Fig 1: RBFOX2 depletion in H9c2 myoblasts affects mitochondrial gene expression and health(A) Mitochondrial membrane potential (Ψm) was determined by JC-1 staining of the H9c2 cells transfected with scrambled or Rbfox2 siRNA. Shown are the ratios of fluorescence intensity of J-aggregates (red fluorescence) to J-monomers (green fluorescence). Statistical significance was calculated using unpaired t test to compare two different groups in five replicates of two independent experiments. Data represent means ± SD. For mitochondrial membrane potential in untreated control versus RBFOX2-KD H9c2 cells, ****p < 0.0001; in control versus RBFOX2-KD H9c2 cells treated with rotenone, ***p = 0.0004; in control versus RBFOX2-KD H9c2 cells treated with antimycin A, ***p = 0.0009; in control versus RBFOX2-KD H9c2 cells treated with FCCP, **p = 0.0017; in control versus RBFOX2-KD H9c2 cells treated with H2O2, ****p < 0.0001.(B) Representative WB images of mitochondrial proteins cytochrome b (Cytb), cytochrome c oxidase 4 (COX4) (n = 3 for control and n = 4 for RBFOX2 KD samples from three independent experiments), OPA1 (n = 3 for control and n = 4 for RBFOX2 KD samples from three independent experiments), and MFN1 (n = 6 for each group from three independent experiments) in control siRNA or RBFOX2-siRNA-treated cells expressing empty vector or FLAG-ANT1 (n ≥ 3, three independent experiments).(C) Quantification of mitochondrial protein levels in control or RBFOX2 KD H9c2 cells expressing empty vector or FLAG-ANT1 plasmid with respect to their respective loading controls. COX4 si-Ctrl versus si-Rbfox2 **p = 0.0068, Cytb si-Ctrl versus si-Rbfox2 **p = 0.0046, si-Ctrl versus si-Rbfox2+ FLAG-ANT1 *p = 0.033, si-RBFOX2 versus si-Rbfox2 +FLAG-ANT1 **p = 0.0020.(D) Representative transmission electron microscopy images of mitochondria in control versus RBFOX2-KD H9c2 cells. Black arrows mark the mitochondria. n = 3 (three independent experiments). Scale bar: 0.1 micron.(E) Surface area of mitochondria in control and RBFOX2-depleted myoblasts was calculated using ImageJ. n = 234 mitochondria for control, n = 216 mitochondria for RBFOX2 KD myoblasts from three independent KD experiments, ****p < 0.0001.
Fig 2: RBFOX2 binding sites near dPASs are critical for APA regulation of the Slc25a4 gene(A) Cartoon representation of luciferase-Slc25a4 poly(A) reporter constructs that harbor both pPASs and dPASs of Slc25a4 in the 3′UTR with or without mutated RBFOX2 binding motifs (underlined) located downstream of the dPAS.(B) qRT-PCR analysis of dPAS usage when normalized to total mRNA levels in HEK293 cells expressing WT or RBFOX2 binding site mutant poly(A) luciferase-Slc25a4 3′UTR constructs. n = 24 for WT, n = 13 for mutant samples from three independent experiments, ****p < 0.0001.(C) Relative firefly luciferase levels in HEK293 cells expressing WT or RBFOX2 binding site mutant poly(A) luciferase-Slc25a4 3′UTR constructs. n = 12 for WT samples, n = 14 for mutant samples from three independent experiments, ****p < 0.0001.
Fig 3: RBFOX2 regulates tandem APA and expression of essential mitochondrial gene Slc25a4(A) Slc25a4 3′UTR lengthening mediated via tandem APA in RBFOX2-depleted H9c2 myoblasts identified by PAC-seq. The number of reads mapped to Slc25a4 transcripts is shown on the right side. See also Figure S5.(B) Nanopore sequencing analysis of full-length Slc25a4 transcripts with different 3′UTR lengths in control versus RBFOX2 KD myoblasts. See also Figure S5.(C) RT-PCR analysis of endogenous Slc25a4 dPAS/total mRNA ratio in RBFOX2 KD myoblasts ectopically expressing GFP or GFP-RBFOX2. n = 8, ****p < 0.0001 (three independent experiments). See Table S2 for primer information.(D) WB analysis of ANT1 protein (Slc25a4) in control versus RBFOX2 KD myoblasts determined by Bio-Rad ChemiDoc Imager. Even protein loading was monitored by imaging stain-free gels. ANT1 protein levels were normalized to loading control, and fold change in ANT1 protein levels was quantified using Bio-Rad ChemiDoc software. n = 4, ***p = 0.0002 (three independent experiments).
Supplier Page from Sino Biological, Inc. for Human SLC25A4 Gene ORF cDNA clone expression plasmid, N-Flag tag