Isoginkgetin and Madrasin are poor splicing inhibitors
Fig 4
Isoginkgetin is a poor splicing inhibitor.
(A) RT-PCR with primers amplifying the intron located between exons 2 and 3 of DNAJB1 or the intron located between exons 4 and 5 of BRD2. HeLa cells were treated with DMSO, 30 μM Isoginkgetin for 1h to 6h, or 1 μM PlaB for 6h. The locations of the spliced and unspliced RNA are shown on the right of the panel. The percentages of unspliced RNA compared to total (spliced + unspliced) are shown below. (B) Percentage of spliced reads in total RNA-seq treated with DMSO (blue) or Isoginkgetin (red) for the time indicated on the figure. Each point represents a biological replicate. Statistical test: Wilcoxon rank sum test. P-value: n.s. not significant, * < 0.05, **** < 0.0001. (C) Representative images of immunofluorescence analysis or 5’EU incorporation in HeLa cells treated with DMSO or 30 μM Isoginkgetin for 1h to 6h. EU (green), DAPI (blue), scale bars: 50 μm. (D) Quantification of 5’EU intensity per nucleus for DMSO and Isoginkgetin. Boxplot settings are: min to max values with the box showing 25–75 percentile range. 8,466 nuclei were quantified per condition. Statistical test: Kruskal-Wallis test. P-value: **** < 0.0001. (E) qRT-PCR with primers amplifying a region from the pol I transcribed pre-rRNA. HeLa cells were treated with DMSO or 30 μM Isoginkgetin for 1h to 6h. cDNA was generated with random hexamers. Values are normalised to the 7SK snRNA and shown as relative to DMSO, mean ± SEM, n = 3 biological replicates. Statistical test: two-tailed unpaired t-test. P-value: n* < 0.05.