tnfr1  (Proteintech)

Figure Legend Snippet: Autophagy limits CAR‐T cell‐mediated cytotoxicity by suppressing TNF‐α induced apoptosis. (A) Volcano plot showing varied genes in Nalm6 cells with the addition of vehicle (as control) and autophinib (log 2 FC > 1 and P < 0.05). (B) Volcano plot showing varied genes between sgControl and indicated RB1CC1 KO Nalm6 cells (log 2 FC > 1 and P < 0.05). (C) KEGG pathway enrichment analysis of varied genes identified in RNA sequencing for Nalm6 cells treated with vehicle (as control) and autophinib. (D) Western blotting showing the expression levels of Caspase‐8, Cleaved caspase‐8, Caspase‐9, Cleaved caspase‐9 and p62 proteins after the addition of vehicle (as control), autophinib and SAR405 in Nalm6 and Raji cells when co‐cultured with or without CD19 CAR‐T cells. GAPDH was used as a loading control. (E) Western blotting showing the expression levels of Caspase‐8, Cleaved caspase‐8, Caspase‐9, Cleaved caspase‐9 and p62 proteins in sgControl and indicated gene‐KO (sgBECN1, sgRB1CC1) Nalm6 and Raji cells when co‐cultured with or without CD19 CAR‐T cells. GAPDH was used as a loading control. (F) Expression of TNFRSF1A mRNA by RT‐qPCR and TNFR1 protein by western blotting in Nalm6 and Raji cells after addition of vehicle (as control), autophinib and SAR405 ( n = 3). Values are shown as the mean ± SD. Statistical differences among three groups in each cell line are calculated with one‐way ANOVA with tests. (G‐H) Cytotoxic analysis of sgControl and indicated gene‐KO (sgTNFRSF1A) Nalm6 and Raji cells co‐cultured with CD19 CAR‐T cells (E:T ratio = 1:4) when treated with vehicle (as control), autophinib and SAR405 and then with or without TNF‐block ( n = 3). Values are shown as the mean ± SD. Statistical differences are calculated with two‐way ANOVA with tests. (I) Cytotoxic analysis of sgControl and indicated gene‐KO (sgBECN1, sgRB1CC1, sgTNFRSF1A) Nalm6 and Raji cells co‐cultured with CD19 CAR‐T cells (E: T ratio = 1: 4) when treated with or without TNF block ( n = 3). Values are shown as the mean ± SD. Statistical differences are calculated with two‐way ANOVA with tests. * P < 0.05; ** P < 0.01; *** P < 0.001; **** P < 0.0,001; ns: not significant. Abbreviations: ANOVA analysis of variance; CAR‐T chimeric antigen receptor T; E:T effector:target; FC fold change; FC fold change; KO knockout; KEGG Kyoto Encyclopedia of Genes and Genomes; RT‐qPCR real‐time quantitative polymerase chain reaction; ns: not significant; SD standard deviation; sg single guide; TNF tumor necrosis factor; TNFR1 tumor necrosis factor receptor 1.

Techniques Used: Control, RNA Sequencing, Western Blot, Expressing, Cell Culture, Quantitative RT-PCR, Blocking Assay, Knock-Out, Real-time Polymerase Chain Reaction, Standard Deviation

Figure Legend Snippet: STAT1/IRF1 axis mediates the upregulation of CXCL10 and CXCL11 induced by autophagy targeting. (A‐B) Western blotting showing the expression levels of STAT1, pSTAT1, and IRF1 proteins in Nalm6 and Raji cells after the addition of autophagy inhibitors or the knockout of RB1CC1 ( n = 3). GAPDH was used as a loading control. The histograms showing the expression of STAT1 and IRF1 mRNA by RT‐qPCR quantification. Values are shown as the mean ± SD. Statistical differences are calculated with one‐way ANOVA tests. (C‐D) Western blotting showing the expression levels of STAT1 and IRF1 proteins in Nalm6 and Raji cells after the addition of autophagy inhibitors and the silencing of STAT1 ( n = 3). GAPDH was used as a loading control. The histograms showing the expression of STAT1 and IRF1 mRNA by RT‐qPCR quantification. Values are shown as the mean ± SD. Statistical differences are calculated with one‐way ANOVA tests. (E‐F) Expression of CXCL10 and CXCL11 mRNA by RT‐qPCR and ELISA quantification of CXCL10 and CXCL11 protein levels in the supernatants of shControl, shSTAT1 and shIRF1 Nalm6 and Raji cells ( n = 3). Values are shown as the mean ± SD. Statistical differences are calculated with two‐way ANOVA tests. (G‐H) ChIP‐qPCR showing the binding of STAT1 and IRF1 to the promoter region of CXCL10 and CXCL11 ( n = 3). Values are shown as the mean ± SD. Statistical differences for each cell line are calculated with unpaired Student's t tests. (I) Graphic abstract: in the proposed model, inhibition of cancer cell‐autonomous autophagy leads to accumulation of cytosolic DNA, which thereby not only suppresses cancer cell survival by inducing TNFR1‐TNF‐α mediated apoptosis but also promotes the CAR‐T cell recruitment in tumor microenvironment via STAT1/IRF1‐dependent activation of chemokine signaling. * P < 0.05; ** P < 0.01; *** P < 0.001; **** P < 0.0,001; ns: not significant. Abbreviations: ANOVA analysis of variance; CAR‐T, chimeric antigen receptor T; ChIP, chromatin immunoprecipitation; CXCL CXC, chemokine ligand; ELISA, enzyme‐linked immunosorbent assay; FC, fold change; IRF, interferon regulatory factor; ns: not significant; RT‐qPCR, real‐time quantitative polymerase chain reaction; SD, standard deviation; sh short hairpin; STAT, signal transducers and activators of transcription.

Techniques Used: Western Blot, Expressing, Knock-Out, Control, Quantitative RT-PCR, Enzyme-linked Immunosorbent Assay, ChIP-qPCR, Binding Assay, Inhibition, Activation Assay, Chromatin Immunoprecipitation, Real-time Polymerase Chain Reaction, Standard Deviation