Journal: Nature Communications

Article Title: Hypoxia induces HIF1α-dependent epigenetic vulnerability in triple negative breast cancer to confer immune effector dysfunction and resistance to anti-PD-1 immunotherapy

doi: 10.1038/s41467-022-31764-9

Figure Lengend Snippet: a Heatmap showing Pearson’s correlation between hypoxic signature genes expression and immune-related genes expression in basal TNBC samples ( n = 98) in TCGA dataset. b Scatter plots (upper panel) and Pearson’s correlation coefficients (lower panel) showing the expression of hypoxic gene signatures and immune-related genes in breast cancers in TCGA dataset (Basal, n = 98; HER2, n = 58; Luminal A, n = 231; Luminal B, n = 129). Regression lines with a 95% confidence interval (gray fill) are shown in the scatter plots. c Images of fluorescent staining of human TNBC samples. Scale bar, 50 µm. Data were representative of 30 independent experiments. d Quantification of infiltrating IFNγ + CD8 + T cell number in HIF1α − and HIF1α + regions of human TNBC sample ( n = 30). P values were determined with paired two-tailed t -test. e Correlation between infiltrating IFNγ + CD8 + T cell count and HIF1α fluorescent intensity in human TNBC samples ( n = 30). The simple linear regression R 2 and P values (two-tailed) are calculated. Dot plot is shown with regression line and 95% confidence interval. f Representative images of fluorescent staining of mouse 4T1 tumor samples. Scale bar, 50 µm. Data represents three independent experiments. g Flow cytometry (left panel) demonstrating the gating strategy of activated-PIM high (H) and activated-PIM low (L) populations in living cells dissociated from 4T1 tumors. The CD8 + T cell percentage and IFNγ expression in CD8 + T cells was quantified (right panel, n = 6). Data were presented as box and whiskers, with median value and whiskers of minimum and maximum values. P values were determined with an unpaired two-tailed t -test. h Kaplan–Meier overall survival (OS) and distant metastasis-free survival (DMFS) analysis of the indicated gene signatures in TNBC patients. The publicly available data used in Fig. 1a, b are available in the TCGA database under accession code BRCA.exp.547.med.txt [ https://gdc.cancer.gov/about-data/publications/brca_2012 ]. The publicly available data used in h are available in the KM-Plotter-Breast Cancer [ https://kmplot.com/analysis/index.php?p=service&cancer=breast ]. For the remaining data, source data are provided in Source Data file.

Article Snippet: The following antibodies were used for staining, anti-activated pimonidazole FITC antibody (Hypoxyprobe, CAT# HP2-200kit, dilution 1:200), anti-mouse HIF1α APC antibody (R&D Systems, CAT# IC1935A, dilution 1:50), anti-mouse CD3 BV421 antibody (BD Biosciences, CAT# 564008, dilution 1:100), anti-mouse CD45 Percp-Vio700 antibody (Miltenyi Biotec, CAT# 130-110-663, dilution 1:100) anti-mouse CD8 APC-Vio770 antibody (Miltenyi Biotec, CAT# 130-120-737, dilution 1:100), anti-mouse Nkp46 APC antibody (Miltenyi Biotec, CAT# 130-112-202, dilution 1:100), anti-mouse CD4 BV650 antibody (Biolegend, CAT# 563747, dilution 1:100), anti-mouse TIM-3 BV711 antibody (Biolegend, CAT# 119727, dilution 1:100), anti-mouse PD-1 PE-Vio770 (Miltenyi Biotec, CAT# 130-120-391, dilution 1:100), anti-mouse IFNγ PE (Miltenyi Biotec, CAT# 130-117-352, dilution 1:100), anti-mouse TNFα BV711 (BD Biosciences, CAT# 563944, dilution 1:100), anti-mouse/human granzyme B FITC (Miltenyi Biotec, Cat#130-118-430, dilution 1:100), anti-mouse PD-L1 BV786 antibody (BD Biosciences, CAT# 741014, dilution 1:100), anti-mouse PD-L2 FITC antibody (Miltenyi Biotec, Cat# 130-102-222, dilution 1:100), anti-human CD45 FITC antibody (BD Biosciences, CAT# 304006, dilution 1:100), anti-human CD3 PE antibody (Biolegend, CAT# 300308, dilution 1:100) anti-human CD8 APC-Cy7 antibody (BD Biosciences, CAT# 557834, dilution 1:100), anti-human CD56 BV711 antibody (Biolegend, CAT# 318336, dilution 1:100), anti-human CD4 APC antibody (Biolegend, CAT# 300514, dilution 1:100), anti-human IFNγ BV785 (Biolegend, CAT# 502542, dilution 1:100), anti-human TNFα BV650 (Biolegend, CAT# 502398, dilution 1:100), anti-human Granzyme B BV421 (BD Biosciences, Cat# 563389, dilution 1:100), anti-human PD-L1 PE-Cy7 antibody (Biolegend, CAT# 374506, dilution 1:100), anti-human PD-L2 PE antibody (Miltenyi Biotec, CAT# 130-098-530, dilution 1:100).

Techniques: Expressing, Staining, Two Tailed Test, Cell Counting, Flow Cytometry

Journal: Nature Communications

Article Title: Hypoxia induces HIF1α-dependent epigenetic vulnerability in triple negative breast cancer to confer immune effector dysfunction and resistance to anti-PD-1 immunotherapy

doi: 10.1038/s41467-022-31764-9

Figure Lengend Snippet: a Schematic graph demonstrating the coculture model. b Representative flow cytograms (upper panel) gated from human pan-T cell culture and quantification (lower panel, n = 3) of differentiated CD8 + T cell subtypes: Tn (naïve T cells), Tcm (central memory T cells), Tem (effector memory T cells), Teff (effector T cells). c Schematic graph demonstrating the normoxia (20% O 2 ) and hypoxia (1% O 2 ) culture condition of T cells coculturing with human TNBC cell line. d Heatmap of the differentially expressed genes (DEGs) in hypoxic cultured human T cells compared to normoxia group. DEGs were identified in edgeR (|logFC| > 1, adjusted P < 0.01). P values were adjusted using Benjamini–Hochberg method in edgeR. DEGs identified in the indicated GO gene clusters are marked in the heatmap. e GSEA analysis of human T cells in hypoxic versus normoxic conditions. Analysis was based on ranked logFC from edgeR. FDR and adjusted p value are shown in the graph. P values were adjusted using Benjamini–Hochberg method in GSEA analysis. f Flow cytometry quantifications of immune effector molecules and exhaustion markers in CD8 + T cells gated from human pan-T cells cultured under the indicated conditions ( n = 4). g Representative flow cytograms of PD-1 and TIM-3 expression in CD8 + T cells gated from human pan-T cells culture. h Flow cytometric quantification of terminally exhausted T cells (PD-1 + TIM-3 + ) in CD8 + T cells gated from human pan-T cells culture ( n = 3). i Flow cytometric quant i fication of proliferating cells (Ki76 + ) in CD8 + and CD4 + T cells gated from human T cells cocultured with TNBC ( n = 3). All flow cytometry data ( b , f , h , and i ) are presented as the mean ± SD of samples from three to four donors. For all flow cytometry data, P values were determined by one-way ANOVA ( f , h ) or two-way ANOVA ( b ) with Turkey’s test, or paired two-tailed t -test ( i ). Raw RNA-seq data i s available in the GEO database with accession number GSE179885 . For the remaining data, source data are provided in Source Data file.

Article Snippet: The following antibodies were used for staining, anti-activated pimonidazole FITC antibody (Hypoxyprobe, CAT# HP2-200kit, dilution 1:200), anti-mouse HIF1α APC antibody (R&D Systems, CAT# IC1935A, dilution 1:50), anti-mouse CD3 BV421 antibody (BD Biosciences, CAT# 564008, dilution 1:100), anti-mouse CD45 Percp-Vio700 antibody (Miltenyi Biotec, CAT# 130-110-663, dilution 1:100) anti-mouse CD8 APC-Vio770 antibody (Miltenyi Biotec, CAT# 130-120-737, dilution 1:100), anti-mouse Nkp46 APC antibody (Miltenyi Biotec, CAT# 130-112-202, dilution 1:100), anti-mouse CD4 BV650 antibody (Biolegend, CAT# 563747, dilution 1:100), anti-mouse TIM-3 BV711 antibody (Biolegend, CAT# 119727, dilution 1:100), anti-mouse PD-1 PE-Vio770 (Miltenyi Biotec, CAT# 130-120-391, dilution 1:100), anti-mouse IFNγ PE (Miltenyi Biotec, CAT# 130-117-352, dilution 1:100), anti-mouse TNFα BV711 (BD Biosciences, CAT# 563944, dilution 1:100), anti-mouse/human granzyme B FITC (Miltenyi Biotec, Cat#130-118-430, dilution 1:100), anti-mouse PD-L1 BV786 antibody (BD Biosciences, CAT# 741014, dilution 1:100), anti-mouse PD-L2 FITC antibody (Miltenyi Biotec, Cat# 130-102-222, dilution 1:100), anti-human CD45 FITC antibody (BD Biosciences, CAT# 304006, dilution 1:100), anti-human CD3 PE antibody (Biolegend, CAT# 300308, dilution 1:100) anti-human CD8 APC-Cy7 antibody (BD Biosciences, CAT# 557834, dilution 1:100), anti-human CD56 BV711 antibody (Biolegend, CAT# 318336, dilution 1:100), anti-human CD4 APC antibody (Biolegend, CAT# 300514, dilution 1:100), anti-human IFNγ BV785 (Biolegend, CAT# 502542, dilution 1:100), anti-human TNFα BV650 (Biolegend, CAT# 502398, dilution 1:100), anti-human Granzyme B BV421 (BD Biosciences, Cat# 563389, dilution 1:100), anti-human PD-L1 PE-Cy7 antibody (Biolegend, CAT# 374506, dilution 1:100), anti-human PD-L2 PE antibody (Miltenyi Biotec, CAT# 130-098-530, dilution 1:100).

Techniques: Cell Culture, Flow Cytometry, Expressing, Two Tailed Test, RNA Sequencing

Journal: Nature Communications

Article Title: Hypoxia induces HIF1α-dependent epigenetic vulnerability in triple negative breast cancer to confer immune effector dysfunction and resistance to anti-PD-1 immunotherapy

doi: 10.1038/s41467-022-31764-9

Figure Lengend Snippet: a RT-qPCR analysis assessing IFNG expression in T/NK cells in an epigenetic-drug screening. Both T cells and NK cells were cultured under 1% O 2 with indicated treatments. Data were presented as the log2 fold change of IFNG mRNA level normalized to vehicle control, mean ± SD of technical triplicates, representative of two independent experiments ( n = 2). b , c Representative histograms (left panel) and flow cytometric quantifications (right panel) of IFNγ expression in human CD8 + T cells ( b n = 4) and NK cells ( c n = 3) with indicated treatments. Quantification data were presented as the mean ± SD of samples from three to four donors. P values were determined by two-way ANOVA with Turkey’s test. d ChIP-qPCR analysis of HDAC1, HDAC2, HDAC3, EZH2, and SUZ12 occupancy on IFNG promoter of human T cells. Four primers were designed to span the promoters of IFNG , with P1 at −1448 to −1354b, P2 at −707 to −628b, P3 at −257 to −171b, P4 at +350 to +461b, relative to TSS. For ChIP analysis of EZH2 and SUZ12 occupancy, RPL30 serves as the negative control and CCND2 as the positive control. e , f ChIP-qPCR analysis of H3K27ac and H3K27me3 enrichment on IFNG promoter of human T cells under indicated conditions. All ChIP-qPCR data ( d – f ) are presented as fold enrichment relative to IgG and expressed as mean ± SD of technical triplicates, representative of two independent experiments ( n = 2). For ChIP-qPCR data of d , e , statistics were performed to analyze bindings of indicated markers across different sites in IFNG promoter ( RPL30 and CCND2 excluded) between hypoxia and normoxia. P values were determined by two-way ANOVA analysis. g RT-qPCR analysis of human T cell with indicated gene knockdown. Data were presented as the fold change of mRNA level normalized to the control group under normoxia (1% O2), mean ± SD of technical triplicates, representative of two independent experiments ( n = 2). Source data are provided as a source data file.

Article Snippet: The following antibodies were used for staining, anti-activated pimonidazole FITC antibody (Hypoxyprobe, CAT# HP2-200kit, dilution 1:200), anti-mouse HIF1α APC antibody (R&D Systems, CAT# IC1935A, dilution 1:50), anti-mouse CD3 BV421 antibody (BD Biosciences, CAT# 564008, dilution 1:100), anti-mouse CD45 Percp-Vio700 antibody (Miltenyi Biotec, CAT# 130-110-663, dilution 1:100) anti-mouse CD8 APC-Vio770 antibody (Miltenyi Biotec, CAT# 130-120-737, dilution 1:100), anti-mouse Nkp46 APC antibody (Miltenyi Biotec, CAT# 130-112-202, dilution 1:100), anti-mouse CD4 BV650 antibody (Biolegend, CAT# 563747, dilution 1:100), anti-mouse TIM-3 BV711 antibody (Biolegend, CAT# 119727, dilution 1:100), anti-mouse PD-1 PE-Vio770 (Miltenyi Biotec, CAT# 130-120-391, dilution 1:100), anti-mouse IFNγ PE (Miltenyi Biotec, CAT# 130-117-352, dilution 1:100), anti-mouse TNFα BV711 (BD Biosciences, CAT# 563944, dilution 1:100), anti-mouse/human granzyme B FITC (Miltenyi Biotec, Cat#130-118-430, dilution 1:100), anti-mouse PD-L1 BV786 antibody (BD Biosciences, CAT# 741014, dilution 1:100), anti-mouse PD-L2 FITC antibody (Miltenyi Biotec, Cat# 130-102-222, dilution 1:100), anti-human CD45 FITC antibody (BD Biosciences, CAT# 304006, dilution 1:100), anti-human CD3 PE antibody (Biolegend, CAT# 300308, dilution 1:100) anti-human CD8 APC-Cy7 antibody (BD Biosciences, CAT# 557834, dilution 1:100), anti-human CD56 BV711 antibody (Biolegend, CAT# 318336, dilution 1:100), anti-human CD4 APC antibody (Biolegend, CAT# 300514, dilution 1:100), anti-human IFNγ BV785 (Biolegend, CAT# 502542, dilution 1:100), anti-human TNFα BV650 (Biolegend, CAT# 502398, dilution 1:100), anti-human Granzyme B BV421 (BD Biosciences, Cat# 563389, dilution 1:100), anti-human PD-L1 PE-Cy7 antibody (Biolegend, CAT# 374506, dilution 1:100), anti-human PD-L2 PE antibody (Miltenyi Biotec, CAT# 130-098-530, dilution 1:100).

Techniques: Quantitative RT-PCR, Expressing, Drug discovery, Cell Culture, Control, ChIP-qPCR, Negative Control, Positive Control, Knockdown

Journal: Nature Communications

Article Title: Hypoxia induces HIF1α-dependent epigenetic vulnerability in triple negative breast cancer to confer immune effector dysfunction and resistance to anti-PD-1 immunotherapy

doi: 10.1038/s41467-022-31764-9

Figure Lengend Snippet: a ChIP-qPCR analysis of HIF1α and HIF2α occupancy on IFNG promoter in human T cells. VEGFA served as a positive control. b Co-immunoprecipitation shows the physical interaction between HDAC1 and HIF1α, and the interaction between HDAC1 and SUZ12 in human T cells. Data is representative of two independent experiments ( n = 2). c Representative western blot images ( n = 2) to demonstrate knockdown of HIF1α in human T cells. d ChIP-qPCR analysis of HDAC1 occupancy on IFNG promoter in human T cells. e ChIP-qPCR analysis of H3K27ac and H3K27me3 enrichment on IFNG promoter in human T cells with indicated treatments. All ChIP-qPCR data ( a , d , e ) are presented as fold enrichment relative to IgG and expressed as mean ± SD of technical triplicates, representative of two independent experiments ( n = 2). For ChIP-qPCR data of a , statistics were performed to analyze bindings of indicated markers across different sites in IFNG promoter ( VEGFA excluded) between hypoxia and normoxia. P values were determined by two-way ANOVA analysis. f Flow cytometric quantifications of IFNγ in CD8 + T cells gated from human pan-T cells cultured under the indicated conditions. Data were presented as the mean ± SD of three independent experiments ( n = 3). P values were determined by one-way ANOVA with Turkey’s test. g Representative western blot images ( n = 2) to demonstrate the inhibition of HIF1α level by indicated compounds in human T cells. h Representative histograms (left panel) and flow cytometric quantifications (right panel) of IFNγ expression in human CD8 + T cells with indicated treatments. Quantification data were presented as the mean ± SD of samples from four donors ( n = 4). P values were determined by two-way ANOVA with Turkey’s test. Source data are provided as a source data file.

Article Snippet: The following antibodies were used for staining, anti-activated pimonidazole FITC antibody (Hypoxyprobe, CAT# HP2-200kit, dilution 1:200), anti-mouse HIF1α APC antibody (R&D Systems, CAT# IC1935A, dilution 1:50), anti-mouse CD3 BV421 antibody (BD Biosciences, CAT# 564008, dilution 1:100), anti-mouse CD45 Percp-Vio700 antibody (Miltenyi Biotec, CAT# 130-110-663, dilution 1:100) anti-mouse CD8 APC-Vio770 antibody (Miltenyi Biotec, CAT# 130-120-737, dilution 1:100), anti-mouse Nkp46 APC antibody (Miltenyi Biotec, CAT# 130-112-202, dilution 1:100), anti-mouse CD4 BV650 antibody (Biolegend, CAT# 563747, dilution 1:100), anti-mouse TIM-3 BV711 antibody (Biolegend, CAT# 119727, dilution 1:100), anti-mouse PD-1 PE-Vio770 (Miltenyi Biotec, CAT# 130-120-391, dilution 1:100), anti-mouse IFNγ PE (Miltenyi Biotec, CAT# 130-117-352, dilution 1:100), anti-mouse TNFα BV711 (BD Biosciences, CAT# 563944, dilution 1:100), anti-mouse/human granzyme B FITC (Miltenyi Biotec, Cat#130-118-430, dilution 1:100), anti-mouse PD-L1 BV786 antibody (BD Biosciences, CAT# 741014, dilution 1:100), anti-mouse PD-L2 FITC antibody (Miltenyi Biotec, Cat# 130-102-222, dilution 1:100), anti-human CD45 FITC antibody (BD Biosciences, CAT# 304006, dilution 1:100), anti-human CD3 PE antibody (Biolegend, CAT# 300308, dilution 1:100) anti-human CD8 APC-Cy7 antibody (BD Biosciences, CAT# 557834, dilution 1:100), anti-human CD56 BV711 antibody (Biolegend, CAT# 318336, dilution 1:100), anti-human CD4 APC antibody (Biolegend, CAT# 300514, dilution 1:100), anti-human IFNγ BV785 (Biolegend, CAT# 502542, dilution 1:100), anti-human TNFα BV650 (Biolegend, CAT# 502398, dilution 1:100), anti-human Granzyme B BV421 (BD Biosciences, Cat# 563389, dilution 1:100), anti-human PD-L1 PE-Cy7 antibody (Biolegend, CAT# 374506, dilution 1:100), anti-human PD-L2 PE antibody (Miltenyi Biotec, CAT# 130-098-530, dilution 1:100).

Techniques: ChIP-qPCR, Positive Control, Immunoprecipitation, Western Blot, Knockdown, Cell Culture, Inhibition, Expressing

Journal: Nature Communications

Article Title: Hypoxia induces HIF1α-dependent epigenetic vulnerability in triple negative breast cancer to confer immune effector dysfunction and resistance to anti-PD-1 immunotherapy

doi: 10.1038/s41467-022-31764-9

Figure Lengend Snippet: a Cell lysis of TNBC cells cocultured with human T cells from two different healthy donors. Human T cells were stimulated with TNBC cell lysate-primed DC cells. Data were presented as mean ± SD of three independent experiments ( n = 3). P values were determined by two-way ANOVA. b Western blot analysis of IFNγ–regulated proteins in TNBC cells cocultured with human T cells. Data were representative of two independent experiments ( n = 2). c Cell lysis of TNBC cells cocultured with human T cells. Human T cells were stimulated with TNBC cell lysate-primed DC cells and pretreated with indicated compounds. Data presented as mean ± SD of three independent experiments ( n = 3). P values were determined by one-way ANOVA with Dunnett’s test. d Western blot analysis of IFNγ–regulated proteins in TNBC cells cocultured with human T cells. Human T cells were stimulated with TNBC cell lysate-primed DC cells and pretreated with indicated compounds. Data were representative of two independent experiments ( n = 2). e Cell lysis of TNBC cells cocultured with human T cells. Data were presented as mean ± SD of three independent experiments ( n = 3). P values were determined by two-way ANOVA with Dunnett’s test. f Flow cytometric quantifications of immune effector molecules in human CD8 + T cells cultured under the indicated conditions. Data were presented as the mean ± SD of samples from three donors ( n = 3). P values were determined by two-way ANOVA with Turkey’s test. Source data are provided as a source data file.

Article Snippet: The following antibodies were used for staining, anti-activated pimonidazole FITC antibody (Hypoxyprobe, CAT# HP2-200kit, dilution 1:200), anti-mouse HIF1α APC antibody (R&D Systems, CAT# IC1935A, dilution 1:50), anti-mouse CD3 BV421 antibody (BD Biosciences, CAT# 564008, dilution 1:100), anti-mouse CD45 Percp-Vio700 antibody (Miltenyi Biotec, CAT# 130-110-663, dilution 1:100) anti-mouse CD8 APC-Vio770 antibody (Miltenyi Biotec, CAT# 130-120-737, dilution 1:100), anti-mouse Nkp46 APC antibody (Miltenyi Biotec, CAT# 130-112-202, dilution 1:100), anti-mouse CD4 BV650 antibody (Biolegend, CAT# 563747, dilution 1:100), anti-mouse TIM-3 BV711 antibody (Biolegend, CAT# 119727, dilution 1:100), anti-mouse PD-1 PE-Vio770 (Miltenyi Biotec, CAT# 130-120-391, dilution 1:100), anti-mouse IFNγ PE (Miltenyi Biotec, CAT# 130-117-352, dilution 1:100), anti-mouse TNFα BV711 (BD Biosciences, CAT# 563944, dilution 1:100), anti-mouse/human granzyme B FITC (Miltenyi Biotec, Cat#130-118-430, dilution 1:100), anti-mouse PD-L1 BV786 antibody (BD Biosciences, CAT# 741014, dilution 1:100), anti-mouse PD-L2 FITC antibody (Miltenyi Biotec, Cat# 130-102-222, dilution 1:100), anti-human CD45 FITC antibody (BD Biosciences, CAT# 304006, dilution 1:100), anti-human CD3 PE antibody (Biolegend, CAT# 300308, dilution 1:100) anti-human CD8 APC-Cy7 antibody (BD Biosciences, CAT# 557834, dilution 1:100), anti-human CD56 BV711 antibody (Biolegend, CAT# 318336, dilution 1:100), anti-human CD4 APC antibody (Biolegend, CAT# 300514, dilution 1:100), anti-human IFNγ BV785 (Biolegend, CAT# 502542, dilution 1:100), anti-human TNFα BV650 (Biolegend, CAT# 502398, dilution 1:100), anti-human Granzyme B BV421 (BD Biosciences, Cat# 563389, dilution 1:100), anti-human PD-L1 PE-Cy7 antibody (Biolegend, CAT# 374506, dilution 1:100), anti-human PD-L2 PE antibody (Miltenyi Biotec, CAT# 130-098-530, dilution 1:100).

Techniques: Lysis, Western Blot, Cell Culture

Journal: Nature Communications

Article Title: Hypoxia induces HIF1α-dependent epigenetic vulnerability in triple negative breast cancer to confer immune effector dysfunction and resistance to anti-PD-1 immunotherapy

doi: 10.1038/s41467-022-31764-9

Figure Lengend Snippet: a Schematic diagram showing the establishment of humanized mice (humice) with human immune system reconstituted in NIKO mice. The presence of human CD45 + cells, NK cells, CD4 + and CD8 + T cells in the mice’s peripheral system was validated by flow cytometry. b Primary LM2 tumor size in humice (control, n = 14; Keytruda, n = 14; ENT, n = 12; PX478, n = 14; ENT + Keytruda, n = 16; PX478 + Keytruda, n = 16) and NIKO mice (control, n = 10; ENT + Keytruda, n = 10; PX478 + Keytruda, n = 10), at Day 21 of treatments. c Lung metastasis of humice (control, n = 6; Keytruda, n = 6; ENT, n = 6; PX478, n = 6; ENT + Keytruda, n = 7; PX478 + Keytruda, n = 7) and NIKO mice (control, n = 5; ENT + Keytruda, n = 5; PX478 + Keytruda, n = 5) bearing LM2 tumors at Day 35 assessed by bioluminescence (BLI) measurement. d Representative bioluminescence (BLI) images showing the lung metastasis of humice and NIKO mice. e Flow cytometric analysis of LM2 tumors harvested from humanized mice. IFNγ, TNFα, and granzyme B expression was examined in tumor-infiltrating human CD8 + T cells and NK cells. N = 5 for each group. f Flow cytometry analysis of LM2 tumors harvested from humanized mice. Expressions of human PD-L1 and PD-L2 were examined in total living cells dissociated from LM2 tumors. N = 5 for each group. Quantification data of flow cytometry ( e , f ) are presented as a box and whiskers, with median values and whiskers of minimum and maximum values. Data for b and c were presented as mean ± SD . P values were determined by one-way ( e , f ) or two-way ( b , c ) ANOVA with Turkey’s test. Source data are provided as a source data file.

Article Snippet: The following antibodies were used for staining, anti-activated pimonidazole FITC antibody (Hypoxyprobe, CAT# HP2-200kit, dilution 1:200), anti-mouse HIF1α APC antibody (R&D Systems, CAT# IC1935A, dilution 1:50), anti-mouse CD3 BV421 antibody (BD Biosciences, CAT# 564008, dilution 1:100), anti-mouse CD45 Percp-Vio700 antibody (Miltenyi Biotec, CAT# 130-110-663, dilution 1:100) anti-mouse CD8 APC-Vio770 antibody (Miltenyi Biotec, CAT# 130-120-737, dilution 1:100), anti-mouse Nkp46 APC antibody (Miltenyi Biotec, CAT# 130-112-202, dilution 1:100), anti-mouse CD4 BV650 antibody (Biolegend, CAT# 563747, dilution 1:100), anti-mouse TIM-3 BV711 antibody (Biolegend, CAT# 119727, dilution 1:100), anti-mouse PD-1 PE-Vio770 (Miltenyi Biotec, CAT# 130-120-391, dilution 1:100), anti-mouse IFNγ PE (Miltenyi Biotec, CAT# 130-117-352, dilution 1:100), anti-mouse TNFα BV711 (BD Biosciences, CAT# 563944, dilution 1:100), anti-mouse/human granzyme B FITC (Miltenyi Biotec, Cat#130-118-430, dilution 1:100), anti-mouse PD-L1 BV786 antibody (BD Biosciences, CAT# 741014, dilution 1:100), anti-mouse PD-L2 FITC antibody (Miltenyi Biotec, Cat# 130-102-222, dilution 1:100), anti-human CD45 FITC antibody (BD Biosciences, CAT# 304006, dilution 1:100), anti-human CD3 PE antibody (Biolegend, CAT# 300308, dilution 1:100) anti-human CD8 APC-Cy7 antibody (BD Biosciences, CAT# 557834, dilution 1:100), anti-human CD56 BV711 antibody (Biolegend, CAT# 318336, dilution 1:100), anti-human CD4 APC antibody (Biolegend, CAT# 300514, dilution 1:100), anti-human IFNγ BV785 (Biolegend, CAT# 502542, dilution 1:100), anti-human TNFα BV650 (Biolegend, CAT# 502398, dilution 1:100), anti-human Granzyme B BV421 (BD Biosciences, Cat# 563389, dilution 1:100), anti-human PD-L1 PE-Cy7 antibody (Biolegend, CAT# 374506, dilution 1:100), anti-human PD-L2 PE antibody (Miltenyi Biotec, CAT# 130-098-530, dilution 1:100).

Techniques: Flow Cytometry, Control, Expressing

Journal: Journal of neuroendocrinology

Article Title: GHR−/− Mice are Protected from Obesity-Related White Adipose Tissue Inflammation

doi: 10.1111/jne.12854

Figure Lengend Snippet: Flow Cytometry Antibodies (24 Months)

Article Snippet: Results were analyzed with FlowJo V10 software. table ft1 table-wrap mode="anchored" t5 Table 1. caption a7 Target Conjugate Manufacturer Catalog # RRID CD206 AF488 BioRad MCA2235A488 AB_324891 Ly-6C PerCP-Cy5.5 Thermo Fisher 45–5932-80 AB_1518762 MHC-II Biotin Thermo Fisher 13–5321-81 AB_466661 F4/80 PE-Cy7 Thermo Fisher 25–4801-82 AB_469653 CD11c APC Thermo Fisher 117–0114-81 AB_469345 CD45 APC-eFluor 780 Thermo Fisher 47–0451-82 AB_1548781 CD11b Alexa Fluor 700 Thermo Fisher 56–0112-80 AB_657586 CCR2 Phycoerythrin R&D Systems FAB5538P AB_10718414 NK1.1 FITC Thermo Fisher 11–5941-81 AB_465317 NKT Phycoerythrin BD Biosciences 550082 AB_393552 CD44 Biotin Thermo Fisher 13–0441-81 AB_466441 CD4 PerCP-Cy5.5 Thermo Fisher 45–0042-80 AB_906231 CD3 PE-Cy7 Thermo Fisher 25–0031-81 AB_469571 CD25 APC Thermo Fisher 17–0251-81 AB_469365 CD62L Alexa Fluor 700 Thermo Fisher 56–0621-80 AB_494004 CD45 Alexa Fluor 700 Thermo Fisher 56–0451-80 AB_891456 CD16/CD32 Thermo Fisher 14–0161-82 AB_467133 Streptavidin eFluor 615 Thermo Fisher 42–4317-80 AB_11218079 Open in a separate window Flow Cytometry Antibodies (8 Months) table ft1 table-wrap mode="anchored" t5 Table 2. caption a7 Target Conjugate Manufacturer Catalog # RRID CD11b VioGreen Miltenyi Biotec 130–113-811 AB_2726328 CD11c APC-Vio770 Miltenyi Biotec 130–110-841 AB_2654715 CD206 APC BioLegend 141707 AB_10896057 CD25 PE Miltenyi Biotec 130–108-996 AB_2656655 CD3 APC-Vio770 Miltenyi Biotec 130–109-840 AB_2657087 CD38 PE-Vio770 Miltenyi Biotec 130–109-258 AB_2657842 CD4 VioGreen Miltenyi Biotec 130–109-413 AB_2657964 CD44 PE-Vio770 Miltenyi Biotec 130–110-085 AB_2658157 CD45 VioBlue Miltenyi Biotec 130–110-802 AB_2658222 CD62L PerCP-Vio700 Miltenyi Biotec 130–107-046 AB_2660523 CD80 PE Miltenyi Biotec 130–116-460 AB_2727557 CD8a FITC Miltenyi Biotec 130–102-490 AB_2659883 F4/80 PerCP-Vio700 Miltenyi Biotec 130–102-161 AB_2651711 GITR APC Miltenyi Biotec 130–116-427 AB_2727531 Open in a separate window Flow Cytometry Antibodies (24 Months) 2.5.

Techniques: Flow Cytometry