Figure 1 | Scientific Reports

Figure 1

From: Unbiased shRNA screening, using a combination of FACS and high-throughput sequencing, enables identification of novel modifiers of Polycomb silencing

Figure 1

Characterisation of mouse F9 cells for screening purposes. (A) A scatter plot of candidate reporter genes in F9 cells. Upon RA treatment, 28 Polycomb-target genes were strongly upregulated. X-axis, log2-fold change (log2-fc) in gene expression after RA treatment; Y-axis, mean log2- count per million (log2-cpm) representing relative expression levels. (B) RT-qPCR of Col4a1 mRNA expression in differentiating F9 cells. RA was titrated in the presence of db-cAMP. Data presented as mean ± s.d. relative to Gapdh mRNA expression. (C) Immunofluorescence analysis of type IV collagen expression before (−) or after (+) RA-induced differentiation. Bars, 50 µm. (D) RT-qPCR f Col4a1 and Hoxb4 mRNA expression at the indicated RA concentration. Data presented as mean ± s.d. relative to Gapdh mRNA expression. *P < 0.05. (E) ChIP-qPCR analysis of histone H3K27me3, H3K4me3 or H2AK119ub levels in chromatin containing the indicated gene promoter regions (around +300 bases from the transcription start site) in the presence or absence of RA. Data presented as mean ± s.d. relative to input. ChIP signals from the promoter region of Il2ra (control region) were indicated by interrupted lines. *P < 0.05. (F) Conventional RT-PCR of indicated mRNA expression in either Suz12 or Ring1b knockdown (KD) cells. (G) Western blot analysis of KD cells using the indicated antibodies. (H) RT-qPCR of Col4a1 mRNA expression in the indicated KD cells. Data presented as mean ± s.d. relative to Gapdh mRNA expression. *P < 0.05. (I) Immunofluorescence analysis of type IV collagen expression in the indicated KD cells. Bars, 50 µm.

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