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, Quantitative RT-PCR, Transduction, shRNA, Injection, Fluorescence

Journal: BioMed Research International

Article Title: LncRNA CAR10 Upregulates PDPK1 to Promote Cervical Cancer Development by Sponging miR-125b-5p

doi: 10.1155/2020/4351671

Figure Lengend Snippet: CAR10 is the target gene of miR-125b-5p. (a, b) The effect of CAR10 overexpression on miR-125b-5p, miR-1249, miR-2277-5p, miR-3192, miR-3663-5p, and miR-3692-5p expression level was detected by RT-qPCR in C33A and HeLa cells, # p < 0.05, compared with pcDNA-NC. (c) Detection of miR-125b-5p expression levels in cervical cancer tissues and paracancerous tissues collected by RT-qPCR, # p < 0.05, compared with adjacent controls. (d) The miR-125b-5p inhibitor (anti-miR-125b-5p) and the negative control Mock were transfected into C33A and HeLa, and the expression level of miR-125b-5p was detected by RT-qPCR, # p < 0.05, compared with Mock. (e, f) The effect of anti-miR-125b-5p on the activity of pGLO-CAR10 WT and pGLO-CAR10 Mut was examined by luciferase assay, # p < 0.05, compared with Mock. (g, h) The interaction of miR-125b-5p and CAR10 was detected by Ago2-RIP-qPCR assay, # p < 0.05, compared with IgG.

Article Snippet: Luciferase reporter plasmids include wild-type and mutant pGLO-CAR10, as well as wild-type and mutant pGLO-PDPK1 3′UTR, both constructed by GenScript Company (Nanjing, China).

Techniques: Over Expression, Expressing, Quantitative RT-PCR, Negative Control, Transfection, Activity Assay, Luciferase

Journal: BioMed Research International

Article Title: LncRNA CAR10 Upregulates PDPK1 to Promote Cervical Cancer Development by Sponging miR-125b-5p

doi: 10.1155/2020/4351671

Figure Lengend Snippet: CAR10 acted as a ceRNA and upregulated the expression of PDPK1 by sponging miR-125b-5p. (a) The binding site of miR-125b-5p to the 3′UTR of PDPK1 was predicted by bioinformatics, and the binding site was mutated to construct luciferase reporter gene plasmids. (b, c) The effect of anti-miR-125b-5p on the activity of PDPK1 3′UTR WT and PDPK1 3′ UTR Mut was examined by luciferase assay in C33A and HeLa cells, # p < 0.05, compared with Mock. (d, e, f) The effects of anti-miR-125b-5p on the expression of PDPK1 mRNA and protein were detected by RT-qPCR and western blot, # p < 0.05, compared with Mock. (g) The mRNA expression level of PDPK1 was detected by RT-qPCR in the collected cervical cancer tissues and adjacent tissues, # p < 0.05, compared with adjacent controls. The miR-125b-5p mimic (miR-125b-5p) and the negative control Mock were synthesized and transfected into C33A and HeLa cells, and (h) the expression level of miR-125b-5p was detected by RT-qPCR, # p < 0.05, compared with Mock. (i, j) The effect of miR-125b-5p and/or CAR10 overexpression on the activity of PDPK1 3′UTR WT and PDPK1 3′ UTR Mut was examined by luciferase assay, # p < 0.05, compared with CON. Ф p < 0.05, compared with CAR10 + miR-125b-5p. (k) The effect of miR-125b-5p and/or CAR10 overexpression on PDPK1 expression was observed by western blot and RT-qPCR, # p < 0.05, compared with CON. Ф p < 0.05, compared with CAR10 + miR-125b-5p.

Article Snippet: Luciferase reporter plasmids include wild-type and mutant pGLO-CAR10, as well as wild-type and mutant pGLO-PDPK1 3′UTR, both constructed by GenScript Company (Nanjing, China).

Techniques: Expressing, Binding Assay, Construct, Luciferase, Activity Assay, Quantitative RT-PCR, Western Blot, Negative Control, Synthesized, Transfection, Over Expression