Journal: Nature Communications

Article Title: Spatial tumor immune heterogeneity facilitates subtype co-existence and therapy response in pancreatic cancer

doi: 10.1038/s41467-024-55330-7

Figure Lengend Snippet: Fig. 3 | JUNB-HDAC1 complex represses inflammatory signals and cJUN. a Heatmap showing expression of cytokines present in the core enrichment of the gene sets shown in Supplementary Fig. 3a–d, for JUNB silencing (siJUNB; n = 3 biological replicates) versus control siRNA (siCtrl; n = 2 biological replicates) in CAPAN1 cells. Cell color indicates z score. b qRT-PCR analysis for indicated target genes in siJUNB conditions (red), normalized to siCtrl (gray), in CAPAN1. Relative mRNA expression with mean ± s.d. shown. n = 3 biological replicates. Two-tailed Student’s t-test with Welch’s correction. Coverage of JUNB ChIP-seq data in CAPAN121, as well as publicly available H3K27ac25 data, for loci of cJUN (c), IL1A/B (d), and CXCL9/10/11 (e). ChIP-qPCR validation regions are indicated. f Gene set enrichment analysis for curated signatures (C2) of the Molecular Signature Data- base (MSigDB) for siJUNB versus siCtrl in CAPAN1 cells. Normalized enrichment score (NES) and FDR q value are indicated. Immunoblot for JUNB, HDAC1, and β- actin after JUNB pulldown, IgG isotype control or input in CAPAN1 (g) and CAPAN2 (h). n = 3 biological replicates. i, j, ChIP-qPCR for regions indicated in (c–e), showing signal relative to input for JUNB (i) and HDAC1 (j) pulldown with

Article Snippet: Patient-derived primary cell line GCDX62 was maintained in a 3:1 mixture of Keratinocyte-SFM (KSF; Thermo Fisher Scientific; supplemented with 2% (v/v) FCS, 1% (v/v) Penicillin-streptomycin, bovine pituitary extract (BPE), and human epidermal growth factor) and RPMI 1640 containing 10% (v/v) FCS. cJUN overexpression (cJUN-OE; construct pMSCV-cJUN, no. 34898, addgene) and empty vector (EV; construct MSCV, no. 68469, addgene) control cell lines of CAPAN2 and GCDX62 were generated previously21, and were maintained in their normal growth medium supplemented with 1μg/mL puromycin. siRNA transfection For transient knockdownexperiments, 5 × 105 cellswere seeded in 6-well plates and immediately transfected with a mixture of 10μL Lipofectamine2000 (Thermo Fisher Scientific), 6μL of 20μM target-specific siRNA (or non-targeting siRNA as control), and 200μL Opti-MEM (Thermo Fisher Scientific) after 15min incubation of the transfection mixture at room temperature (RT).

Techniques: Expressing, Control, Quantitative RT-PCR, Two Tailed Test, ChIP-sequencing, ChIP-qPCR, Biomarker Discovery, Western Blot