Fig 2: The involvement of ASS1 in the proliferation promoted by PSMD2. A, B The impact of ASS1 knockdown on the proliferation of KYSE30 cells (A) and KYSE450 cells (B) with PSMD2 overexpression. The cells were fluorescently stained with EdU (red). The nucleus was stained with DAPI (blue). The percentage of EdU-positive cells was calculated and shown in the right panel. Scale bar: 100 µm. C, D The impact of ASS1 knockdown on the proliferation of KYSE30 cells (C) and KYSE450 cells (D) with PSMD2 overexpression. E, F The impact of ASS1 overexpression on the proliferation of KYSE30 cells (E) and KYSE450 cells (F) with PSMD2 knockdown. The cells were fluorescently stained with EdU (red). The nucleus was stained with DAPI (blue). The percentage of EdU-positive cells was shown in the right panel. N = 3. G-H The impact of ASS1 overexpression on the proliferation of KYSE30 cells (G) and KYSE450 cells (H) with PSMD2 depletion. N = 3. Data were presented as mean ± SD, ***p < 0.001, and****p < 0.0001

Fig 3: The involvement of ASS1 in PSMD2-mediated inhibition of autophagy. A–D The impact of ASS1 knockdown on the formation of LC3B puncta in KYSE30 cells (A) and KYSE450 cells (B) with PSMD2 overexpression. The efficiency of ASS1 knockdown was shown in the right panel. Protein levels, upper right panel. RNA levels, bottom right panel. Autophagy was induced by FBS deprivation then stained with the DAPGreen (green). The nucleus was stained with Hoechst 33342 (blue). Scale bar: 30 µm. Quantitative analysis of the autophagic puncta of KYSE30 cells (C) and KYSE450 cells (D) was shown. N = 3.E–H The impact of ASS1 overexpression on the formation of LC3B puncta in KYSE30 cells (E) and KYSE450 cells (F) with PSMD2 knockdown. The treatment conditions were the same as those in (A, B). The efficiency of ASS1 overexpression was shown in the right panel. Protein levels, upper right panel. RNA levels, bottom right panel. Quantitative analysis of the autophagic puncta of KYSE30 cells (G) and KYSE450 cells (H) was shown. N = 3. Data were presented as mean ± SD, *p < 0.05, **p < 0.01, and****p < 0.0001

Fig 4: PSMD2 inhibits autophagy by inducing ASS1. A Differential expression analysis of proteomics data of KYSE30 cells with or without PSMD2 knockdown. B Functional enrichment of the differentially expressed proteins in KYSE30 cells with or without PSMD2 knockdown by KEGG. C Functional enrichment of the differentially expressed proteins in KYSE30 cells with or without PSMD2 knockdown by Metascape. D Statistical analysis of the protein levels of of ASS1 detected by IHC of tissue microarray consisting of 144 ESCC and normal samples. E Box plots showing ASS1 expression levels in the early and advanced stage of ESCC. Data were derived from tissue microarray. F The positive correlation between the protein levels of PSMD2 and ASS1 proteins in ESCC tissues. N = 144. G-H KYSE450 cells with PSMD2 overexpression were examined for PSMD2 mRNA levels (G) and ASS1 mRNA levels (H) by qPCR.I, J KYSE450 cells with PSMD2 knockdown were examined for PSMD2 mRNA levels (I) and ASS1 mRNA levels (J) by qPCR.K KYSE30 cells with PSMD2 overexpression or vector were treated with CHX (100 µg/ml). The protein level of ASS1 was examined at 0, 2, 4, 8 h by immunoblotting (left panel) and protein half-life calculated (right panel).L, M ASS1 overexpression reduced the formation of LC3B puncta. Autophagy was induced in KYSE30 cells (L) and KYSE450 cells (M) with ASS1 overexpression by FBS deprivation. The cells were then stained with the DAPGreen (green). The nucleus was stained with Hoechst 33342 (blue). Scale bar: 30 µm. Quantitative analysis of the autophagic puncta was shown in the right panel. N = 3. Data were presented as mean ± SD. N–O ASS1 knockdown in KYSE30 cells (N) and KYSE450 cells (O) increased the formation of LC3B puncta. N = 3. Data were presented as mean ± SD

Fig 5: PSMD2 inhibits autophagy and promotes the proliferation of KYSE30. A The overexpression of PSMD2 reduced the formation of LC3B puncta. B The knockdown of PSMD2 increased the formation of LC3B puncta. Autophagy was induced in KYSE30 cells with PSMD2 overexpression or knockdown by FBS deprivation for 3 h or 20 h. The cells were then stained with the DAPGreen (green) with nucleus counterstained with Hoechst 33342 (blue). Scale bar: 30 µm. C, D PSMD2 inhibited autophagy under FBS deprivation. KYSE30 cells with PSMD2 overexpression (PSMD2) (C) or PSMD2 knockdown (shPSMD2) (D) were treated with CQ as indicated. The cell lysate was examined by immunoblotting to detect p62, LC3 and GAPDH. The LC3-II: LC3-I ratio was qualified with ImageJ software. E Effect of ATG7 knockdown on the proliferation of ESCC cells with PSMD2 depletion was examined by fluorescently staining 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. Scale bar: 100 µm. F Effect of ATG7 knockdown on colony formation of ESCC cells with PSMD2 depletion. N = 3. The data were presented as mean ± SD. G Effect of ATG7 knockdown in ESCC cells with PSMD2 depletion on cell proliferation. The cells were used to determine the cell proliferation rate by CCK-8 assay at the indicated time point. N = 3. The data were presented as mean ± SD, * for p < 0.05, ** for p < 0.01, *** for p < 0.001 and **** for p < 0.0001