mouse antibodies against pd 1  (Proteintech)

Journal: Integrative Cancer Therapies

Article Title: Sennoside A Modulates the Ferroptosis and Immune Evasion of Oral Squamous Cell Carcinoma Cells Through Inhibiting the NF-κB Pathway

doi: 10.1177/15347354251375464

Figure Lengend Snippet: SA reduced immune escape of OSCC. (A) The expression of PD-L1 was examined by western blot. Data were expressed after being normalized with GAPDH. (B) CD8 + T cell viability was detected by CCK-8 assays. (C) Measurement of IFN-γ, IL-2 and TNF-α by ELISA. * P < .05 and ** P < .01 versus 0 μM SA.

Article Snippet: Then, sections were stained with a rabbit polyclonal antibody against GPX4 (1:500 dilution, Abcam, Cat# ab231174, RRID: AB_3073732), a mouse monoclonal antibody against PD-L1 (1:2500 dilution, Proteintech, Wuhan, China, Cat# 66248-1-Ig, RRID: AB_2756526), and a rabbit polyclonal antibody against IFN-γ (1:100 dilution, Invitrogen, Carlsbad, CA, USA, Cat# PA5-95560, RRID: AB_2807362) at 4°C overnight.

Techniques: Expressing, Western Blot, CCK-8 Assay, Enzyme-linked Immunosorbent Assay

Journal: Integrative Cancer Therapies

Article Title: Sennoside A Modulates the Ferroptosis and Immune Evasion of Oral Squamous Cell Carcinoma Cells Through Inhibiting the NF-κB Pathway

doi: 10.1177/15347354251375464

Figure Lengend Snippet: SA repressed cell viability and immune escape, but induced ferroptosis by inactivating NF-κB pathway in OSCC. (A) SCC7 cell viability was determined by CCK-8 assays. (B) The relative protein expression of GPX4, xCT and PD-L1 were detected by western blot. Data were expressed after being normalized with GAPDH. (C) The relative lipid ROS level was examined after SCC7 cells were tread with DCFH-DA. Scale bar = 100 µm. (D) Examination of the relative Fe 2+ level. (E) CD8 + T cell viability was detected by CCK-8 assays. (F) Measurement of IFN-γ by ELISA. ** P < .01 and *** P < .001 versus 0 μM SA; #P < 0.05 and ##P < 0.01 versus 100 μM SA.

Article Snippet: Then, sections were stained with a rabbit polyclonal antibody against GPX4 (1:500 dilution, Abcam, Cat# ab231174, RRID: AB_3073732), a mouse monoclonal antibody against PD-L1 (1:2500 dilution, Proteintech, Wuhan, China, Cat# 66248-1-Ig, RRID: AB_2756526), and a rabbit polyclonal antibody against IFN-γ (1:100 dilution, Invitrogen, Carlsbad, CA, USA, Cat# PA5-95560, RRID: AB_2807362) at 4°C overnight.

Techniques: CCK-8 Assay, Expressing, Western Blot, Enzyme-linked Immunosorbent Assay

Journal: Integrative Cancer Therapies

Article Title: Sennoside A Modulates the Ferroptosis and Immune Evasion of Oral Squamous Cell Carcinoma Cells Through Inhibiting the NF-κB Pathway

doi: 10.1177/15347354251375464

Figure Lengend Snippet: SA regulated OSCC growth, ferroptosis and immune escape involving in the NF-κB pathway in vivo. Mice were subcutaneously inoculated with a total of 2 × 10 5 of SCC7 cells into the right flank of nude mice, and then intraperitoneally received with 10 mg/kg SA and the same dose of PBS every day for 10 days. (A) Representative images of neoplasms from nude mice (Left). Monitor of tumor volume every 1 week for successive 4 weeks. Tumor volume was quantified by the following formula: volume = 0.5 × length × width 2 (Right). (B) Supervision of mice body weight every 1 week for successive 4 weeks. (C) Measurement of tumor weight after 4 weeks. (D and E) Pathological examination by HE staining, and the expression levels of GPX4, PD-L1, and IFN-γ in tumor tissues were detected by immunohistochemistry. (F) The relative protein expression of p-NF-κB/NF-κB and p-IkBα/IkBα was examined by western blotting. Data were expressed after being normalized with GAPDH. ** P < .01 versus sham.

Article Snippet: Then, sections were stained with a rabbit polyclonal antibody against GPX4 (1:500 dilution, Abcam, Cat# ab231174, RRID: AB_3073732), a mouse monoclonal antibody against PD-L1 (1:2500 dilution, Proteintech, Wuhan, China, Cat# 66248-1-Ig, RRID: AB_2756526), and a rabbit polyclonal antibody against IFN-γ (1:100 dilution, Invitrogen, Carlsbad, CA, USA, Cat# PA5-95560, RRID: AB_2807362) at 4°C overnight.

Techniques: In Vivo, Staining, Expressing, Immunohistochemistry, Western Blot

Journal: Nature Communications

Article Title: Targeting pathogenic CD8 + tissue-resident T cells with chimeric antigen receptor therapy in murine autoimmune cholangitis

doi: 10.1038/s41467-024-46654-5

Figure Lengend Snippet: a Heatmap of mRNA expression level of TCR signaling, cytokine production and cytotoxicity-associated genes of CD8 + Tcm, Tem and Trm cells from liver of DKO mice. b Representative FACS histogram of Granzyme A (GzmA), Granzyme B (GzmB), Perforin, FasL and CD107a expression on CD8 + Tcm, Tem and Trm cell subsets. The ranged gate and the percentages of the positive population are shown in the figure. c Heatmap of mRNA expression level of co-inhibitory moleculars associated genes of CD8 + Tcm, Tem and Trm cells from liver of DKO mice. d Representative FACS histogram of selected immune-checkpoint molecule expression on CD8 + Tcm, Tem and Trm cell subsets. The ranged gate and the percentages of the positive population are shown in the figure. e UMAP plots of DKO liver CD8 + Trm cells, colored by cell clusters. f Violin plots showing mRNA level of Cd244a and Cd200r1 of different Trm subset. g Differentiation trajectory of Trm subset by monocle analysis. Blue arrow indicating the probable direction of differentiation. h Representative flow plots of CD200R and 2B4 expression on CD8 + Trm cells. i Heatmap of mRNA expression level of exhaustion, stemness, cytotoxicity and costimulatory molecule associated genes of CD8 + Trm subsets. j Representative FACS plot of Trm clusters and representative FACS histogram graphs of TCF-1, CD127, PD-1, Annexin V, IFN-γ and Perforin expression on Trm subsets. The ranged gate, the percentages of the positive population and gMFI of Perforin are shown in the figure. k Representative flow plots of Annexin V and DAPI in PIBECs co-cultured with corresponding CD8 + T cell subsets. l Percentage of hepatic DAPI + or Annexin V + cells in PIBECs cocultured with corresponding CD8 + T cell subset from DKO mice liver. n = 3 cells examined over three independent experiments. Data in ( b , d , h , j ) are representative results of at least three independent experiments. The p values were determined by a one-way ANOVA with Tukey’s multiple comparisons test ( l ). Data are shown as Means ± SEM.

Article Snippet: Antibodies against mouse PD-1 (1:100, EPR20665), and CK19 (1:1500, EP1580Y) were purchased from Abcam.

Techniques: Expressing, Cell Culture

Journal: Nature Communications

Article Title: Targeting pathogenic CD8 + tissue-resident T cells with chimeric antigen receptor therapy in murine autoimmune cholangitis

doi: 10.1038/s41467-024-46654-5

Figure Lengend Snippet: a Volcano plot shows the differentially expressed genes between CD8 + Trm and non-Trm cells in DKO liver. b Dot plot shows PD-1 expression of DKO liver CD8 + T cell subsets. The color of dots represents the average expression of genes and size for the percent of cells expressed the genes. c Representative flow plots of CXCR6 and PD-1 expression on DKO liver CD8 + Tcm, Tem and Trm cell subsets. d Representative multi-colored immunohistochemistry graph of CXCR6 GFP DKO liver stained with DAPI (blue), GFP (green), CD8 (red) and PD-1 (yellow). e Representative multi-colored immunohistochemistry graph of DKO liver stained with DAPI (blue), CK19 (pink), CD8 (green) and PD-1 (red). The white arrows pointed to PD-1 + CD8 + T cells. f Statistics of the distance between PD-1 +/− CD8 + T cells and the nearest CK19 + bile duct epithelial cells in DKO liver ( n = 3). Data in ( d , e ) are representative results of at least two independent experiments. Box-and-whisker plots show the median (center line), 25th, and 75th percentile (lower and upper boundary), whiskers extend to the farthest data points. The p values were determined by Wilcoxon test, Bonferroni p value correction ( a ), a two-tailed unpaired t -test ( f ).

Article Snippet: Antibodies against mouse PD-1 (1:100, EPR20665), and CK19 (1:1500, EP1580Y) were purchased from Abcam.

Techniques: Expressing, Immunohistochemistry, Staining, Whisker Assay, Two Tailed Test

Journal: Nature Communications

Article Title: Targeting pathogenic CD8 + tissue-resident T cells with chimeric antigen receptor therapy in murine autoimmune cholangitis

doi: 10.1038/s41467-024-46654-5

Figure Lengend Snippet: a Schematic representation of the retroviral vector expressing the anti-PD-1 CAR. (TM transmembrane). The diagram was created with BioRender.com. b The experimental procedure of anti-PD-1 CAR-T preparation. c Level of CD69 expression 5 days after transduction. n = 3 cells examined over two independent experiments. d Percentage of 7-AAD + cells 5 days after transduction. n = 3 cells examined over two independent experiments. e Level of CD69 expression of anti-PD-1 CAR-T and Ctrl-T cells under the stimulation of PD-1 FC and the blockade of anti-PD-L1 antibody. n = 3 cells examined over three independent experiments. Level of CD69 expression ( f ) and proliferation ( g ) of anti-PD-1 CART and Ctrl-T cells 1 day after coculture with αCD3/28 stimulated PD-1 + T cells with/without the PD-L1 blockade. The ranged gate and the percentages of the positive population are shown in the figure. n = 3 cells examined over three independent experiments. Cytotoxicity assay of anti-PD-1 CAR-T and Ctrl-T cells against ( h , i ) αCD3/28 stimulated PD-1+ T cells or ( j ) sorted Trm/non-Trm cells from liver of DKO mice at indicated effector/target ratio, with/without the PD-L1 blockade. n = 3 cells examined over three independent experiments. Data in ( g ) are representative results of three independent experiments. The p values were determined by a one-way ANOVA with Tukey’s multiple comparisons test ( c – f , i , j ), two-tailed unpaired t -test ( h ). Data are shown as Means ± SEM.

Article Snippet: Antibodies against mouse PD-1 (1:100, EPR20665), and CK19 (1:1500, EP1580Y) were purchased from Abcam.

Techniques: Plasmid Preparation, Expressing, Transduction, Cytotoxicity Assay, Two Tailed Test

Journal: Nature Communications

Article Title: Targeting pathogenic CD8 + tissue-resident T cells with chimeric antigen receptor therapy in murine autoimmune cholangitis

doi: 10.1038/s41467-024-46654-5

Figure Lengend Snippet: a Number of hepatic mononuclear cells, ( b ) percentage of hepatic PD-1 + CD8 + T cells and ( c ) Trm cells in DKO mice after anti-PD-1 CAR-T ( n = 5) or Ctrl-T infusion ( n = 5). Absolute number of liver CD8 + Trm ( d ), Tcm ( e ) or Tem ( f ) after anti-PD-1 CAR-T ( n = 5) or Ctrl-T infusion ( n = 5). g Representative H&E staining pictures of liver from DKO mice treated with anti-PD-1 CAR-T or Ctrl-T infusion, magnification showing the portal area. h Pathological score of portal inflammation of DKO liver after anti-PD-1 CAR-T ( n = 5) or Ctrl-T infusion ( n = 5). i PCA plots of liver transcriptome data of DKO mice after anti-PD-1 CAR-T ( n = 5) or Ctrl-T infusion ( n = 5) or ctrl mice ( n = 7). j GO analysis of the upregulated genes in DKO mice liver after anti-PD-1 CAR-T infusion comparing to Ctrl-T infusion. k Dot plot shows gene sets NES (normalized enrichment score) and p value of DKO mice liver after anti-PD-1 CAR-T ( n = 5) or Ctrl-T infusion ( n = 5), comparing to ctrl mice liver respectively. The color of dots represents the NES score and size for −log10( p value). The p values were determined by a two-tailed unpaired t -test ( a – f , h ). The p values of GO analysis was generated in R using a Fisher’s exact test ( j ). The p values of GSEA was generated in GSEA software using an empirical phenotype-based permutation test ( k ). All experiments were repeated for 2–3 times. Data are shown as Means ± SEM.

Article Snippet: Antibodies against mouse PD-1 (1:100, EPR20665), and CK19 (1:1500, EP1580Y) were purchased from Abcam.

Techniques: Staining, Two Tailed Test, Generated, Software