Fig 2: The five host factors affect the abundance of multiple HBV molecules(A) Kinetics of the decrease in extracellular HBsAg produced by HepG2.2.15 cells transfected with siRNAs. The amount of HBsAg produced was measured in 12-h intervals after siRNA transfection and normalized to the level produced by mock-treated cells. The siRNAs were transfected at a final concentration of 10 nM. REP 2139 was at 20 nM. (B) siRNAs that knock down the five host factors reduced the levels of intracellular HBsAg. The western blot image contains the signal for the large S antigen. The LC is of the ß-actin protein. The knockdown of the host factors is shown in Figure S3. (C) The five host factor siRNAs can reduce the HBV polymerase levels. The western blot image shows the amounts of the HBV polymerase identified by a monoclonal antibody specific to the HBV polymerase. The L.C. shows the amount of ß-actin protein. Additional western blots of cellular proteins are shown in Figure S6. HepG2.2.15 cells were transfected with a final concentration of10 nM siRNA, 5 nM ALG-20002, 8 nM ALG-10000, or 20 nM REP 2139. (D) The host factors have differential effects on the amounts of HBsAg, HBeAg, and core. ALG-10000 was transfected into HepG2.2.15 cells at 8 nM and the siRNAs at 10 nM. (E) Total HBV RNAs are modestly reduced by the knockdown of SRSF1 and HNRNPA2B1, but not by knockdown of RPLP1 or RPLP2. The cells were harvested 76 h after transfection. The amount of HBV RNA was normalized to the amount of GAPDH in the cells. In the graphs on panel A and D, each error bar represents one standard deviation of uncertainty.

Fig 3: Pool of the TAO2–SRSF1 complex is localized at the periphery of nuclear speckles. (A) A549 cells grown on coverslips were fixed and subjected to PLAs with the indicated primary antibodies. (Scale bars, 5 µm.) (B) PLA signals were quantified in 100 cells for each condition. Values are means ± SD. n = 3. (C and D) A549 cells were subjected to PLA to detect the TAO2–SRSF1 complex followed by immunofluorescence microscopy to stain nuclear speckles with an antibody against SC35. n = 3. (C) Two-dimensional images of PLA signals merged with nuclear speckles and DNA. (Scale bars, 2 µm.) (D) Three-dimensional projection of PLA signals and nuclear speckles. (I) Low magnification of the maximum-intensity projection (MIP) is shown. PLA signal of the TAO2–SRSF1 complex (red), nuclear speckles (SC35, green), and chromatin (blue). (II and III) High magnification of the MIP surfaces is shown for PLA signal of the TAO2–SRSF1 complex (red) and nuclear speckles are stained in green. (Scale bars, 2 µm.) (IV) Pairwise merged surface created for PLA signal of the TAO2–SRSF1 complex (red) and nuclear speckles (green) is shown. (V–VII) Views of a chromatin merged surface created for (V) PLA signal of the TAO2–SRSF1 complex (red), (VI) nuclear speckles (green), and (VII) PLA signal of TAO2–SRSF1 (red) and nuclear speckles (green) are shown. (Scale bars, 2 µm.) The squared marked area in VII is enlarged and shown in the view of 3D isometric MIP-rendering volume (Top) or 3D isometric blend-rendering volume (Bottom). Both 3D rendered volumes are presented in 90° alternative views to provide visual and spatial emphasis for the colocalization (yellow) of the TAO2–SRSF1 complex (red) and nuclear speckles (green). To calculate the percentage of the TAO2–SRSF1 complex colocalization with nuclear speckles, 156 cells were analyzed and 42% of the PLA signal was found at nuclear speckles.

Fig 4: Srsf1 depletion inhibits growth of human colorectal cancer-derived organoids.a A sample of cores taken from a human colon cancer tissue microarray (TMA), with the same respective core stained for SRSF1 or PROX1 on different sections. Scale bar 500 µm. b Linear regression analysis showing the correlation of SRSF1 and PROX1 staining (based on histoscore) on the TMA shown in a, with each datapoint representing a core taken from a patient. c Relationship between SRSF1 immunohistochemistry staining and tumour stage in human patients, using TMA CO2081b, n = 54 vs 56 biologically independent tumour cores. d Representative images of patient-derived organoids (PDOs) treated with control or Srsf1 shRNAs. MD175 (polyp), MD20853 (CRC), MD20910 (CRC), MD19648 (FAP rectum Tumour), MD20043 (rectal carcinoma), C-002 (liver metastasis). Scale bar 1000 µm. e Number of surviving organoid clones after shRNA treatment and f size of indicated PDOs, n = 3 vs 3 independent experiments. g Viability of PDO MD20043 after Srsf1 shRNA treatment and h mRNA expression (qPCR) of indicated genes, n = 3 vs 3 independent experiments. i Model outlining the role of SRSF1 in modulating tumour cell plasticity and invasion in colorectal cancer. Data in bar charts are represented as mean and error bars are SD with data analysed with two-tailed, unpaired t-tests, p values are indicated in figure panels. All biological replicates are shown as individual value plots and n > 3. See also Figure S7.

Fig 5: Wnt-induced SRSF1 levels enforce splicing dysregulation.a Alternative splicing events in Apc;fl/fl Srsf1fl/+ compared to Apcfl/fl in small intestine isolated 5 days postinduction. b Gene set enrichment analysis showing the intersecting alternative splicing events discovered in the wild-type versus Apc;fl/fl Srsf1fl/+ dataset compared to the Apcfl/fl versus Apc;fl/fl Srsf1fl/+ dataset. c RT-PCR validation of a selection of alternative splicing events and d quantification, n = 3 vs 3 biologically independent mice. e Images of wild-type, Apcfl/fl and KPN organoids treated with deltarasin or control vehicle DMSO. Scale bar 500 µm. f Relative cell viability following deltarasin treatment in wild-type, Apcfl/fl and KPN organoids, n = 3 vs 3 (Wt and Apcfl/fl) and 3 vs 4 (KPN) independent experiments. g Images showing Apcfl/fl organoids following treatment with a doxycycline (dox) inducible Srsrf1 shRNA + /- dox, and with GFP or Kras4b-GFP overexpression. RFP is co-expressed with the shRNA following addition of dox. Scale bar 500 µm. h Quantification of organoid size under the stated conditions, n = 3 vs 3 vs 3 vs 3 independent experiments. All images are representative. Data in bar charts are represented as mean and error bars are SD with data analysed with two-tailed, unpaired t-tests, p values are indicated in figure panels. All biological replicates are shown as individual value plots. See also Fig. S5.