Fig 2: Inhibition of miR-21 and WIP1 sensitizes HER2+ breast cancer cells to the treatment of trastuzumab. a, b Parental or trastuzumab-resistant HER2+ breast cancer cells (HER18 or BT-474) were incubated with trastuzumab at the indicated concentrations for 72 h. The cell viability was then measured and these results are presented as % vehicle ± SD. The cell lysates were subjected to western blot analyses with the indicated antibodies (right panels). c, d HER18R (c) or BT-474R (d) cells with or without Dox-induced miR-21 knockdown were incubated with indicated concentrations of GSK2830371 for 72 h. The cell viability was then measured and these results are presented as % vehicle ± SD. The cell lysates were subjected to western blot analyses with the indicated antibodies (right panels). e HER18R cells with or without Dox-induced miR-21 knockdown were incubated with GSK2830371 (0.2 µM) and/or trastuzumab (1 µg ml−1) for 72 h. The cell viability was then measured and these results are presented as % vehicle ± SD. f, g Gross tumor images (f) and tumor growth curves (g) of xenograft tumors derived from orthotopically implanted parental or trastuzumab-resistant HER18 cells expressing Dox-inducible WIP1 or DDX5 shRNA or anti-miR21 oligonucleotides with trastuzumab treatment (5 mg kg−1, twice per week). *p < 0.05; **p < 0.01; ***p < 0.001; unpaired 2-tailed t-test (c–e) and one-way ANOVA followed by Tukey’s t test (g) were used. Data are presented as mean ± SD and are representative of 3 independent experiments in a–e

Fig 3: RNA helicases DDX5 and DDX17 stabilize long-term chromatin loops.(a) MS/ChIP of dimerized CLOuD9 cells after 72 h and 10 day treatments demonstrate differential enrichment of novel proteins at the induced looping loci after 10 days. (b) Immunoprecipitation of CLOuD9 complexes demonstrates that CTCF and cohesin were not found to be localized to the induced loops. (c) shRNA knockdown of DDX5 and DDX17 in K562s containing CLOuD9 constructs. (d,e) 3C demonstrates that while DDX5 and DDX17 knockdown do not affect induction of β-globin-LCR contacts, the induced chromatin loops are no longer stabilized following 10 days of dimerization and subsequent washout. 3C values were normalized to tubulin, and interaction frequencies between the anchor fragment and the fragment encompassing the β/HS fragment were set to zero. Error bars indicate s.d. n=3. (f) Endogenous β-globin expression is restored following short- and long-term dimerization and subsequent ligand washout in DDX5 and DDX17 knockdown cells. Significance given relative to control treated cells. Two-tailed student’s t-tests *P<0.05, t=2.538, df=6; **P<0.001, t=3.791, df=6; ***P<0.0001, shDDX5 t=12.6, df=6, shDDX17 t=19.35, df=6; n.s. non-significant. All error bars indicate s.d.

Fig 4: SARS-CoV-2 N binds to host cellular RNA helicases. (A) Vero E6 TMPRSS2 cells (5 × 105 cells/well) were infected with SARS-CoV-2 at an MOI of 0.5. The cell lysates were collected at 24 h postinfection. (B) 293T cells (2 × 105 cells/well) were transfected with 4 μg of pcDNA3.1-SARS-CoV-2 N. The cell lysates were immunoprecipitated with anti-SARS-CoV-2 nucleocapsid (GTX632269 [6H3]), anti-DDX21 (A300-627A), anti-DDX6 (A300-460A), or anti-MOV10 (A301-571A) antibody, followed by immunoblotting analysis using anti-SARS-CoV-2 nucleocapsid, anti-DDX21, anti-DDX6, and anti-MOV10 antibodies, respectively. Both short-exposure and long-exposure images are shown. (C) 293T cells (2 × 106 cells in a 10-cm dish) were cotransfected with 10 μg of pcDNA3-HA-DDX1, pHA-DDX3, pcDNA3-HA-DDX5, pcDNA3-HA-DDX6, pcDNA3-HA-DDX21, or pcDNA3-HA-MOV10 and/or 15 μg of pcDNA3.1-SARS-CoV-2 N. The cell lysates were immunoprecipitated with anti-SARS-CoV-2 nucleocapsid (GTX632269 [6H3]) antibody, followed by immunoblotting analysis using anti-SARS-CoV-2 nucleocapsid and anti-HA (3F10) antibodies, respectively. WCL, whole-cell lysate; IP, immunoprecipitation.

Fig 5: DDX3X affects the stability of 3'-tracer tRNAs in vitro and in vivo. (A) Western blotting for DDX3X, DDX5 and eIF2a (serine-51 phospho) before and 48 h after transfection with siRNA pools (non-targeting as controls versus targeting each RNA helicase alone or in combination). Antibodies against vinculin were used as a loading control. Lane marked with (*) points at background eIF2a-P signal, while lanes marked with (**) indicate siRNA-mediated induction of eIF2a-P signal in RNA helicase knockdowns. (B) 3'-tracer tRNAs (8 nM final) were incubated with cytoplasmic protein extracts (2 or 4 µg) obtained from HeLa cells transfected with siRNAs targeting DDX3X or DDX3X in combination with DDX5. Reactions were separated by nPAGE and 3' tsRNA signals were collected as described above. Black arrowhead, 3'-tracer tRNAs; green arrowhead, 3' tsRNAs. (C) Western blotting for DDX3X in total protein extract obtained from BMDF after 72 h of 4-hydroxytamoxifen (4-OHT)-mediated deletion of the DDX3X gene locus. ß-actin was used as a loading control. (D) NB using sequential probing of the same membrane against the 5' and 3' portions of tRNA-GlyGCC/CCC on total RNA purified from BMDF after 72 h of 4-OHT-mediated deletion of the DDX3X gene locus before and after As[III] exposure (0.5 mM for 1 h). Black arrowheads, mature tRNAs; red arrowhead, 5' tsRNAs; green arrowhead, 3' tsRNAs. (E) 3'-tracer tRNAs (8 nM final) were incubated with cytoplasmic protein extracts (5 or 10 µg) obtained from BMDF before or after 4-OHT-mediated deletion of the DDX3X. Reactions were separated by nPAGE and 3' tsRNA signals were collected as described above. Black arrowhead, 3'-tracer tRNAs; green arrowhead, 3' tsRNAs; grey arrowhead, signals from partial 3' tsRNA degradation. (F) NB of total RNA purified from SEC fractions originating from As[III]-exposed BMDF before (upper panels), after 4-hydroxytamoxifen (4-OHT) mediated deletion of DDX3X (middle panels), and in the presence of ectopic expression of wild type DDX3X (pCMV-DDX3X, lower panels) using sequential probing of the same membrane. Probes for the 5' (left panels) and 3' (right panels) moieties of tRNA-GlyGCC/CCC were used. Black arrowheads, mature tRNAs; red arrowhead, 5' tsRNAs; green arrowhead, 3' tsRNAs.