Fig. 3

Interaction of HDAC6 and RBPs in regulating LINC00461 stability. A Expression levels of LINC00461 from transcriptome data, including two microarray data as shown in Fig. 1A and one microarray analysis using Sp1 knockdown cells. B Parental and TMZ-resistant U87MG cells were treated with either si-Sp1 to silence Sp1 expression or si-NC as a control. Sp1 protein levels were detected by western blotting. LINC00461 expression was detected by qRT-PCR. Unpaired Student’s t-test. C The amount of LINC00461 was measured in TMZ-resistant Pt#3 and Pt#5 cells in either the presence (green lines) or the absence (gray lines) of MPT0B291 following actinomycin D (ActD) treatment at 0, 20, 40, 60, and 120 min. Two-way ANOVA. The red dashed line indicates the time at which 50% of the lncRNA remained. D A172 cells were treated with either si-HDAC6 to silence HDAC6 expression or si-NC as a control. HDAC6 protein levels were detected by western blotting. LINC00461 expression was detected by qRT-PCR. Unpaired Student’s t-test. E Expression levels of LINC00461 in parental and TMZ-resistant GBM cells (Pt#3 and Pt#5). Unpaired Student’s t-test. F Ingenuity pathway analysis (IPA) in U87MG cells was performed using the antibody against HDAC6 or IgG as a negative control. The IP product was examined by western blotting with CONT6 and HDAC6 antibodies. G Desthiobiotin-labeled LINC00461 was captured by streptavidin magnetic beads. PCR (upper panel) and qRT-PCR (lower panel) detected the amount of LINC00461 binding to beads. H Proteins associated with LINC00461 in TMZ-resistant Pt#3 cells were identified by iTRAQ-based proteomic analysis. Interaction between LINC00461-binding proteins and HDAC6 was further evaluated by IPA analysis. I Effect of MPT0B291 on the protein expression of RBPs (CNOT6 and FUS) at 48 h in parental and TMZ-resistant Pt#3 cells. J Effect of HDAC6 knockdown by siRNA on the protein expression of RBPs (CNOT6 and FUS) in parental and TMZ-resistant Pt#3 cells. The results are shown as mean ± SEM for triplicate samples in each group. K The amount of LINC00461 was measured in TMZ-resistant Pt#3 cells treated with either si-FUS to silence FUS expression (green lines) or si-NC as a control (gray lines) following actinomycin D (ActD) treatment at 0, 20, 40, 60, 120, and 240 min. FUS protein levels were detected by western blotting. Two-way ANOVA. The red dashed line indicates the time at which 50% of lncRNA remained