Journal: BMC Cancer

Article Title: IL-17 triggers PD-L1 gene transcription in NSCLC cells via TRIM31-dependent MEF2C K63-linked polyubiquitination

doi: 10.1186/s12885-025-13473-w

Figure Lengend Snippet: Effects of IL-17 stimulation and MEF2C overexpression or knockdown on PD-L1 promoter activity, gene transcription and expression. (A-C) H1299 or PC9 cells were transfected with pGL3-PD-L1-FL and pRL-SV40 plasmids for 48 h followed by 50 ng/ml IL-17 for 3 h (A) , cotransfected with pIRES2-EGFP or pIRES2-MEF2C along with pGL3-PD-L1-FL and pRL-SV40 for 48 h (B) , or cotransfected with shCTR or shMEF2C along with pGL3-PD-L1-FL and pRL-SV40 for 48 h plus IL-17 for 3 h (C) . PD-L1-FL promoter activity was examined by luciferase reporter assay (* p < 0.05, ** p < 0.01 vs. DMEM, pIRES2-EGFP or shCTR; Δ p < 0.05, ΔΔ p < 0.01 vs. shCTR + IL-17). (D-G) H1299 and PC9 cells were transfected with pIRES2-EGFP or pIRES2-MEF2C for 48 h (D , F) or with shCTR or shMEF2C for 48 h plus IL-17 for 3 h (E , G) . PD-L1 mRNA and protein expression levels were detected by real-time PCR and IB (* p < 0.05, ** p < 0.01 vs. pIRES2-EGFP or shCTR; Δ p < 0.05, ΔΔ p < 0.01 vs. shCTR + IL-17). Data from three independent experiments are presented as the means ± SDs and were analysed by one-way ANOVA or t tests, n = 3

Article Snippet: A reverse transcription reagent kit (Cat. no. R222-01), a chromatin immunoprecipitation (ChIP) kit (Cat. no. P2078), anti-β-actin antibody (Cat. no. AF0003), a nuclear and cytoplasmic protein extraction kit (Cat. no. P0027), and a cell counting kit-8 (Cat. no. C0037) were from Beyotime Biotech (Shanghai, China).

Techniques: Over Expression, Knockdown, Activity Assay, Expressing, Transfection, Luciferase, Reporter Assay, Real-time Polymerase Chain Reaction

Journal: BMC Cancer

Article Title: IL-17 triggers PD-L1 gene transcription in NSCLC cells via TRIM31-dependent MEF2C K63-linked polyubiquitination

doi: 10.1186/s12885-025-13473-w

Figure Lengend Snippet: Effects of TRIM31 overexpression or knockdown on PD-L1 promoter activity, gene transcription and expression or PD-L1 knockdown on MEF2C and TRIM31 expression induced by IL-17. (A , B) H1299 or PC9 cells were cotransfected with pIRES2-EGFP or pIRES2-TRIM31 and pGL3-PD-L1-FL plus pRL-SV40 for 48 h or with shCTR or shTRIM31 and pGL3-PD-L1-FL plus pRL-SV40 for 48 h followed by 50 ng/ml IL-17 for 3 h. PD-L1 promoter activity was measured by luciferase reporter assay (* p < 0.05, ** p < 0.01 vs. pIRES2-EGFP or shCTR; ΔΔ p < 0.01 vs. shCTR + IL-17). (C-F) H1299 or PC9 cells were transfected with pIRES2-EGFP or pIRES2-TRIM31 for 48 h or with shCTR or shTRIM31 for 48 h plus 50 ng/ml IL-17 for 3 h. PD-L1 mRNA (C , D) and protein (E , F) expression levels were detected by real-time PCR and IB ( p < 0.05, ** p < 0.01 vs. pIRES2-EGFP or shCTR; Δ p < 0.05, ΔΔ p < 0.01 vs. shCTR + IL-17). (G) H1299 or PC9 cells were transfected with shPD-L1 for 48 h followed by 50 ng/ml IL-17 for 3 h, and MEF2C and TRIM31 expression was detected by IB (** p < 0.01 vs. shCTR; ΔΔ p < 0.01 vs. shCTR + IL-17). Representative images are shown, and the data from triplicate experiments are presented as the means ± SDs and were analysed by one-way ANOVA or t tests; n = 3

Article Snippet: A reverse transcription reagent kit (Cat. no. R222-01), a chromatin immunoprecipitation (ChIP) kit (Cat. no. P2078), anti-β-actin antibody (Cat. no. AF0003), a nuclear and cytoplasmic protein extraction kit (Cat. no. P0027), and a cell counting kit-8 (Cat. no. C0037) were from Beyotime Biotech (Shanghai, China).

Techniques: Over Expression, Knockdown, Activity Assay, Expressing, Luciferase, Reporter Assay, Transfection, Real-time Polymerase Chain Reaction

Journal: BMC Cancer

Article Title: IL-17 triggers PD-L1 gene transcription in NSCLC cells via TRIM31-dependent MEF2C K63-linked polyubiquitination

doi: 10.1186/s12885-025-13473-w

Figure Lengend Snippet: Roles of IL-17 stimulation, TRIM31 overexpression or knockdown or TRIM31 enzyme activity deficiency in MEF2C K63-linked or K48-linked polyubiquitination as well as PD-L1 gene transcription and expression. (A) H1299 or PC9 cells were stimulated with 50 ng/ml IL-17 for 0, 1, 2, 3, 6 and 12 h, and the levels of MEF2C K63- and K48-linked polyubiquitination were measured by IP and IB. (B) H1299 or PC9 cells were transfected with pIRES2-TRIM24, pIRES2-TRIM31, pIRES2-TRIM32 or pIRES2-TRIM59 for 48 h, and the levels of MEF2C K63- and K48-linked polyubiquitination were determined by IP and IB. (C , D) H1299 or PC9 cells were transfected with pIRES2-TRIM31 for 48 h (C) or with shTRIM31 for 48 h plus IL-17 for 3 h (D) , and the levels of MEF2C K63- and K48-linked polyubiquitination were measured by IP and IB. (E) H1299 or PC9 cells were transfected with pIRES2-EGFP, pIRES2-TRIM31 or the pIRES2-TRIM31 mutant (C53A, C56A) for 48 h, and the levels of MEF2C K63- and K48-linked polyubiquitination were examined by IP and IB. (F) H1299 or PC9 cells were cotransfected with pIRES2-TRIM31 or pIRES2-TRIM31 (C53A, C56A) for 48 h, and the level of MEF2C binding to PD-L1 promoter sites was detected by ChIP. (G-I) H1299 or PC9 cells were cotransfected with pIRES2-EGFP, pIRES2-TRIM31 or pIRES2-TRIM31 (C53A, C56A) and the PD-L1-FL promoter plus pRL-SV40 for 48 h, and PD-L1 promoter activity (G) and mRNA (H) and protein (I) expression levels were determined by luciferase reporter assay, real-time PCR and IB. ** p < 0.01 vs. pIRES2-EGFP; ΔΔ p < 0.01 vs. pIRES2-TRIM31. Data from triplicate experiments are presented as the means ± SDs and were analysed by one-way ANOVA, n = 3

Article Snippet: A reverse transcription reagent kit (Cat. no. R222-01), a chromatin immunoprecipitation (ChIP) kit (Cat. no. P2078), anti-β-actin antibody (Cat. no. AF0003), a nuclear and cytoplasmic protein extraction kit (Cat. no. P0027), and a cell counting kit-8 (Cat. no. C0037) were from Beyotime Biotech (Shanghai, China).

Techniques: Over Expression, Knockdown, Activity Assay, Expressing, Transfection, Mutagenesis, Binding Assay, Luciferase, Reporter Assay, Real-time Polymerase Chain Reaction

Journal: BMC Cancer

Article Title: IL-17 triggers PD-L1 gene transcription in NSCLC cells via TRIM31-dependent MEF2C K63-linked polyubiquitination

doi: 10.1186/s12885-025-13473-w

Figure Lengend Snippet: Site identification of MEF2C K63-linked polyubiquitination by TRIM31 and effect of the site mutation on PD-L1 transcription and expression. (A) HEK293T cells were cotransfected with HA-MEF2C and Flag-TRIM31 or the mutant plasmids of the 12 lysines of MEF2C, including the united mutant plasmid, for 48 h, and MEF2C K63-linked ubiquitination was examined by IP and IB. (B) H1299 or PC9 cells were cotransfected with HA-MEF2C and Flag-TRIM31 or MEF2C K23 mutant (K23R) or K25 mutant (K25R) or K30 mutant (K30R) or K31 mutant (K31R) for 48 h, and MEF2C K63-linked ubiquitination was detected by IP and IB. (C) MEF2C amino acid sequences of different species are highly conserved at K25. (D) H1299 or PC9 cells were cotransfected with Flag-TRIM31 and HA-MEF2C (WT) or HA-MEF2C (K25R) for 48 h, and HA-MEF2C and HA-MEF2C K63-linked ubiquitination in the cytoplasm and nucleus were measured by IP and IB. (E) H1299 or PC9 cells were cotransfected with HA-MEF2C, Flag-TRIM31 or MEF2C (K25R) for 48 h, a ChIP assay was performed with anti-MEF2C (with anti-IgG as the control), and purified DNA was amplified by real-time PCR to measure the level of MEF2C binding to the regions of the PD-L1 promoter. (F-G) H1299 or PC9 cells were cotransfected with the PD-L1-FL promoter, HA-MEF2C, MEF2C-K25R and Flag-TRIM31 for 48 h, and PD-L1 promoter activity (F) and mRNA (G) and protein (H) expression levels were determined by luciferase reporter assay, real-time PCR and IB. ** p < 0.01 vs. Flag-TRIM31; Δp < 0.01 vs. HA-MEF2C + Flag-TRIM31. The data from triplicate experiments are presented as the means ± SDs and were analysed by one-way ANOVA, n = 3

Article Snippet: A reverse transcription reagent kit (Cat. no. R222-01), a chromatin immunoprecipitation (ChIP) kit (Cat. no. P2078), anti-β-actin antibody (Cat. no. AF0003), a nuclear and cytoplasmic protein extraction kit (Cat. no. P0027), and a cell counting kit-8 (Cat. no. C0037) were from Beyotime Biotech (Shanghai, China).

Techniques: Mutagenesis, Expressing, Plasmid Preparation, Ubiquitin Proteomics, Control, Purification, Amplification, Real-time Polymerase Chain Reaction, Binding Assay, Activity Assay, Luciferase, Reporter Assay

Journal: BMC Cancer

Article Title: IL-17 triggers PD-L1 gene transcription in NSCLC cells via TRIM31-dependent MEF2C K63-linked polyubiquitination

doi: 10.1186/s12885-025-13473-w

Figure Lengend Snippet: Putative scheme for the PD-L1 gene transcription mechanism in IL-17-stimulated H1299 or PC9 cells. MEF2C and TRIM31 are synchronously increased by IL-17-IL-17RA, and the upregulated TRIM31 can mediate MEF2C K63-linked polyubiquitination at Lys 25. The ubiquitinated MEF2C can easily translocate into the cell nucleus, where it can bind to two sites (-778 to -475 nt and − 336 to -97 nt) within the PD-L1 promoter, ultimately increasing PD-L1 gene transcription and expression

Article Snippet: A reverse transcription reagent kit (Cat. no. R222-01), a chromatin immunoprecipitation (ChIP) kit (Cat. no. P2078), anti-β-actin antibody (Cat. no. AF0003), a nuclear and cytoplasmic protein extraction kit (Cat. no. P0027), and a cell counting kit-8 (Cat. no. C0037) were from Beyotime Biotech (Shanghai, China).

Techniques: Expressing