Fig. 5: SWI/SNF complex fine-tunes expression of CDKN1C.

a Representative immunofluorescence images showing expression and localisation of GR (green), treated with glucocorticoids (GC) or control (Veh), using DAPI as nuclear staining (blue) (n = 3). Scale bar, 10 μm. b Heatmap of ChIP-sequencing signal around peak midpoint for all sites detected across the genome (top), and average signal of GR ChIP-seq experiments across all sites called over input control (bottom), for untreated (Veh) and glucocorticoid-treated (GC) cells. c Scatter plot depicting differential gene expression changes upon GC treatment in RNA sequencing. Genes significantly (Padj ≤ 0.01) up- or downregulated by GCs are depicted in red (n = 2). Adjusted P values were determined by DESeq2 (Wald test P values corrected for multiple testing using Benjamini and Hochberg method). d Scatter plot depicting enrichment over IgG control in a GR-RIME experiment. Proteins considered to be recruited by GR are 2.5 LFQ enriched over IgG (dotted line) and significant (−log(P value)>2; red) (n = 3). P values were determined by two-sided t test. e Volcano plot depicting differentially enriched interactors in GR-RIME experiments between three cell lines with active and two cell lines with inactive GR (n = 3). P values were determined by two-sided t test. f GSEA enrichment profiles for RNA polymerase II transcription factor complex (M17103; blue). Nuclear transcription factor complex (M17532; purple), SWI/SNF complex (M17713; red) and mediator complex (M17759; green) gene sets based on A549/H2122/H1944 and H1975/H460 comparison GR-RIME dataset (n = 3). Nominal P values were determined by GSEA software. g Box plot depicting GR activity (z score of 253 genes) in SWI/SNF WT (n = 786) and mutant (n = 91) human lung adenocarcinoma tumours. The central mark indicates the median, and the bottom and top edges of the box indicate the 25th and 75th percentiles, respectively. The notch displays a confidence interval around the median. The maximum whisker lengths are specified as 1.5 times the interquartile range and outliers are depicted as filled circles. P values were determined by Wilcoxon rank-sum test with continuity correction. h (left) Normalised mRNA expression level relative to shControl for SMARCD3, ARID2, SMARCD2, SMARCC2, SMARCB1, SMARCA2, SMARCE1 and ARID1A, in cell lines with shRNA targeting respective genes. Mean values with ± SEM depicted. ≥2 shRNAs per gene in biological duplicates (n = 2). h (middle) Normalised (relative to untreated condition) CDKN1C mRNA expression level of GC-treated shSMARCD3, shARID2, shSMARCD2, shSMARCC2, shSMARCB1, shSMARCA2, shSMARCE1 and shARID1A H2122 cell lines. Mean values with ±SEM depicted. ≥2 shRNAs per gene in biological duplicates (n = 2). h (right) Protein expression index (number of positive cells * average signal intensity) depicting quantified expression of p57 in immunofluorescence experiments using shSMARCD3, shARID2, shSMARCD2, shSMARCC2, shSMARCB1, shSMARCA2, shSMARCE1 and shARID1A H2122 cell lines. Mean values with ±SEM depicted. ≥2 shRNAs per gene in biological duplicates (n = 2), ≥10,000 cells quantified.