Fig 2: SHMT2 knockdown in ccRCC cells induced the autophagy-lysosome pathway and affected autophagic flux.A Top enriched KEGG pathways based on the differentially expressed proteins between shSHMT2 and shNC ACHN cells. The abscissa represented the number of differentially expressed proteins in each pathway. The color of the bar chart indicated the significance of the enriched KEGG pathways. B Heat-map of 37 differentially expressed proteins involved in the lysosome pathway. Red represented significantly upregulated proteins and blue represented significantly downregulated proteins after SHMT2 knockdown. Red arrows indicated the LAMP1 and LAMP2. A represented shNC, and B represented shSHMT2. C The levels of autophagy-lysosome-related proteins after knockdown or overexpression of SHMT2 in ccRCC cells. D Immunofluoresence analysis of LC3 (green) in ACHN cells after SHMT2 knockdown alone or in combination with CQ (20uM) or 3-MA (5 mM) for 48 h. Nuclei were stained with DAPI. E Double immunofluoresence analysis of the co-localization of LC3 (green) and LAMP1 (red) in ACHN cells after SHMT2 knockdown alone or in combination with CQ (20uM) or 3-MA (5 mM) for 48 h. Nuclei were stained with DAPI. F ACHN cells were transfected with adenovirus harboring mRFP-GFP-LC3 (green and red puncta indicate GFP and mRFP, respectively) to follow autophagic flux, after SHMT2 knockdown following treatment with CQ (20uM) or 3-MA (5 mM). Nuclei were stained with DAPI (left). Quantitative analysis of autophagosomes (yellow dots) and autolysosomes (red dots) (right). G TEM analysis revealed that autolysome formation was induced after SHMT2 knockdown in ACHN cells. Red arrows indicated autolysosomes or autophagosomes. The scale bars in the original image and the enlarged image represented 5 μm and 1 μm, respectively. *P < 0.05, **P < 0.01.

Fig 3: Knockdown of SHMT2/MTHFD2 promotes liver fibrosis resolution in NASH mice.A, schematic illustrating animal experimental design. B and C, liver Shmt2 and Mthfd2 mRNA levels. Ad-shRNA–medicated Shmt2 and Mthfd2 knockdown in mouse liver tissue (n = 4). D, schematic representation of vitamin A–coupled liposomes nanoparticle for the delivery of siRNA. E, representative image of H&E, Masson, and sirius red–stained paraffin-embedded sections of mouse liver tissues (scale bar represents 50 μm). F, Ishak fibrosis stage scores at indicated treatment conditions (n = 6). G, hepatic hydroxyproline content (n = 6). H and I, ALT and AST levels of plasma (n = 6). J and K, hepatic mRNA expression of fibrogenic genes Acta2 and Col1a1 (n = 5). The statistical significance (∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001) was tested by student’s unpaired t test. AST, aspartate aminotransferase; ALT, alanine aminotransferase; H&E, hematoxylin-eosin staining; MTHFD, methylenetetrahydrofolate dehydrogenase; NASH, nonalcoholic steatohepatitis; SHMT, serine hydroxymethyltransferase.

Fig 4: SHMT2 knockdown inhibited GC cell invasion and metastasis. (A) Representative images of SHMT2 knockdown’s effect on cell colony formation (plate clones and soft agar clones) and quantitative analysis of colony formation. (B) Representative images and quantitative analysis of the effect of SHIN1 (1 μM) on cell colony formation. (C) Transwell assay detected the effect of SHMT2 knockdown on the cell migration ability and the representative images. Scale bar, 100 μm. (D) Representative images and quantitative analysis of SHMT2 knockdown’s effect on the cell migration ability detected using a 24 h wound-healing assay in SGC7901. Scale bar, 100 μm. (E) Western blot examined SHMT2 knockdown’s effect on the expression of cellular EMT markers. * p < 0.05, ** p < 0.01, and *** p < 0.001; ns: no significance.

Fig 5: SHMT2 was aberrantly upregulated in ccRCC tissues and associated with patient survival.A Relative expression of SHMT2 and SHMT1 in ccRCC specimens and controls based on the TCGA database. B qRT-PCR analysis was performed to evaluate the SHMT2 mRNA levels in 12 pairs of ccRCC tissues and controls. C Western blot analysis was performed to evaluate the SHMT2 protein levels in 12 pairs of ccRCC tissues and controls. β-actin expression was detected as a loading control. T meaned tumors and N meaned paired controls. D TMA IHC scores showing that high, middle and low SHMT2 expression were observed in 21, 13, and 41 of the 75 tumor specimens. The corresponding numbers were 1, 21, and 53 in the 75 paired control tissues (left). Representative IHC staining of SHMT2 protein in human ccRCC tissues (right). E Overall survival curves of ccRCC patients with different SHMT2 expression levels based on the GEPIA database. *P < 0.05, **P < 0.01, ****P < 0.0001.