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. Author manuscript; available in PMC: 2021 Jan 27.
Published in final edited form as: Nat Cell Biol. 2020 Jul 27;22(8):960–972. doi: 10.1038/s41556-020-0550-8

Fig. 2. AKAP95 directly regulates splicing of key transcripts for cancer.

Fig. 2.

a, CCNA2 expression in MDA-MB-231 cells upon AKAP95 KD. Left, relative mRNA levels of indicated cyclins were determined by RT-qPCR and normalized to GAPDH, and presented as mean ± SD from n = 3 biological repeats. Right, immunoblotting for Cyclin A1/A2.

b, Co-overexpression of AKAP95 and CCNA2 in breast cancer tissues of TNBC patients. Left, each box represents a patient. Right, correlation of their mRNA levels in the TNBC patients with indicated Pearson correlation coefficient. From cBioPortal. n = 82 patient samples.

c,f, RNA immunoprecipitation-sequencing (RIP-seq) profiles for CCNA2 (c) and SMAD6 (f) based on our previous work33. Blue, anti-FLAG RIP-seq in control or 293 cells expressing the FLAG-HA-tagged AKAP95 WT or mutants. Red, anti-AKAP95 RIP-seq in control or AKAP95-KD 293 cells. Black, profiles of total input RNAs. All profiles have the same Y-axis scale. Arrows indicate AKAP95-binding sites at intron 1. One representative RIP-seq analysis from 2 repeats.

d, Total RNAs were used for RT-PCR, in the absence (- RT) or presence (+ RT) of reverse transcriptase, for CCNA2 intron 1 in MDA-MB-231 cells with indicated siRNAs. Top, PCR products on agarose gel. Asterisk, an unknown amplification product. Repeated 3 times. Bottom, relative ratios of the signal for the intron 1- retaining transcript over the intron 1-spliced transcript, as mean ± SD from n = 3 independent experiments.

e, Assay for CCNA2 mRNA stability. Control (Scr) and AKAP95-KD MDA-MB-231 cells were treated starting from 0 min with Actinomycin D (+A, to block RNA synthesis) and cycloheximide (+C, to block NMD) or not as indicated. Total RNA at indicated times were used for RT-PCR and normalized to ACTB, as mean ± SD from n = 3 biological repeats.

g, mRNA-seq profiles for SMAD6 in MDA-MB-231 cells expressing control or AKAP95 shRNA #1 (KD) and vector or AKAP95-expressing construct. Asterisk, a stop codon.

P values by two-sided Student’s t-test for a and e and one-way ANOVA followed by Tukey’s post hoc test for d. Uncropped blots and statistical source data are provided as in source data fig 2.