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. 2021 May 14;40(23):3974–3988. doi: 10.1038/s41388-021-01815-4

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© The Author(s) 2021

Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/.

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Fig. 2 — A The effect of SHMT2 on autophagy. To establish stable cell lines, HCT116 cells were infected with Scramble-sh (Control) or SHMT2 knockdown (SHMT2-sh-1, -sh-2, or -sh-3) lentivirus for 72 h and selected with puromycin (1 mg/ml). The protein levels of endogenous SHMT2, p62, LC3, and β-actin (as the internal standard) were examined by western blotting. B Identification of SHMT2-KO monoclonal HCT116 cell lines. C The protein levels of endogenous SHMT2, p62, LC3, and β-actin were evaluated in control and SHMT2-KO HCT116 cells. D The effect of SHMT2 on autophagy under glucose deprivation (GD) was assessed. E GFP-LC3 puncta were induced in SHMT2 knockdown cells. Control and SHMT2-sh stable HCT116 cell lines were transfected with the GFP-LC3 plasmid and cultured in complete medium for 24 h. Scale bar, 10 μm. F The percentage of cells exhibiting accumulation of GFP-LC3 in puncta (GFP-LC3^vac) is shown (mean ± s.d., n = 3; **P < 0.01). G Ultrastructural evidence of autophagic vacuolization induced by SHMT2 depletion. H The numbers of autophagosomes (APs) and autolysosomes (ALs) were determined in at least 50 cells in three independent experiments (mean ± s.d.; **P < 0.01). I Effect of SHMT2 on LC3 maturation in SW480 cells. To establish stable cell lines, SW480 cells were infected with Scramble-sh (Control) or SHMT2 knockdown (SHMT2-sh-1 or -sh-2) lentivirus for 72 h and selected with puromycin (1 mg/ml). The protein levels of endogenous SHMT2, p62, LC3, and β-actin (as the internal standard) were examined by western blotting. J Autophagy levels were increased in SHMT2-sh cells and prevented by the autophagy inhibitor chloroquine (CQ). K–M Representative images and quantification of HCT116 cells expressing mCherry-GFP-LC3B and the indicated shRNA. APs and ALs were identified as yellow and red puncta, respectively. The numbers of puncta are shown as the mean ± s.e.m. values. Statistical significance was determined by Poisson regression. ns nonsignificant, *P < 0.05, ***P < 0.001.

Fig. 2 — A The effect of SHMT2 on autophagy. To establish stable cell lines, HCT116 cells were infected with Scramble-sh (Control) or SHMT2 knockdown (SHMT2-sh-1, -sh-2, or -sh-3) lentivirus for 72 h and selected with puromycin (1 mg/ml). The protein levels of endogenous SHMT2, p62, LC3, and β-actin (as the internal standard) were examined by western blotting. B Identification of SHMT2-KO monoclonal HCT116 cell lines. C The protein levels of endogenous SHMT2, p62, LC3, and β-actin were evaluated in control and SHMT2-KO HCT116 cells. D The effect of SHMT2 on autophagy under glucose deprivation (GD) was assessed. E GFP-LC3 puncta were induced in SHMT2 knockdown cells. Control and SHMT2-sh stable HCT116 cell lines were transfected with the GFP-LC3 plasmid and cultured in complete medium for 24 h. Scale bar, 10 μm. F The percentage of cells exhibiting accumulation of GFP-LC3 in puncta (GFP-LC3^vac) is shown (mean ± s.d., n = 3; **P < 0.01). G Ultrastructural evidence of autophagic vacuolization induced by SHMT2 depletion. H The numbers of autophagosomes (APs) and autolysosomes (ALs) were determined in at least 50 cells in three independent experiments (mean ± s.d.; **P < 0.01). I Effect of SHMT2 on LC3 maturation in SW480 cells. To establish stable cell lines, SW480 cells were infected with Scramble-sh (Control) or SHMT2 knockdown (SHMT2-sh-1 or -sh-2) lentivirus for 72 h and selected with puromycin (1 mg/ml). The protein levels of endogenous SHMT2, p62, LC3, and β-actin (as the internal standard) were examined by western blotting. J Autophagy levels were increased in SHMT2-sh cells and prevented by the autophagy inhibitor chloroquine (CQ). K–M Representative images and quantification of HCT116 cells expressing mCherry-GFP-LC3B and the indicated shRNA. APs and ALs were identified as yellow and red puncta, respectively. The numbers of puncta are shown as the mean ± s.e.m. values. Statistical significance was determined by Poisson regression. ns nonsignificant, *P < 0.05, ***P < 0.001.