Fig 1: Example of CCIVR analysis II: identification of cis-NATs with TGFß stimulation. (A) Schematic representation for TGFß stimulation of Huh-7 cells. For details, see also Figure S3. (B) CCIVR analysis with genes that demonstrate differential expression with TGFß stimulation [upgenes: fold change = 1.5, padj < 0.05; downgenes: fold change = 0.67, padj < 0.05]. The numbers of four kinds of cis-NAT pairs with the different combinations of expression pattern including down-down, down-up, up-down, and up-up (sense-antisense) are shown. (C) GO analysis of biological processes with genes from differentially expressed cis-NAT pairs. The top 15 biological processes are shown. Processes related to TGFß signaling in up-regulated genes are in red and, among them, those related to EMT are indicated with an asterisk (*). Processes related to cell growth in down-regulated genes are in green. (D) Heatmap analysis of cis-NAT pairs with inverse-correlated gene expression (down-up). Three kinds of cis-NATs, embedded, fully-overlapped, and head-to-head are shown. Fold change (f.c.) with an asterisk (*) indicates significant differential expression after TGFß stimulation for 12 h and the other values are for 48 h after TGFß stimulation. (E–G) Confirmation of SETD2 expression by RT-qPCR (E) and western blotting (F) and the level of H3K36me3 by western blotting (G) in SETD2 knockdown samples. Images for western blotting were cropped for improving the clarity and conciseness. Original blots are presented in Supplementary Figure S5. (H) Maps of cis-NAT pairs, BCAR3-AS1/BCAR3 and GPX2/CHURC1. *: BCAR3-AS1 replaced AL109613.1 by revisiting the latest version of Ensembl, Human GRCh38p13. (I) Expression dynamics of BCAR3-AS1/BCAR3 and GPX2/CHURC1 cis-NAT pairs upon TGFß stimulation for 48 h. (J, K) RT-qPCR analysis for sense transcripts (J) and their cis-NATs (K) upon TGFß stimulation with or without SETD2 knockdown. (L) ChIP-qPCR analysis of H3K36me3 accumulation at the promoter of sense transcripts. For comparison, blue and red dashed lines representing the level of H3K36me3 accumulation in “TGFß 0 h siCtrl” and “TGFß 48 h siCtrl” are shown. (E, I–L) Means ± SD across biological replicates are shown (E, I–K: n = 3; L: n = 5). *p < 0.05, **p < 0.01, ***p < 0.001, comparisons as indicated (E, I–K) and by dashed lines (L); Student’s t-test. Normalized to GAPDH (E, J–K).