Isoginkgetin and Madrasin are poor splicing inhibitors
Fig 1
Isoginkgetin and Madrasin are poor splicing inhibitors.
(A) Chemical structure of Madrasin and Isoginkgetin. (B) RT-PCR with primers amplifying the intron located between exons 2 and 3 of DNAJB1 or exons 4 and 5 of BRD2. HeLa cells were treated with DMSO, 1 μM PlaB, 1 μM HB, 30 μM Isoginkgetin, or 90 μM Madrasin for 1h. The location of the spliced and unspliced RNA is shown on the left of the panel. The percentages of unspliced RNA compared to total (spliced + unspliced) are shown below. (C) Representative images of immunofluorescence analysis or 5’EU incorporation in HeLa cells treated with DMSO, 100 μM DRB, a CDK9 inhibitor, 1 μM PlaB, 1 μM HB, 30 μM Isoginkgetin, or 90 μM Madrasin for 1h. EU (green), DAPI (blue), scale bars: 50 μm. (D) Quantification of 5’EU intensity per nucleus for DMSO, DRB, PlaB, HB, Isoginkgetin, and Madrasin. Boxplot settings are: min to max values with the box showing 25–75 percentile range. > 10,000 nuclei were quantified per condition. Statistical test: Kruskal-Wallis test. P-value: n.s. not significant, **** < 0.0001. (E) Western blots of total pol II, SF3B1, α-tubulin (loading control), and histone H3 (loading control) from chromatin, nucleoplasm, and cytoplasm fractions of HeLa cells treated with DMSO, 1 μM PlaB, 1 μM HB, 30 μM Isoginkgetin, or 90 μM Madrasin for 1h.