Figure 6: PANDA regulates PRC target gene expression and senescence entry.
From: Long noncoding RNA PANDA and scaffold-attachment-factor SAFA control senescence entry and exit
(a) Pre-senescent proliferating BJ fibroblasts were transiently transfected with indicated siRNA duplexes. qRT–PCR on total RNA prepared from respective samples at day 2 of siRNA treatment for indicated genes. Error bars represent the s.d. of three independent experiments; P≤0.05, t-test. (b) SAFA and BMI1 qChIP–PCR of indicated gene promoter regions in siScramble control (siC), siSAFA (siS) and siPANDA (siP)-treated BJ fibroblasts. Error bars represent the s.d. of three independent experiments performed in triplicates; P≤0.05, t-test. (c) PANDA-qChIRP for indicated gene promoters using biotinylated lacZ control and PANDA-specific antisense DNA probes in pre-senescent (PS) and RAS-senescent (S) BJ fibroblasts. Affinity-purified material was analysed by qPCR with gene-specific primers for indicated genes. Error bars represent the s.d. of three independent experiments performed in triplicates; P≤0.05. (d–g) Pre-senescent proliferating BJ fibroblasts were transiently transfected with siScramble control (siC), siPANDA-1 (siP-1), siPANDA-2 (siP-2) or a pool of siPANDA-1 and -2 (siP-1/P-2) for a duration of 4 days. (d) Relative (rel.) cumulative cell numbers at day 0, 2 and 4 post siRNA treatment. Error bars represent the s.d. of three independent experiments performed in triplicates; P≤0.05, t-test. (e) Representative micrographs 4 days post siRNA treatment showing BJ fibroblast cell morphology and percentage of SABG-positive cells (upper panel; shown is the result for the siPANDA pool only; scale bar, 20 μm) and bar chart for percentage of Edu positive (pos.) staining cells (lower panel). Error bars represent the s.d. of three independent experiments; P≤0.05, t-test. (f) qRT–PCR on total RNA prepared from respective samples at day 4 of siRNA treatment for p53 and CDKN1A. Shown is the result for the siPANDA pool only. Error bars represent the s.d. of three independent experiments; P≤0.05, t-test. (g) CDKN1A loss rescues siPANDA-induced senescence. CDKN1A knock-out LF1 fibroblasts were transiently transfected either with siScramble control (siC) or siPANDA pool (siP) for a duration of 7 days. Relative cell numbers at day 0, 2, 4 and 7 post siRNA treatment are depicted. Error bars represent the s.d. of three independent experiments; P≤0.05, t-test.
