Journal: Advanced Science

Article Title: Phosphorylated SHMT2 Regulates Oncogenesis Through m 6 A Modification in Lung Adenocarcinoma

doi: 10.1002/advs.202307834

Figure Lengend Snippet: STUB1 ubiquitinates and degrades dephosphorylated SHMT2‐Ser90. A) The E3 ligases that specifically target SHMT2‐S90A identified by mass spectrometry. B) HA‐SHMT2‐WT and HA‐SHMT2‐S90A were transfected into A549 cells, and immunoprecipitation was used to examine the protein interactions. C) A549 cells were cotransfected with the HA‐SHMT2‐S90A plasmid and negative control siRNA or with HA‐SHMT2‐S90A and STUB1‐specific siRNAs. 48 h later, the ubiquitination of SHMT2‐S90A was detected via western blotting. D) A549 cells were cotransfected with the HA‐SHMT2‐S90A plasmid and the Flag‐STUB1 plasmid. 48 h later, the ubiquitination of SHMT2‐S90A was detected via western blotting. E,F) HA‐SHMT2‐S90A and Flag‐STUB1 plasmids were transfected into 293T cells, which were subsequently immunoprecipitated using anti‐HA (E) or anti‐Flag (F) antibodies. Western blotting was used to evaluate protein interactions. G,H) HA‐SHMT2‐WT (G) and HA‐SHMT2‐S90A (H) plasmids were transfected with or without the Flag‐STUB1 plasmid into A549 cells. CHX was used to treat the cells for the indicated times, and protein degradation was detected via western blotting (Top panels). The relative HA‐SHMT2 expression level compared with that of β‐actin was quantified. The data represent the average of three independent experiments (mean ± SD). ns, p > 0.05; *, p < 0.05; **, p < 0.01; ***, p < 0.001 (Bottom panels). I,J) HA‐SHMT2‐S90A plasmids with or without the Flag‐STUB1 plasmid were transfected into A549 cells, which were subsequently immunoprecipitated using an anti‐HA antibody. Western blotting was used to detect K48‐linked (I) and K63‐linked (J) ubiquitination. K,L) Flag‐SHMT2‐S90A‐K103R with or without the HA‐STUB1 plasmid was transfected into A549 cells, which were immunoprecipitated using an anti‐Flag antibody. Western blotting was used to detect total (K) and K48‐linked (L) ubiquitination.

Article Snippet: The UBR5 siRNAs were purchased from OriGene (SR309739).

Techniques: Mass Spectrometry, Transfection, Immunoprecipitation, Plasmid Preparation, Negative Control, Western Blot, Expressing

Journal: Advanced Science

Article Title: Phosphorylated SHMT2 Regulates Oncogenesis Through m 6 A Modification in Lung Adenocarcinoma

doi: 10.1002/advs.202307834

Figure Lengend Snippet: MAPK1 inhibition reduces the protein stability of SHMT2. A) A549 cells transfected with HA‐SHMT2 were separately treated with 1 µ m BDP5290, 1 µ m LY3214996, 1 µ m PIM447, 10 µ m seliciclib, 1 µ m tomivosertib, and 10 µ m DMAT for 24 h. The ubiquitination of SHMT2 was detected using western blotting. B) A549 cells transfected with HA‐SHMT2 were separately treated with 1 µ m LY3214996, 10 µ m seliciclib, 1 µ m tomivosertib, or 10 µ m DMAT. K48‐linked ubiquitination of SHMT2 was detected using western blotting. C) A549 cells transfected with the SHMT2 mutants were treated with 1 µ m LY3214996. K48‐linked ubiquitination of SHMT2 was detected using western blotting. D,E) H1299 (D) and A549 (E) cells were treated with 1 µ m LY3214996 for 24 h. Western blotting was used to detect protein expression. F) H1299 and A549 cells were treated with 1 µ m LY3214996 for 24 h. Q‐PCR was used to detect mRNA expression. The data represent the average of three independent experiments (mean ± SD). ns, p > 0.05. G) A549 cells were treated with CHX alone or with CHX or LY3214996 for the indicated times. Protein degradation was detected using western blotting (Top panel). The relative SHMT2 expression compared with that of β‐actin was quantified. The data represent the average of three independent experiments (mean ± SD). ns, p > 0.05; *, p < 0.05 (Bottom panels). H) A549 and H1299 cells transfected with HA‐SHMT2 were treated with 1 µ m LY3214996. Immunoprecipitation was used to concentrate HA‐SHMT2. The phosphorylation of SHMT2‐Ser90 was detected using an antibody specifically targeting SHMT2‐Ser90. I) A549 cells were transfected with the indicated plasmids and treated with or without LY3214996. Western blotting was used to detect protein ubiquitination. J) A549 cells were transfected with the indicated plasmid and siRNAs and then treated with or without LY3214996. Western blotting was used to detect protein ubiquitination.

Article Snippet: The UBR5 siRNAs were purchased from OriGene (SR309739).

Techniques: Inhibition, Transfection, Western Blot, Expressing, Immunoprecipitation, Plasmid Preparation

Journal: Advanced Science

Article Title: Phosphorylated SHMT2 Regulates Oncogenesis Through m 6 A Modification in Lung Adenocarcinoma

doi: 10.1002/advs.202307834

Figure Lengend Snippet: MAPK1 regulates the phosphorylation of SHMT2‐Ser90. A) A549 cells were transfected with MYC‐MAPK1. Western blotting was used to detect protein expression. B) A549 cells were transfected with MAPK1 siRNAs. Western blotting was used to detect protein expression. C) A549 cells were transfected with HA‐SHMT2. Immunoprecipitation was used to concentrate HA‐SHMT2. Western blotting was used to detect protein expression. D) A549 cells were transfected with MYC‐MAPK1. Immunoprecipitation was used to concentrate MYC‐MAPK1. Western blotting was used to detect protein expression. E) A549 cells were transfected with the indicated plasmids. Immunoprecipitation was used to concentrate HA‐SHMT2. Western blotting was used to detect protein expression. F) A549 cells were transfected with HA‐SHMT2 and MAPK1 siRNAs. Immunoprecipitation was used to concentrate HA‐SHMT2. Western blotting was used to detect protein expression. G) HA‐SHMT2 was concentrated from BEAS‐2B cells, and MYC‐MAPK1 was concentrated from A549 cells for use in an in vitro kinase assay. Western blotting was used to detect protein expression. H) A549 cells were transfected with HA‐SHMT2 and MAPK1 siRNAs. Immunoprecipitation was used to concentrate HA‐SHMT2, and western blotting was used to detect protein expression. I) A549 cells were transfected with control or MAPK1 siRNAs. The cells were treated with CHX for the indicated times, and western blotting was used to detect protein expression (Top panel). The relative SHMT2 expression compared with that of β‐actin was quantified. The data represent the average of three independent experiments (mean ± SD). #, siRNA1 versus Control; *,siRNA2 versus Control. ns, p > 0.05; *, p < 0.05; #, p < 0.05 (Bottom panels).

Article Snippet: The UBR5 siRNAs were purchased from OriGene (SR309739).

Techniques: Transfection, Western Blot, Expressing, Immunoprecipitation, In Vitro, Kinase Assay, Control

Journal: Advanced Science

Article Title: Phosphorylated SHMT2 Regulates Oncogenesis Through m 6 A Modification in Lung Adenocarcinoma

doi: 10.1002/advs.202307834

Figure Lengend Snippet: PTPMT1 dephosphorylates SHMT2 at Ser90 and regulates the protein stability of SHMT2. A,B) 293T cells were treated with the indicated plasmids. Immunoprecipitation combined with western blotting was used to detect protein interactions. C) A549 cells were transfected with the indicated plasmids. Immunoprecipitation was used to concentrate HA‐SHMT2. Western blotting was used to detect protein expression. D,E) A549 (D) and H1299 (E) cells were transfected with HA‐SHMT2 and PTPMT1 siRNAs. Immunoprecipitation was used to concentrate HA‐SHMT2, and western blotting was used to detect protein expression. F) A549 cells were transfected with the indicated plasmids. Western blotting was used to detect protein expression. G,H) A549 cells transfected with SHMT2‐WT (G) or SHMT2‐S90A (H) were cotransfected with Flag‐PTPMT1. The cells were treated with CHX for the indicated times, and western blotting was used to detect protein expression (Top panels). The relative HA‐SHMT2 expression level compared with that of β‐actin was quantified. The data represent the average of three independent experiments (mean ± SD). ns, p > 0.05; *, p < 0.05; ***, p < 0.001 (Bottom panels). I) A549 cells were transfected with the indicated plasmids. Immunoprecipitation combined with western blotting was used to detect protein expression. J,K) A549 cells were transfected with the indicated plasmids. Immunoprecipitation was used to concentrate HA‐SHMT2. Western blotting was used to detect K48‐linked (J) and K63‐linked (K) ubiquitination.

Article Snippet: The UBR5 siRNAs were purchased from OriGene (SR309739).

Techniques: Immunoprecipitation, Western Blot, Transfection, Expressing