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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. 3 — A–C Effect of SHMT2 and p53 on LC3 maturation. The protein levels of SHMT2, p53, p62, LC3, and β-actin (as the internal standard) were assessed by western blotting using anti-Flag and anti-p53, anti-p62, anti-LC3, and anti-β-actin antibodies, respectively. A HCT116^p53+/+ cells and HCT116^p53-/- cells were transfected with Flag-SHMT2 for 24 h. B HCT116 cells were infected with Scramble-sh (Control) or p53 knockdown (Sh) lentivirus for 72 h and transfected with Flag-SHMT2 for 24 h. C Stable control and SHMT2-sh cells were transfected with Flag-WT, nuclear (NES-) and cytosolic p53 (NLS-) plasmids for 24 h. The protein levels of p53, SHMT2, p62, LC3, and β-actin were assessed by western blotting using anti-Flag and anti-p53, anti-SHMT2, anti-p62, anti-LC3, and anti-β-actin antibodies, respectively. D GFP-LC3 puncta formation induced by SHMT2-sh or p53 mutants. Control and SHMT2-sh stable HCT116 cell lines were transfected with Flag-WT p53, nuclear (NES-) p53, cytosolic p53 (NLS-), or GFP-LC3 plasmids and cultured in complete medium for 24 h. Scale bar, 10 μm. E The percentage of HCT116 and SW480 cells exhibiting accumulation of GFP-LC3 in puncta (GFP-LC3^vac) is shown (mean ± s.d., n = 3; **P < 0.01). Puncta were quantified from 100 cells. F SHMT2 disrupted the binding of cytosolic p53 to HDM2. HCT116 cells transfected with GFP-HDM2, HA-SHMT2, and Flag-cytosolic p53 (NLS-) plasmids were immunoprecipitated with FLAG-M2 beads. Western blotting for p53, GFP, and HA was then performed. G SHMT2 maintained the stability of cytosolic p53. Western blot analysis of lysates of cells with stable SHMT2 overexpression and knockdown that were transfected with Flag-WT, nuclear (NES-), and cytosolic p53 (NLS-) plasmids and treated with the translation inhibitors cycloheximide (CHX, 50 μg/ml) and MG132 (25 μM, 4 h) for the indicated durations. H, I HCT116 cells transfected with Flag-cytosolic p53 (NLS-), GFP-SHMT2 or GFP-HDM2 were immunoprecipitated with FLAG-M2 beads. Western blotting for p53 and GFP was then performed. H 5-FU disrupted the binding of cytosolic p53 to SHMT2. I 5-FU promoted the binding of cytosolic p53 to HDM2. J Western blot analysis of lysates of HCT116 cells transfected with Flag-cytosolic p53 (NLS-) or SHMT2 plasmids and treated with CHX and MG132 for the indicated durations with or without 5-FU. K Nuclear-cytosolic separation shows that SHMT2 affects the stability of endogenous cytosolic p53. Cyt cytosolic, Nuc nuclear.