iscript cdna synthesis supermix reverse transcriptase  (Bio-Rad)

Journal: Cancer Immunology, Immunotherapy : CII

Article Title: Loss of type I IFN responsiveness impairs natural killer cell antitumor activity in breast cancer

doi: 10.1007/s00262-021-02857-z

Figure Lengend Snippet: Loss of NK cell IFNAR1 impairs NK cell function, may impact adaptive immune regulation of tumorigenesis and increases lung metastasis. 1 × 105 EO771.LMB cells were injected into the fourth mammary fat pad (IMFP) of controliCre and Ifnar−/−NKp46 C57BL/6 mice at day 0 and subsequent alterations to circulating and primary tumor cells assessed. Peripheral blood (PB) was taken at day 7 for FACS analysis and for intracellular staining (ICS) of monocytes was performed at endpoint. a Primary tumor weights (mg) of control (n = 7) and Ifnar−/−NKp46 mice (n = 7) mice at experimental endpoint. Flow cytometric analysis of b absolute number (cell #) of primary tumor NK cells (n = 6/group). c PB CD69+ NK (NK1.1+) cell frequency (%). Primary tumor d CD69+ (e. representative plots shown) and f IFNγ+ NK cell frequency (%) and g IFNAR1 expression, represented as MFI (n = 5/group). Flow cytometric analysis of h CD8+ T cell IFNAR1 (MFI; n = 5/group) and i. PB CD69+ CD8+ T cells, j primary tumor CD69+ CD8+ T cells, k CD8+ IFNγ+ T cells, l. PB CD69+ CD4+ T cells and m primary tumor CD4+ IFNγ+ T cells, expressed as frequency (%). n RT-qPCR of mCherry (EO771.LMB) DNA expression in the lungs post-intravenous (IV) injection of EO771.LMB cells into WT (n = 9), controliCre (n = 5) and Ifnar−/−NKp46 (n = 8) mice at day 24. Representative fluorescence imaging of mCherry (EO771.LMB) in lungs shown for each group. All PB analysis (n = 7/group) and primary tumor analysis (n = 6/group). p values * < 0.05, ** < 0.005, *** < 0.0005. **** < 0.0001 determined by Student’s t test. Errors bars, SEM

Article Snippet: When required, cells were transfected with poly I:C (10 μg/ml) overnight, prior to RNA extraction. cDNA was generated using the iScript Reverse Transcriptase Supermix cDNA for RT-qPCR kit (BioRad).

Techniques: Cell Function Assay, Injection, Staining, Expressing, Quantitative RT-PCR, IV Injection, Fluorescence, Imaging

Journal: EMBO Reports

Article Title: Prostate cancer cell‐intrinsic interferon signaling regulates dormancy and metastatic outgrowth in bone

doi: 10.15252/embr.202050162

Figure Lengend Snippet: A–C Bulk RNA‐seq analysis of DE genes (DEG) significantly suppressed (FDR < 0.05; generalized linear model extraction (GLM) by edgeR) in proliferating RM1 bone‐derived (BD) cells compared to parental RM1 and lung metastases. Tumor‐intrinsic type I IFN signaling is suppressed in proliferating bone lesions ( n = 1) compared to parental RM1 ( n = 2) cells as shown by (A) camera analysis of Hallmark gene set responses associated with bone metastasis (bar length is the –log 10 FDR, false discovery rate). Color indicates mean log 2 fold change (FC) of all genes in gene set. Bar width is relative gene set size. Dashed line shows FDR = 0.05; (B) barcode plot showing enrichment of the Hallmark IFN‐α response gene set in genes suppressed in bone metastases, using the signed log‐likelihood ratio statistic (EdgeR). Bars indicate value of the statistic for each gene in the gene set; (C) log 2 FC values of key downregulated IRGs, with genes retained in dormant cells and classical downstream IFN signaling targets indicated. Altered IRGs directly involved in IFN‐α/β production (light blue) compared to downstream IFN targets (dark blue) are segregated. P ‐values represented as * < 0.05, ** < .005, and *** < 0.0005; GLM by edgeR. D Schematic of proliferating RM1 cell isolation from bone and lung metastases following IC injection for preliminary RNA‐seq and orthogonal validation by qRT–PCR. E Heatmap of mean normalized voom expression of IRGs suppressed in RM1 bone metastases ( n = 1) compared to lung metastases ( n = 1) and parental cells ( n = 2). F qRT–PCR validation of Irf7 and Irf9 downregulation in RM1 cells from bone metastases (RMI BD) compared to parental RM1 cells, lung metastases (RM1 lung), and naïve bone marrow (BM) ( n = 3 mice per group). P ‐values represented as ** < 0.005, and *** < 0.0005 (Student's t ‐test). G qRT–PCR of Irf7 and Irf9 downregulation in parental RM1 cells and RM1 cells from bone metastases (RM1 BD) in WT and Ifnar1 ‐deficient (−/−) mice, with naïve BM from WT and Ifnar −/− animals for reference. ( n = 3 mice per group, n = 1 for Ifnar −/− BM control). P ‐values were represented as * < 0.05 and ** < 0.005 (Student's t ‐test). Data information: All error bars ± SEM. Source data are available online for this figure.

Article Snippet: When required, cells were transfected with poly I:C (10 μg/ml) overnight or treated with HDACi (entinostat [MS275], 1 μM [Selleckchem]; romidepsin [depsipeptide], 50 nM [Selleckchem]; or vorinostat [suberanilohydroxamic acid, SAHA], 2.5 μM [Selleckchem]) for 48 h prior to RNA extraction. cDNA was generated using the iScript Reverse Transcriptase Supermix cDNA for qRT–PCR kit (Bio‐Rad). qRT PCR was performed using SsoAdvanced Universal SYBR Green Supermix (Bio‐Rad) to quantify murine Irf7 , Irf9 , and Oas2 transcript expression on the CFX96 (Bio‐Rad) cycler per manufacturer's guidelines.

Techniques: RNA Sequencing Assay, Derivative Assay, Cell Isolation, Injection, Quantitative RT-PCR, Expressing

Journal: EMBO Reports

Article Title: Prostate cancer cell‐intrinsic interferon signaling regulates dormancy and metastatic outgrowth in bone

doi: 10.15252/embr.202050162

Figure Lengend Snippet: Stability of Irf7 and Irf9 mRNA suppression by qRT–PCR in ex vivo bone‐derived cells (RM1 BD Irf − , n = 7) in culture compared to a bone‐derived line that showed initial loss during early passage (RM1 BD REV (EP), n = 3), then reverted to parental expression levels at late passage (RM1 BD REV (LP), n = 4) compared to parental RM1 ( n = 3). ELISA of IFN‐α production by RM1 parental ( n = 3), RM1 bone‐derived Irf‐low (RM1 BD Irf − , n = 4), and RM1 BD REV ( n = 3) cells subsequent to poly I:C stimulation. qRT–PCR analysis of Irf7 and Irf9 expression in RM1 BD Irf − cells ± 48 h treatment with MS275 (1 μM) ( n = 7–9). Schematic of contact and transwell co‐culture systems. qRT–PCR analysis of Irf7 and Irf9 expression in parental RM1 cells ( n = 4) 48 h post‐contact culture with naïve BM ( n = 6). qRT–PCR analysis of Irf9 expression in parental RM1 cells ± 48 h co‐culture with naïve BM under contact (non‐transwell; NT) and transwell (0.4‐μm filters that prevent cell contact) conditions ( n = 6–8 per condition). qRT–PCR analysis of Irf9 expression in parental RM1 cells ± 48 h contact co‐culture with naïve BM ± MS275 (1 μM) ( n = 3–6 per condition). Data information: P ‐values represented as * < 0.05, ** < 0.005, and *** < 0.0005 (Student's t ‐test). All error bars ± SEM.

Article Snippet: When required, cells were transfected with poly I:C (10 μg/ml) overnight or treated with HDACi (entinostat [MS275], 1 μM [Selleckchem]; romidepsin [depsipeptide], 50 nM [Selleckchem]; or vorinostat [suberanilohydroxamic acid, SAHA], 2.5 μM [Selleckchem]) for 48 h prior to RNA extraction. cDNA was generated using the iScript Reverse Transcriptase Supermix cDNA for qRT–PCR kit (Bio‐Rad). qRT PCR was performed using SsoAdvanced Universal SYBR Green Supermix (Bio‐Rad) to quantify murine Irf7 , Irf9 , and Oas2 transcript expression on the CFX96 (Bio‐Rad) cycler per manufacturer's guidelines.

Techniques: Quantitative RT-PCR, Ex Vivo, Derivative Assay, Expressing, Enzyme-linked Immunosorbent Assay, Co-Culture Assay

Journal: EMBO Reports

Article Title: Prostate cancer cell‐intrinsic interferon signaling regulates dormancy and metastatic outgrowth in bone

doi: 10.15252/embr.202050162

Figure Lengend Snippet: Assessment of tumor‐intrinsic IFN suppression stability over passage (P; number indicated) in culture by qRT–PCR analysis of mean Irf7 and Irf9 mRNA expression in bone‐derived RM1 Irf‐low (RM1 BD Irf − ) cells and a reverted (REV) bone‐derived cell line compared to RM1 parental cells. Values are means ± SEM of three independent experiments. HDACi impact on RM1 BD Irf − proliferation over time by SRB assay. Mean OD at 550 nm ( n = 3). Flow cytometry characterization of bone marrow lymphocyte (%) populations ( n = 3). qRT–PCR of Irf9 expression in parental RM1 cells 48, 72, and 96 h post‐contact co‐culture with FACS‐isolated naïve CD11b + Ly6G + BM cells ( n = 3 mice per time point). qRT–PCR of Irf7 expression in RM1 parental cells ± co‐culture with naïve BM ± 48 h treatment with MS275 ( n = 3–6). P ‐values represented as * < 0.05, ** < 0.005, and *** < 0.0005 (Student's t‐ test). All error bars ± SEM.

Article Snippet: When required, cells were transfected with poly I:C (10 μg/ml) overnight or treated with HDACi (entinostat [MS275], 1 μM [Selleckchem]; romidepsin [depsipeptide], 50 nM [Selleckchem]; or vorinostat [suberanilohydroxamic acid, SAHA], 2.5 μM [Selleckchem]) for 48 h prior to RNA extraction. cDNA was generated using the iScript Reverse Transcriptase Supermix cDNA for qRT–PCR kit (Bio‐Rad). qRT PCR was performed using SsoAdvanced Universal SYBR Green Supermix (Bio‐Rad) to quantify murine Irf7 , Irf9 , and Oas2 transcript expression on the CFX96 (Bio‐Rad) cycler per manufacturer's guidelines.

Techniques: Quantitative RT-PCR, Expressing, Derivative Assay, Sulforhodamine B Assay, Flow Cytometry, Co-Culture Assay, Isolation