Fig 1: Edited and wild-type CDK13 show differences in nuclear speckle and nucleolus localization. Stably-transduced CDK13-WT, CDK13-Edit or Empty vector Cal62 and TPC1 cell lines were used. A and B Confocal images of immunofluorescence staining of the cells with antibodies against HA (green), the nuclear speckle marker SC35 (red) (A) or the nucleolus marker Fibrillarin (Pink) (B). Nuclei were stained with DAPI (blue). Colocalization was analyzed using Fiji-Coloc2 plugin, and the mean of the Pearson R value from 3 independent experiments is represented in the bottom panels. C Upper panels: Representative image of CDK13-Edit Cal62 and TPC1 cells with nucleolar accumulation of CDK13. The images represent the immunofluorescence staining of cells with antibodies against HA (green), SC35 (red) or Fibrillarin (pink). Bottom panel: Graphs show quantification of the percentage of CDK13-WT or CDK13-Edit cells that present with nucleolar accumulation of CDK13 (n = 3 independent experiments). Error bars indicate standard deviations. Asterisks denote statistical significance as assessed with Student’s t-test (two-tailed). * p < 0.05, ** p < 0.01, *** p < 0.001
Fig 2: Edited CDK13 alters alternative splicing. RT-qPCR of ADAR1-regulated alternative splicing events for indicated genes in Cal62 (upper panel) or TPC1 cells (lower panel) stably expressing CDK13-WT, CDK13-Edit or the empty vector (n = 4). Error bars indicate standard deviations. Asterisks denote statistical significance compared with siControl treatment assessed by Student’s t-test (two-tailed). * p < 0.05, ** p < 0.01, *** p < 0.001
Fig 3: Edited CDK13 rescues the ADAR1-knockdown phenotype. Stably-transduced Cal62 and TPC1 cells with CDK13 Edit or Empty vector were silenced for ADAR1 and were assayed for (A) BrdU incorporation (n = 4 for Cal62 and n = 3 for TPC1 cell line). B and C Viability by XTT dye reduction (n = 3) (B) and crystal violet staining (n = 5) (C). D Matrigel Transwell invasion assay (n = 3 for Cal62 and n = 4 for TPC1 cell line). Error bars indicate standard deviations. Asterisks denote statistical significance compared with siControl treatment as assessed with Student’s t-test (two-tailed). * p < 0.05, ** p < 0.01, *** p < 0.001
Fig 4: Edited CDK13 potentiates the aggressive behavior of thyroid cancer cells. Cal62 and TPC1 cells were stably infected with lentiviruses containing the WT CDK13 or its edited form (Edit CDK13) and were assayed for (A) BrdU incorporation (n = 3). B XTT dye reduction (n = 3 for Cal62 and n = 5 for TPC1) and (C) crystal violet staining (n = 4). D Matrigel Transwell invasion assay (n = 4). Error bars indicate standard deviations. Asterisks denote statistical significance compared with siControl treatment as assessed with Student’s t-test (two-tailed). * p < 0.05, ** p < 0.01, *** p < 0.001
Fig 5: ADAR1 edits CDK13 transcript at position c.308A > G. A Distribution of 12 types of nucleotide changes across the entire transcriptome of Cal62 cells after ADAR1 knockdown, as profiled by RNA-seq. B RNA-seq analysis showing a global reduction in the total number of variants after ADAR1 silencing. C Distribution of A-to-I editing sites over annotated genomic regions. Abbreviations: UTR, untranslated region. D Details of the position and significance of the CDK13 editing event that is downregulated after ADAR1 knockdown in Cal62 cells. Fold-change and FDR refers to the allele frequency change in siControl versus siADAR1. E and F Sanger sequencing chromatograms using a reverse primer illustrate editing of the selected CDK13 event in siControl, siADAR1 #1, siADAR1 #2 and genomic DNA (gDNA) in Cal62 (E) and TPC1 (F) thyroid cancer cell lines. G CDK13 c.308 A > G editing frequency in normal (n = 6) and PTC tumor samples (n = 6) calculated by taking the peak area of G versus A using Indigo. Error bars indicate standard deviations. Asterisks denote statistical significance as assessed with Student’s t-test (two-tailed). * p < 0.05
Supplier Page from Abcam for Anti-CDK13 antibody