Fig 1: Activation-induced DHX36 is required for muscle formation.a Schematic for isolation of quiescent satellite cells (SCT0) after in situ fixation, freshly isolated SCs without prior fixation (SCT8) and SCs cultured for 24, 48, or 72 h. b RNAs from the above cells were subject to RNA-seq and FPKM of Dhx36 mRNA is plotted. c DHX36 protein in the above SCs were examined by western blot. The relative intensity of each band is calculated by normalizing with total proteins stained by Ponceau S. The intensity for SCT8 was set as 1. d Mouse TA muscles were injected with barium chloride (BaCl2) and the muscle homogenates were collected at the designated dpi for qRT-PCR measurement of Dhx36 mRNAs. n = 3 independent experiments; P = 0.0000097 and 0.000015. e Immunofluorescence staining of DHX36 protein in SCT0, SCT8 or SC 24 h. Scale bar: 2 μm. f Breeding strategy to generate Dhx36 cKO mouse. g H&E staining of TA muscle from adult Ctrl or Dhx36 cKO mice. Scale bar = 100 μm (×20 images) or 50 μm (×40 images). h Average fiber size in the above muscles was measured, Ctrl: n = 3 mice; cKO: n = 4 mice; P = 0.003. i Cross-section area (CSA) of each fiber from the above muscles (Ctrl in white and cKO in gray) was quantified and the distribution is shown. From left to right, P = 0.014, 0.027, 0.000013, 0.008, 0.003, 0.026, 0.007. j Grip strength of fore and hindlimb muscles were measured in Ctrl and cKO mice, Ctrl: n = 8 mice; cKO: n = 7 mice; P = 0.023. k Immunohistochemistry staining of eMyHC was performed on sections of TA muscles from Ctrl and cKO mice on 7 dpi, scale bar = 100 μm. l The average fiber size of newly formed fibers was quantified from the above eMyHC+ fibers, Ctrl: n = 3 mice; cKO: n = 4 mice; P = 0.022. m Distribution of CSA of the above fibers (Ctrl in white and cKO in gray). P = 0.011 and 0.033. Data are represented as mean ± s.d., Student’s t test (two-tailed unpaired) was used to calculate the statistical significance (d, h, i, j, l, m): *P < 0.05, **P < 0.01, ***P < 0.001. Source data are provided as a Source Data file.
Fig 2: DHX36 and DHX9 mediate translation of selected cancer genes. a Gene ontology classification for genes, which TE decreases (P < 0.05) upon depletion of DHX36 (red) and DHX9 (green). b Immunoblots of lysates from HeLa cells depleted in DHX36 and DHX9 and probed as indicated. Immunoblots were performed 96 h after siRNA transfection. c Diagram showing a DHX36 / DHX9-dependent mechanism of translational control. (1) Scanning 43S PICs that translate unstructured 5′-UTRs or rG4-containning 5′-UTRs, that are maintained in their unfolded state by the DHX36 and DHX9 helicases, initiate translation at the main ORF (mORF). (2) A fraction of scanning PICs may initiate translation at upstream start codons, present within 5′-UTR in a suboptimal context, affecting the efficiency of the mORF translation. (3) In the absence of the rG4 processing helicases, rG4 motifs folding may slow down PIC scanning, thereby providing more time for the recognition of the upstream start codon and stimulating the translation of the upstream open reading frame (uORF). (4) 80S ribosomes may either dissociate from the mRNA after termination or stall during elongation or termination by the uORF-encoded attenuator peptide, preventing the translation of the mORF. d Schematic of bicistronic reporter genes containing within their 5′-UTRs either an rG4 motif (∆ uORF + rG4), a mutated rG4 (∆ uORF + rG4 mut), an rG4-containing uORF (uORF + rG4) or an rG4-mutated uORF (uORF + rG4 mut). e Relative translation of the different expression vectors showing that an rG4 enhances the repressive effect of a short uORF. f Effect of DHX36 (red) and DHX9 (green) depletion on the relative translation of reporter genes containing within their 5′-UTRs either an rG4-containing uORF or an rG4-mutated uORF as compared to control (non-targeting siRNAs, gray). Data represent the mean and s.d., n = 3 biological replicates. P-values were calculated using an unpaired student’s t-test. ns non-significant, **P < 0.01, ***P < 0.001. Representative flow cytometry profiles are reported in Additional file 1: Figure S15
Fig 3: DHX36 knockdown sensitizes cells to G‐quadruplex‐stabilizing compounds. IMR90 cells expressing shDHX36 or control cells harboring shVector were treated with multiple concentrations of PDS or Phen‐DC3 for 2 days. (a) Western blotting with the indicated antibodies for whole‐cell extracts. (b) Cell viability was determined by measuring the dehydrogenase activity in living cells after PDS (left) or Phen‐DC3 (right) treatment. (c) Dead cells were stained with trypan blue after PDS treatment. (d) The reintroduction of DHX36 rescued the elevated sensitivity against PDS in DHX36‐depleted cells. The same assay as in (b) was performed after PDS treatment. Indicated sh‐insensitive DHX36 variants were expressed under viral promoters using a pMXs vector. AUC was calculated using the cell survival curve shown in the left panel. In all figures, values represent mean ± SEM of data from three experiments. Statistical significance was calculated using one‐way ANOVA with Tukey's correction for multiple comparisons. ***p < .001, *p < .05, ns = not significant.
Fig 4: Dhx36 inactivation in adult SCs impairs muscle regeneration.a Schematic illustration of the strategy to inactivate Dhx36 in inducible knockout (iKO) mice. Dhx36fl/fl mice were mated with Pax7CreER; ROSAEYFP mice; the exon 8 of Dhx36 was deleted in the iKO mice after Tamoxifen (Tmx) injection which also resulted in the removal of the stop signal for YFP at the Rosa26 site to allow the expression of YFP in iKO SCs. b Schematic illustration of five doses of Tmx injection to delete Dhx36 in the iKO mouse. Freshly isolated SCs were collected at day 6 post-injection and cultured for 2 days. c Left: no obvious morphological difference was observed in representative Ctrl vs iKO mice. Right: loss of DHX36 protein was confirmed by western blot with α-Tubulin as the loading control. d Upper panel: schematic illustration of the injury-induced muscle regeneration scheme. BaCl2 was injected into TA muscles of the above Ctrl or iKO mice 6 days post-Tmx injection to induce acute injury. The injected TA muscles were harvested at the designated times for the assessment of the regeneration process. Lower panel: H&E staining of the TA muscles collected at 2.5, 5, 7, and 28 days post injury. Scale bar = 100 μm (×20) or 50 μm (×40). e IF staining of eMyHC (red) and laminin (green) was performed on the TA muscles collected at 5 and 7 days post BaCl2 injury. Nuclei were visualized by DAPI staining (blue). Scale bar = 50 μm. f CSAs of newly formed fibers were quantified from the above-stained sections and the distribution is shown, Ctrl in white bars and iKO in gray bars; n = 3 mice per group. <100 μm, P = 0.013; 100–200 μm, P = 0.038; 200–300 μm, P = 0.016; 500–600 μm, P = 0.009; 600–700 μm, P = 0.036. g Left: Representative images of TA muscles collected at 28 days post injury are shown. Right: The muscle weight from three pairs of mice, P = 0.031. Data are represented as mean ± s.d., Student’s t test (two-tailed unpaired) was used to calculate the statistical significance (f, g): *P < 0.05, **P < 0.01. Source data are provided as a Source Data file.
Fig 5: The protein levels of DNA sensors in human CRC tissues. Protein was extracted from cancer and matched peri-carcinomatous tissues of CRC patients, and then the levels of DNA sensors including STING, IFI16, DAI, DDX41, DHX9, DHX36 and DDX60 were determined by Western-blot. (A) The representative Western-blot results. The triangles point to the specific bands of the molecules. Densitometric analysis of band intensity of STING (B), IFI16(C), DAI(D), DDX41(E), DHX9(F), DHX36(G) and DDX60(H) was shown. Control: matched peri-carcinomatous tissues, CRC: colorectal cancer tissues, n=12. *p < 0.05; **p < 0.01; NS: Not significantly different.
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