Fig. 4

An RNA component stabilizes RCOR2 at nuclear speckles. A Merged representation of RCOR2 (green), SRRM2 (red), and Poly(A)-RNA (magenta) triple immunostaining at super-resolution confocal acquisition in HT22 cells. Right panels show zoomed-in individual nuclear speckles. B Fluorescence correlation plot showing the normalized fluorescence intensity of each mark plotted against the position on the dashed line. C RCOR2 (green), SRRM2 (red), and poly(A)-RNA (magenta) labeling of RNAse A treated HT22 cells visualized at regular confocal acquisition. Arrows indicate a representative cell that lost its RNA content after RNAse A treatment. D Quantitation of normalized cell fluorescence intensity for each channel of cells mock-treated and RNAse A-treated. Quantitations included 20 cells from 3 biological replicates. E Western blot analysis of RCOR2 and GAPDH to test their coprecipitation after poly(A)-pull-down. Increasing concentrations of protein input from HT22 cells were loaded to estimate enrichment after pull down. No loaded extract and extract without beads were subjected to the same procedure as specificity controls. F Real-time quantitative PCR results showing 7SK and MALAT1 fragment amplification after performing RNA immunoprecipitation on HT22 nuclear extracts with an anti-RCOR2 antibody. IgG was used as specificity control and qPCR without reverse transcription (RT(-)) was analyzed to discard eventual DNA concentration after immunoprecipitations. HDAC2 antibody was used as a negative control and the nuclear lncRNA HOTAIR was analyzed as a negative control. Results are representative of two different biological replicates