Fig. 4: RARα divergently regulates TGFβ signaling and proliferation in a cluster-specific manner: transcriptional and functional validation.

a siRARα RT-qPCR on CL1 and CL2 RA FLS. Boxplots of change in mRNA levels under RARα depletion (CL1: p = 0.041, CL2: p = 0.015) for TGFβ1 (CL2: p = 0.004) and canonical CDKN2B (CL1: p = 0.0009, CL2: p = 0.0002)/non-canonical signaling axis genes that undergo transcriptional regulation. Arrows (↑) illustrate activating and blunt ended lines (T) inhibiting/repressive effects. Cyan infill depicts the primary CL1 regulation logic, dark blue representing the primary CL2 regulation logic and black arrows representing both CL1 and CL2 logic. Boxplot of change in CDKN2B protein levels under RARα depletion (CL1: p = 0.048). p-values calculated by paired two-tailed Student’s t-test for siRARA vs CTL *p < 0.05. CL1 n = 3 and CL2 n = 3 biologically independent cell lines. b Heatmap of gene expression (z-score(TPM)) for genes with significant differential expression (p < 0.05 by two-tailed Student’s t-test) between clusters, related to cellular proliferation. Growth rate boxplot for CL1 and CL2 FLS. CL1 n = 5 and CL2 n = 4 biologically independent cell lines. c MTT proliferation assay boxplot for siRARα and siCtrl at 1 and 5 days after plating. CL1 n = 6 and CL2 n = 3 biologically independent cell lines. For all box plots, red center line for median, whiskers represent maximum and minimum values, box width from quartile 1 to quartile 3. Source data are provided as a Source Data file.