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Fig. 2 | BMC Biology

Fig. 2

From: The ESCRT-0 subcomplex component Hrs/Hgs is a master regulator of myogenesis via modulation of signaling and degradation pathways

Fig. 2

Significant perinuclear colocalization of Hrs with Rab5a and Clathrin heavy chain (CHC) in differentiated myotubes. a Representative immunofluorescence images of Hrs distribution in C2C12 in Pro or during differentiation at the indicated time points (in hours). Cells were stained with the anti-HRS (green) and DAPI (blue) for nuclear staining. White dotted squares represent a focus of the selected region (lower panel). White arrowheads/arrows show cytoplasmic/perinuclear Hrs distribution respectively during the course of myogenesis. b Representative immunofluorescence images of C2C12 transfected with the human GFP-HRS (huHRS-GFP) encoding construct in Pro or at 144 h of differentiation. huHRS-GFP signal (green) and DAPI (blue). White dotted squares represent a focus of the selected region (lower panel). c Representative immunofluorescence images of Hrs distribution in primary murine muscle cells in Pro or at 24 and 48 h of differentiation. Cells were stained with anti-HRS (green) and DAPI (blue). White dotted squares represent a focus of the selected region (lower panel). d,e Representative immunofluorescence images of dissociated EDL muscle fibers stained with anti-HRS (green) and DAPI (blue). White dotted squares represent a focus of the selected region (right panel). Perinuclear Hrs (white arrows). f Representative immunofluorescence images of Hrs distribution in C2C12 at 144 h of differentiation. Cells were probed with the anti-HRS (green) and the anti-Rab5a for EE or anti-CHC; anti-Rab7; -KDEL and -GM130 for LE; ER or Golgi respectively (in red). Note the accumulation of Hrs around and at the poles of nuclei. White dotted squares represent a focus of the selected regions (see insets). g Quantifications of colocalization from experiments like in f have been made by creating binary mask between merge channel of Hrs and Rab5a, CHC, Rab7, KDEL, or GM130 signals. Quantification of the signals obtained around nuclei is presented as plot-graphs where one dot corresponds to the signal for one nucleus. Data represent mean +/− SD, n = 33–45 nuclei for each group. n = 3. Significance was assessed using a Mann–Whitney U test (****p < 0.0001). Scale bars, 20 μm

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