Fig. 1: A Cyclin D1-interacting lncRNA DILA1 is overexpressed in tamoxifen-resistant breast cancer cells with upregulated Cyclin D1 protein.
From: LncRNA DILA1 inhibits Cyclin D1 degradation and contributes to tamoxifen resistance in breast cancer
a Western blotting showing Cyclin D1 protein in parental and tamoxifen-resistant MCF-7 (MCF7-Pa, MCF-Re) and T47D (T47D-Pa, T47D-Re) cells. GAPDH was used as a loading control. b MCF-Re cells, transfected with negative control siRNA (NC) or one of the two siRNAs targeting Cyclin D1 (siD1-1 and siD1-2), were treated with tamoxifen for 48 h. Relative cell numbers were determined by a cell counter. c Heatmap of the enriched long noncoding RNAs by RIP–sequencing in MCF-7 cells with ectopically expressed HA-Cyclin D1 and untagged Cyclin D1 control. (fold change >2, p < 0.05). p values were determined by negative binomial generalized linear models. No adjustments were made for multiple comparisons. d RT-qPCR showing the expression of DILA1 in MCF7-Pa and MCF-Re cells. e Binding of DILA1 to CyclinD1 protein in MCF7-Re cells, assayed by RIP, followed by RT-qPCR. IgG and GAPDH were used as negative controls. f The full length of DILA1 (ENST00000435697.1) in UCSC Genome Browser (upper) and determined by 5′ and 3′ RACE (lower). g RT-qPCR showing the nuclear and cytoplasmic fraction of DILA1 in MCF-Re cells, with GAPDH and MALAT1 as cytoplasmic and nuclear control, respectively. h Confocal FISH images showing nuclear localization of DILA1 (green) in MCF7-Pa and MCF-Re cells. i RNAScope showing subcellular localization and relative expression of DILA1 (red) in MCF7-Pa and MCF7-Re cells. j RNA pull-down showing the interaction between Cyclin D1 and DILA in vitro (MCF7-Re cell lysates or recombinant GST-Cyclin D1 protein). Biotin-labeled DILA1 detection by anti-biotin antibody as a control. k Confocal FISH images showing the co-localization of Cyclin D1 (red) and DILA1 (green) in MCF7-Re cells. For a, h–k, representative images of three biologically independent experiments are shown. For b, d, e, g, n = 3 biologically independent experiments, means ± s.d. are shown, and p values were determined by two-tailed Student’s test. For h, i, k, scale bars represented 10 μm.
