Journal: bioRxiv

Article Title: Targeting of Cdc42 GTPase in regulatory T cells unleashes anti-tumor T cell immunity

doi: 10.1101/2021.09.23.461402

Figure Lengend Snippet: Heterozygous loss of Cdc42 induces Treg cell plasticity and inhibits tumor growth through upregulation of CAI. ( A ) Quantitative real-time RT-PCR analysis of the expression of CAI mRNA in Treg cells from Cdc42 +/+ Foxp3 YFP-Cre and Cdc42 Flox/+ Foxp3 YFP-Cre mice. ( B ) Extracellular pH of Cdc42 +/+ Foxp3 YFP-Cre and Cdc42 Flox/+ Foxp3 YFP-Cre Treg cells cultured ex vivo . ( C and D ) Flow cytometry analysis of the expression of Foxp3 ( C ) and IFN-γ ( D ) in Cdc42 +/+ Foxp3 YFP-Cre Treg cells cultured with normal medium (pH 7.40) or medium of pH 7.60. ( E ) Flow cytometry analysis of the expression of IFN-γ in CD4 + effector T cells (ex-Treg) that were converted from Cdc42 +/+ Foxp3 YFP-Cre Treg cells cultured with normal medium or medium of pH 7.60. ( F and G ) Flow cytometry analysis of the expression of Foxp3 ( F ) and IFN-γ ( G ) in Cdc42 +/+ Foxp3 YFP-Cre Treg cells incubated with conditional medium (CM) from Cdc42 +/+ Foxp3 YFP-Cre or Cdc42 Flox/+ Foxp3 YFP-Cre Treg cell culture. (H) Flow cytometry analysis of the expression of IFN-γ in CD4 + effector T cells (ex-Treg) that were converted from Cdc42 +/+ Foxp3 YFP-Cre Treg cells incubated with conditional medium (CM) from Cdc42 +/+ Foxp3 YFP-Cre or Cdc42 Flox/+ Foxp3 YFP-Cre Treg cell culture. ( I ) Extracellular pH of Cdc42 +/+ Foxp3 YFP-Cre and Cdc42 Flox/+ Foxp3 YFP-Cre Treg cells cultured with or without AZA. ( J ) Flow cytometry analysis of the expression of IFN-γ in Cdc42 +/+ Foxp3 YFP-Cre and Cdc42 Flox/+ Foxp3 YFP-Cre Treg cells cultured with or without AZA. ( K ) Flow cytometry analysis of the expression of IFN-γ in CD4 + effector T cells (ex-Treg) that were converted from Cdc42 +/+ Foxp3 YFP-Cre or Cdc42 Flox/+ Foxp3 YFP-Cre Treg cells cultured with or without AZA. ( L ) Quantitative real-time RT-PCR analysis of the expression of CAI mRNA in Cdc42 +/+ Foxp3 YFP-Cre and Cdc42 Flox/+ Foxp3 YFP-Cre Treg cells transduced with or without CAI shRNA. ( M ) Extracellular pH of Cdc42 +/+ Foxp3 YFP-Cre and Cdc42 Flox/+ Foxp3 YFP-Cre Treg cells transduced with or without CAI shRNA. ( N ) Flow cytometry analysis of the expression of IFN-γ in Cdc42 +/+ Foxp3 YFP-Cre and Cdc42 Flox/+ Foxp3 YFP-Cre Treg cells transduced with or without CAI shRNA. ( O ) Flow cytometry analysis of the expression of IFN-γ in CD4 + effector T cells (ex-Treg) that were converted from Cdc42 +/+ Foxp3 YFP-Cre or Cdc42 Flox/+ Foxp3 YFP-Cre T reg cells transduced with or without CAI shRNA. ( P ) MC38 tumor growth in Cdc42 +/+ Foxp3 YFP-Cre and Cdc42 Flox/+ Foxp3 YFP-Cre mice treated with or without AZA. ( A ) Error bars indicate SD of 4 mice. ( B-O ) Error bars indicate SD of triplicates. Data are from 5 mice pooled. ( P ) Error bars indicate SD of 4 mice. Data are representative of two independent experiments. *p < 0.05; **p < 0.01. AZA: acetazolamide. MFI: Mean fluorescence intensity.

Article Snippet: For lentiviral shRNA-mediated knockdown, scramble and CAI shRNA lentiviral supernatant were produced by transfection of 293T cells with packaging lentiviral plasmids and either scramble or CAI shRNA lentiviral vectors (Origene, TL510087) followed by concentrating with Lenti-X concentrator (Takara, 631231).

Techniques: Quantitative RT-PCR, Expressing, Cell Culture, Ex Vivo, Flow Cytometry, Incubation, Transduction, shRNA, Fluorescence

Journal: bioRxiv

Article Title: Targeting of Cdc42 GTPase in regulatory T cells unleashes anti-tumor T cell immunity

doi: 10.1101/2021.09.23.461402

Figure Lengend Snippet: Heterozygous loss of Cdc42 induces Treg cell plasticity and inhibits tumor growth through WASP-GATA3-mediated CAI expression. ( A ) Diagram of GATA3 binding sites on the CAI locus. ( B ) Flow cytometry analysis of the expression of GATA3 in Treg cells from Cdc42 +/+ Foxp3 YFP-Cre and Cdc42 Flox/+ Foxp3 YFP-Cre mice. ( C ) CHIP-qPCR analysis of GATA3 binding to the CAI locus in Cdc42 +/+ Foxp3 YFP-Cre and Cdc42 Flox/+ Foxp3 YFP-Cre Treg cells. ( D ) Quantitative real-time RT-PCR analysis of the expression of CAI mRNA in Cdc42 +/+ Foxp3 YFP-Cre and Cdc42 Flox/+ Foxp3 YFP-Cre Treg cells treated with or without PTG. ( E ) Flow cytometry analysis of the expression of IFN-γ in Cdc42 +/+ Foxp3 YFP-Cre and Cdc42 Flox+ Foxp3 YFP-Cre Treg cells treated with or without PTG. (F) Flow cytometry analysis of the expression of IFN-γ in CD4 + effector T cells (ex-Treg) that were converted from Cdc42 +/+ Foxp3 YFP-Cre or Cdc42 Flox/+ Foxp3 YFP-Cre Treg cells treated with or without PTG. ( G ) Quantitative real-time RT-PCR analysis of the expression of CAI mRNA in Cdc42 +/+ Foxp3 YFP-Cre and Cdc42 Flox/+ Foxp3 YFP-Cre Treg cells transduced with or without GATA3 shRNA. ( H ) Flow cytometry analysis of the expression of IFN-γ in Cdc42 +/+ Foxp3 YFP-Cre and Cdc42 Flox/+ Foxp3 YFP-Cre Treg cells transduced with or without GATA3 shRNA. ( I ) Flow cytometry analysis of the expression of IFN-γ in CD4 + effector T cells (ex-Treg) that were converted from Cdc42 +/+ Foxp3 YFP-Cre or Cdc42 Flox/+ Foxp3 YFP-Cre Treg cells transduced with or without GATA3 shRNA. ( J ) MC38 tumor growth in Cdc42 +/+ Foxp3 YFP-Cre and Cdc42 Flox/+ Foxp3 YFP-Cre mice treated with or without PTG. ( K ) Flow cytometry analysis of the expression of GATA3 in Cdc42 +/+ Foxp3 YFP-Cre Treg cells treated with or without WKS. ( L ) Quantitative real-time RT-PCR analysis of the expression of CAI mRNA in Cdc42 +/+ Foxp3 YFP-Cre Treg cells treated with or without WKS. ( M ) Flow cytometry analysis of the expression of IFN-γ in Cdc42 +/+ Foxp3 YFP-Cre Treg cells treated with or without WKS. ( N ) Flow cytometry analysis of the expression of IFN-γ in CD4 + effector T cells (ex-Treg) that were converted from Cdc42 +/+ Foxp3 YFP-Cre Treg cells treated with or without WKS. ( O ) MC38 tumor growth in Cdc42 +/+ Foxp3 YFP-Cre and Cdc42 Flox+ Foxp3 YFP-Cre mice treated with or without WKS. 1.4 x 10 6 MC38 cells were injected. ( B ) Error bars indicate SD of 4 mice. ( C ) Error bars indicate SD of triplicates. Data are from 8 mice pooled. (D-I and K-N) Error bars indicate SD of triplicates. Data are from 5-6 mice pooled. ( J and O ) Error bars indicate SD of 4 mice. Data are representative of two independent experiments. *p < 0.05; **p < 0.01. PTG: pyrrothiogatain. WKS: wiskostatin. MFI: Mean fluorescence intensity.

Article Snippet: For lentiviral shRNA-mediated knockdown, scramble and CAI shRNA lentiviral supernatant were produced by transfection of 293T cells with packaging lentiviral plasmids and either scramble or CAI shRNA lentiviral vectors (Origene, TL510087) followed by concentrating with Lenti-X concentrator (Takara, 631231).

Techniques: Expressing, Binding Assay, Flow Cytometry, ChIP-qPCR, Quantitative RT-PCR, Transduction, shRNA, Injection, Fluorescence

Journal: PLOS Pathogens

Article Title: Lactobacillus gasseri ATCC33323 affects the intestinal mucosal barrier to ameliorate DSS-induced colitis through the NR1I3-mediated regulation of E-cadherin

doi: 10.1371/journal.ppat.1012541

Figure Lengend Snippet: a. qPCR assay to analyze the differential gene expression of L . gasseri ATCC33323 after 3/6/12 h of coculture with HCT116 and Caco2 colon cancer cells; b. Western blot analysis of NR1I3 and E-cadherin expression in L . gasseri ATCC33323 after 3/6/12 h of coculture with HCT116 and Caco2 colon cancer cells; c. Validation of the knockdown efficiency of NR1I3 in HCT116 and Caco2 colon cancer cells; d. NR1I3 and E-cadherin expression after intracellular NR1I3 knockdown in coculture with L . gasseri ATCC33323 for 3/6/12 h; e.,f. Results of ChIP experiments showing that NR1I3 can act as a transcription factor for CDH1; g. Luciferase reporter gene assay to verify the activity of NR1I3 as a CDH1 transcription factor. The values are expressed as the means ± SDs (n = 3). Superscript letters indicate significant differences at *P < 0.05, **P < 0.01, and ***P < 0.001.

Article Snippet: The types and dilution ratios of the antibodies used were as follows: ZO-1 (1:500; Proteintech, USA), E-cadherin (1:2000; Proteintech, USA), claudin1 (1:500; Proteintech, USA), β-actin (1:1000; Proteintech, USA), p120-catenin (1:1000; Proteintech, USA), β-catenin (1:1000; Proteintech, USA), occludin (1:500; Proteintech, USA), and NR1I3 (1:1000; Proteintech, USA).

Techniques: Gene Expression, Western Blot, Expressing, Biomarker Discovery, Knockdown, Luciferase, Reporter Gene Assay, Activity Assay

Journal: PLOS Pathogens

Article Title: Lactobacillus gasseri ATCC33323 affects the intestinal mucosal barrier to ameliorate DSS-induced colitis through the NR1I3-mediated regulation of E-cadherin

doi: 10.1371/journal.ppat.1012541

Figure Lengend Snippet: a. E-cadherin expression in HCT116 cell lines with or without NR1I3 knockdown by L . gasseri ATCC33323 pretreatment; b. E-cadherin expression in Caco2 cell lines with or without NR1I3 knockdown by L . gasseri ATCC33323 pretreatment; c. IL-1β, IL-6 and TNFα expression in HCT116 cell lines with or without NR1I3 knockdown by L . gasseri ATCC33323 pretreatment; d. IL-1β, IL-6 and TNFα expression in Caco2 cell lines with or without NR1I3 knockdown by L . gasseri ATCC33323 pretreatment. The values are expressed as the means ± SDs (n = 3). Superscript letters indicate significant differences at *P < 0.05, **P < 0.01, and ***P < 0.001.

Article Snippet: The types and dilution ratios of the antibodies used were as follows: ZO-1 (1:500; Proteintech, USA), E-cadherin (1:2000; Proteintech, USA), claudin1 (1:500; Proteintech, USA), β-actin (1:1000; Proteintech, USA), p120-catenin (1:1000; Proteintech, USA), β-catenin (1:1000; Proteintech, USA), occludin (1:500; Proteintech, USA), and NR1I3 (1:1000; Proteintech, USA).

Techniques: Expressing, Knockdown

Journal: Genome Medicine

Article Title: Evolving neoantigen profiles in colorectal cancers with DNA repair defects

doi: 10.1186/s13073-019-0654-6

Figure Lengend Snippet: Molecular, functional characteristics and source of origin of the indicated cell lines

Article Snippet: CAR1 , Stable , – , Stable , 0.36 , 2.47 , 0.28 , JCRB.

Techniques: Functional Assay, In Vitro