Fig 2: Expression and distribution of mRNP granule proteins in isolated skeletal muscle fibers. A Schematic of an isolated myofiber (MF) depicting myonuclei (MN) and an associated muscle satellite cell (MuSC). The longitudinal striations represent orientation of the myofibrils while the cross-striations represent the A-band (A) and Z line (Z). B–D, B’–D’ Depict magnified views of the regions enclosed by brackets (dotted lines) in B–D to visualize subcellular distribution of Fmrp. Arrow heads indicate cytoplasm, double arrows indicate nucleus. Fmrp puncta are observed both in nucleus and cytoplasm of MuSC (B, B’) and as cross-striated staining in myofiber. Puncta also accumulate in a cytoplasmic domain adjacent to the MN, while MN is itself not stained (C, C’). Nuclear accumulation of Fmrp is also seen in the Pax7+ MuSC nucleus (D, D’) but not in an adjacent Pax7- MN. B’, C’, and D’ represent single-channel (488) images. E–G Distribution of Fmrp puncta (green) in myofiber in a cross-striated pattern congruent with Z lines revealed by a-actinin (red). Arrows in G point to Fmrp puncta co-localizing with a-actinin striations. H–I. Secondary antibody controls (mouse and rabbit) do not show either punctate or striated background. K Distinct GW182 bodies are visible in Pax7+ MuSC. Pax7- MN also show distinct perinuclear puncta (arrowheads) and significant punctate staining is observed in MF cytoplasm in a doublet striated pattern likely reflecting A-band localization. K’ Region within brackets in k magnified to show GW182 puncta in the MuSC nucleus (double arrow). L, L’ No enrichment (either nuclear or cytoplasmic) is detected of Dcp1a in MuSC nucleus (marked with the membrane marker Caveolin 1). Faint fibrillar puncta are observed in myofibers. M, M’ Xrn1 is faintly detected in MuSC, but strongly expressed in myofibres in both a longitudinal and cross-striated pattern. K’, L’, and M’ represent single channel (green) images of enlarged areas indicated by brackets in K, L, and M, respectively

Fig 3: pSer25AnxA2 partially colocalises with the P-body marker GW182. PC12 cells were double-stained for immunofluorescence using mono- and polyclonal antibodies against GW182 (green) and pSer25AnxA2 (red) respectively. The insets in the merged confocal image to the left – including DAPI staining (blue) to highlight the nuclei – show higher magnifications of the regions, denoted in 3 and 4, to illustrate the partial colocalisation of pSer25AnxA2 and GW182. Scale bar: 10 µm. The fluorescence intensity profiles (from left to right) of the two proteins correspond to the cross-sections, denoted 1 and 2, shown in the insert in the upper right corner of the merged image.

Fig 4: HOIP and HOIL-1L co-localize with miRISC.a, b AGO2/TNRC6A interacted with LUBAC. HeLa cells were lysed for co-IP with anti-HOIL-1L (a) or anti-TNRC6A (b) antibody, and then the interactions between AGO2/TNRC6A with HOIP and HOIL-1L were detected by WB with indicated antibodies. c, d LUBAC was co-localized with AGO2/TNRC6C of miRISC. GFP-AGO2 and HA-TNRC6C were co-transfected with mCherry-HOIP (c) or mCherry-HOIL-1L (d) into HeLa cells, the co-localization of GFP-AGO2 (green), TNRC6C (purple) with mCherry-HOIP (red) or mCherry-HOIL-1L (red) were observed by immunofluorescence staining. e–h Hypoxia increased the co-localization of HOIP and HOIL-1L to AGO2/TNRC6A miRISC. GFP-AGO2 was transfected into HeLa cells, the co-localization of GFP-AGO2 (green), TNRC6A (purple) with HOIP (red) or HOIL-1L (red) were observed by immunofluorescence staining. The scale of the magnified region (white frame) is 2 µm x 2 µm (e, g). Co-localization between GFP-AGO2/TNRC6A with HOIP or HOIL-1L were analyzed by Pearson’s correlation coefficient. Data were mean ± s.d., and P-values were determined by unpaired two-sided t-test (f, h).

Fig 5: Translation initiation factors are co-expressed or adjacent to OPP foci in differentiating MCCs. All panels: confocal images of a single MCC in ALI day 4 culture. Diagram (right) summarizing the findings. (a, b) Translation initiation factors (EIF3B and p-RPS6) overlap with smaller OPP apical foci (I, shown to be in TNRC6A-enriched granules in Fig. 5). Phalloidin-stained cell membrane (blue) depicts apical MCC region. (c, d) EIF3B and p-RPS6 proteins are found surrounding the large OPP apical foci (II, shown to be in fibrous granules in Fig. 5). High magnification panels on the right are enlarged view of the boxed areas.