Journal: Nature

Article Title: Ras drives malignancy through stem cell crosstalk with the microenvironment

doi: 10.1038/s41586-022-05475-6

Figure Lengend Snippet: a , ATAC sequencing is performed on FACS-purified α6 hi β1 hi basal populations of interfollicular epidermis (IFE, Sca1+), bulge hair follicle stem cells (HFSCs, CD34 + ), and tumour cells (CD44 hi ) either positive or negative for TGFβ-responsiveness (mCherry). Peaks are clustered according to their openness in each population by k -mean clustering. b , Venn diagram showing marked divergence of ATAC peaks from TGFβ-responding tumour basal cells and their non-responding neighbours between papilloma and SCC stages ( n = 2 for each condition, each stage). c , Motif enrichment analysis of the 7 ATAC peak clusters. d , Quantifications of the Lepr cis-regulatory region boxed in Fig. . e , Immunofluorescence images reveal that transcription factors RUNX1 and FOS are not detected in normal homeostatic skin but are enriched progressively during tumorigenesis. Scale bars, 50 µm. See also pSMAD2 immunofluorescence quantifications in Extended Data Fig. . f , Lepr EGFP reporter and TGFβ mCherry reporter show minimal activity in papillomas but co-localize at the invasive fronts of SCC. Note numerous SCC cells marked by EGFP cytoplasm and mCherry nucleus. Integrin (white) denotes invasive fronts. For the original images, scale bars, 20 μm. For the magnified insets, scale bar, 10 μm. The percentages of reporter double-positive (DP) cells in these invasive regions are significantly higher in SCC than in papilloma. Majority of the TGFβ mCherry reporter + cells are these DP cells in SCC compared to the ones in papilloma. ( n = 4 for papilloma, n = 3 for SCC; top right: p = 0.0477; bottom right: p < 0.0001). All statistics were using unpaired two-tailed Student’s t -test: ns, p ≥ 0.05); *, p ≤ 0.05); **, p ≤ 0.01; ***, p ≤ 0.001; ****, p ≤ 0.0001. Data are presented as mean ± s.e.m.

Article Snippet: After blocking, the sections were stained with primary antibodies: ITGA6 (rat, 1:2,000, BD), RFP/mCherry (guinea pig, 1:5,000, E.F.’s laboratory), K14 (chicken, 1:1,000, BioLegend), CD31 (rat, 1:100, BD Biosciences), K5 (guinea pig, 1:2,000, E.F.’s laboratory), K8 (rabbit, 1:1,000, E.F.’s laboratory), mLEPR (goat, 1:200, R&D Systems), hLEPR (rabbit, 1:100, Sigma-Aldrich), RUNX1 (rabbit, 1:100, Abcam), FOS (rabbit, 1:100, Cell Signalling), GFP (chicken, 1:500, BioLegend), pSTAT3-Y705 (rabbit, 1:100, Cell Signalling), pSMAS2-S465/467 (rabbit, 1:1,000, Cell Signalling) or pS6-S240/244 (rabbit, 1:100, Cell Signalling).

Techniques: Sequencing, Purification, Immunofluorescence, Activity Assay, Two Tailed Test

Journal: Molecular Therapy Oncolytics

Article Title: COMMD7 Regulates NF-κB Signaling Pathway in Hepatocellular Carcinoma Stem-like Cells

doi: 10.1016/j.omto.2018.12.006

Figure Lengend Snippet: Overexpression of PIAS4 Influenced the Effect of COMMD7 Knockdown in Nanog + HCSCs (A and B) The expression of PIAS4 was determined using real-time qPCR (A) and western blotting (B) analysis of the protein level of CXCL12, CXCL2, and PIAS4 in Nanog + HCSCs after COMMD7 knockdown. Then the COMMD7-silenced Nanog + HCSCs were transfected with empty vector or pcDNA3.1-PIAS4. (C) The mRNA expression of PIAS4 after PIAS4 overexpression. (D) Western blotting analysis of the protein expression of NEMO, p65, HNF4α, and PIAS4. The proliferative ability, apoptotic rate, migration, and invasion were evaluated using (E) CCK-8 assay, (F) flow cytometry assay, and (G) wound healing and transwell invasion assay in Nanog + HCSCs, respectively. *p < 0.05; ***p < 0.001 versus vector or sh-COMMD7.

Article Snippet: Next, the membranes were blocked in 5% nonfat milk dissolved in Tris-buffered saline with Tween 20 (TBST) solution for 1 h at room temperature followed by incubation with primary antibodies against Nanog (1:1,000, ab153419; Abcam), COMMD7 (1:2,000, #9742; Cell Signaling), COMMD1 (1:1,000, #24640; SAB), NF-κB p65 (1:1,500, 19553-1-AP; Proteintech), HNF4α (1:1,000, #2947; Cell Signaling), NEMO (1:3,000, #23870; SAB), CXCL12 (1:1,000, #36120; SAB), CXCL2 (1:4,000, #24681; SAB), PIAS4 (1:2,500, #8709; Cell Signaling), histone H3.1 (1:2,000, #9265; Cell Signaling), β-actin (1:1,000, #5762; Cell Signaling), and GAPDH (1:10,000, 10494-1-AP; Proteintech) at 4°C overnight.

Techniques: Over Expression, Expressing, Western Blot, Transfection, Plasmid Preparation, Migration, CCK-8 Assay, Flow Cytometry, Transwell Invasion Assay