2 contributes to the progression of esophageal squamous cell carcinoma by repressing autophagy
Creators: name: Liu, Yachen
name: Wu, Meng
name: Xu, Shuxiang
name: Niu, Xiangjie
name: Liu, Weiling
name: Miao, Chuanwang
name: Lin, Ai
name: Xu, Yang
name: Yu, Lili
Publisher: name: figshare
Year: 2024
Subjects: subject: Biochemistry
subject: Medicine
subject: Cell Biology
subject: Genetics
subject: FOS: Biological sciences
subject: Molecular Biology
subject: Biological Sciences not elsewhere classified
subject: Cancer

description: Additional file 1: Fig. S1. PSMD2 inhibits autophagy and promotes proliferation of KYSE450 cells. (A, B) PSMD2 overexpression reduced the formation of LC3B puncta in KYSE450 cells (A). PSMD2 knockdown increased the formation of LC3B puncta in KYSE450 cells (B). Autophagy of KYSE450 cells with PSMD2 overexpression or knockdown was induced by FBS deprivation for 3 h or 20 h. The cells were then stained with the DAPGreen (green). The nucleus was stained with Hoechst 33342 (blue). Scale bar: 30 μm. (C) The impact of ATG7 knockdown on the proliferation of ESCC cells with PSMD2 depletion. The cells were fluorescently stained with EdU (red). The nucleus was stained with DAPI (blue). The efficiency of ATG7 knockdown was shown in the upper right panel. The percentage of EdU-positive cells was shown in the bottom right panel. N=3. The data were presented as mean ± SD. Scale bar: 100 μm. (D) The impact of ATG7 knockdown on the colony formation of ESCC cells with PSMD2 depletion. The cells were seeded into 6-well plates with a density of 1000 cells per well. After being cultured for 14 days, the cells were stained with crystal violet. N=3. The data were presented as mean ± SD. (E) The impact of ATG7 knockdown on cell proliferation of ESCC cells with PSMD2 depletion. Cell numbers were determined by CCK-8 assay at the indicated time points. N=3. The data were presented as mean ± SD. * p &lt; 0.05, ** p &lt; 0.01, *** p &lt; 0.001 and **** p &lt; 0.0001. Fig. S2. PSMD2 knockdown induce autophagic fluxes. (A, B) The autophagic fluxes of KYSE30 cells (A) and KYSE450 cells (B) with PSMD2 knockdown. The cells were stably transduced to express mRFP-GFP-LC3 fusion protein. Scale bar: 10 μm. N=10. The data were presented as mean ± SD, *** p &lt; 0.001 and **** p &lt; 0.0001. Fig. S3. PSMD2 promotes the proliferation of ESCC cells. (A, B) Representative images of the xenograft tumors formed in NOD/scid mice inoculated with KYSE30 cells (A) and KYSE450 cells (B) with or without PSMD2 knockdown. Growth curves of tumors derived from the indicated cell lines were shown. (C) PSMD2 knockdown significantly inhibited the migration and invasion capabilities of ESCC cells. The left panels were representative images of ESCC cells in transwell assays. The right panels were statistical data. N=3. Data were presented as mean ± SD. *p&lt; 0.05, **p &lt; 0.01, ***p&lt; 0.001, and ****p &lt; 0.0001. Fig. S4. ASS1 mediates the PSMD2-dependent inhibition of autophagy. (A) Representative H&amp;E and IHC images of tumors formed by KYSE450 cells with PSMD2 overexpression. The expression levels of PSMD2, ASS1, LC3 and p62 are shown. Right panel, quantitative analyses of the positive cell numbers. N=5. Data were presented as mean ± SD. (B) Representative IHC images of p62 expression in tumors formed by KYSE30 cells with PSMD2 overexpression or knockdown. (C) Quantitative analyses of the expression of PSMD2, ASS1, LC3 and p62 in tumors formed by KYSE30 cells with PSMD2 overexpression. N=5. Data were presented as mean ± SD. *p&lt; 0.05, **p &lt; 0.01, ***p&lt; 0.001, and ****p &lt; 0.0001. (D) Quantitative analyses of the expression of PSMD2, ASS1, LC3 and p62 in tumors formed by KYSE30 cells with PSMD2 knockdown. N=5. Data were presented as mean ± SD. *p&lt; 0.05, **p &lt; 0.01, ***p&lt; 0.001, and ****p &lt; 0.0001. Fig. S5. ASS1 mediates the roles of PSMD2 in the progression of ESCC. (A) The relationship between PSMD2 and ASS1 protein levels in ESCC determined by IHC staining. Showing representative IHC images of PSMD2 and ASS1 expression in serial sections of ESCC tissue array (n = 144). Scale bar in left images = 600 μm. Scale bar in right images = 100 μm. (B, C) Effect of ASS1 knockdown on cell proliferation in KYSE30 cells (B) and KYSE450 cells (C) after the change of PSMD2 expression. The cell proliferation rate was determined by CCK-8 assay. The cells were seeded into 6-well plate at a density of 3000 cells per well for KYSE30 cells or 1000 cells per well for KYSE450 cells. After being cultured for 14 days, the cells were then stained with crystal violet. Quantification of the data is shown. Data were presented as mean ± SD of three independent experiments, **p &lt; 0.01, ***p &lt; 0.001, and ****p &lt; 0.0001. Fig. S6. The effects of ASS1 on ESCC cell proliferation caused by the change of PSMD2 expression levels. (A) Effect of ASS1 knockdown on colony formation in KYSE30 cells (upper panel) and KYSE450 cells (bottom panel) with PSMD2 overexpression. (B) Effect of ASS1 overexpression on colony formation in KYSE30 cells (upper panel) and KYSE450 cells (bottom panel) with PSMD2 depletion. Data were presented as mean ± SD of three independent experiments, **p &lt;0.01, ***p &lt;0.001, and ****p &lt;0.0001. Table S1. Sequences of siRNAs and shRNAs used in this study. Table S2. 26S proteasome gene expression with paried t-test and survival with logrank test in esophageal squamous cell carcinoma data. Table S3. Protein level change in KYSE30 cells with PSMD2 knockdown related to the corresponding control.