Fig 1: The MEK1/2/ERK1/2 pathway is upregulated into Hrs-depleted cells and its inhibition rescues formation of myotubes. a Representative Western blotting of protein extracts from shCT and shHrs#3 C2C12 collected in Pro (lanes 1 and 7) and at 7, 24, 48, 72, and 96 h of differentiation (lanes 2–6 and 8–12) and probed with anti-HRS, -MHC, -pT202/Y204-ERK1/2, and -total-ERK1/2 antibodies. GAPDH was used as a loading control. b Quantification of the phosphorylated-ERK1/2 from similar experiments presented in a. The data are presented as a ratio of pERK1/ERK1 and pERK2/ERK2 and normalized to the Pro starting point condition. Data represent mean +/- SEM, n = 3 experiments. Significance was assessed using a two-way ANOVA test; *p < 0.05; **p < 0.01; ***p < 0.001. ns not significant. c Representative immunofluorescence images of shCT and shHrs#3 transduced C2C12 at 96 h of differentiation upon MEK1/2 inhibitor U0126 (10 µM) or DMSO treatment and stained with anti-MHC antibody as a marker of myotubes (green) and DAPI (red). Scale bar, 200 µm. d Quantification of the myogenic index corresponding to the number of nuclei present in MHC-positive structures in experiments as presented in c. Data are mean +/- SEM, each dot represents one field, n = 27 images (n = 3 experiments). Significance was assessed using a Mann–Whitney U test; *p < 0.05; ****p < 0.0001; ns not significant. e Representative Western blotting of shCT and shHrs#3 cell extracts collected at 3 days of differentiation in presence of DMSO or 10 µM of U0126 and probed with anti-pT202/Y204-ERK1/2 and -total-ERK1/2 antibodies. GAPDH was used as a loading control. f Representative immunofluorescence images of shCT and shHrs#3 transduced C2C12 at 72 h of differentiation upon treatment with either 10 µM of the U0126 MEK1/2 inhibitor or DMSO and stained with the anti-myogenin (red) and DAPI (blue) for nuclear staining. Scale bar, 40 µm. g Quantification of the myogenin-positive nuclei (%). Data represent mean +/- SEM, 10 fields have been counted per experiments, n = 2 independent experiments
Fig 2: Hgs and ALIX are recruited to the ApV and benefit A. phagocytophilum replication and infectious progeny production. (A) and (B) Hgs and ALIX colocalize with the ApV membrane-localized effector, APH0032. RF/6A cells were infected with A. phagocytophilum for 24 h, after which they were fixed and immunolabeled with antibodies against Hgs (A), ALIX (B), and A. phagocytophilum APH0032 (0032) (A) and (B). The cells were stained with DAPI and examined using LSCM and DIC microscopy. The graphs by each set of micrographs represent the relative signal intensity profiles of green and red pixels along the yellow line (moving left to right) normalized to the highest fluorescence intensity per channel. Black arrows demarcate the ApV membrane. Scale bars, 25 μm. (C) to (K) Knockdown of both Hgs and ALIX hinders A. phagocytophilum proliferation and infectious progeny generation. RF/6A cells were treated with siRNA targeting Hgs (siHgs) (C) to (E), ALIX (siALIX) (F) to (H), both Hgs and ALIX (siH+A) (I) to (K), or non-targeting siRNA (siNT) (C) to (K). At 48 h, one set of each condition was collected to confirm knockdown of the protein of interest (KD check). The other two sets were either mock infected (Uninf.) or incubated with A. phagocytophilum DC organisms for 48 h (48 hpi). All samples were examined by Western blotting using antibodies specific for Hgs, ALIX, P44, and GAPDH. The A. phagocytophilum load was quantified as the mean (±SD) normalized ratio of P44:GAPDH densitometric signals from triplicate samples per condition. To assess for infectious progeny production and release, media from siNT- or target-specific siRNA-treated cells that had been infected with A. phagocytophilum was collected at 48 h postinfection and added to naive RF/6A cells that had not been treated with siRNA. This time point corresponds to when infectious DC bacteria would be present in the media if the bacterial developmental cycle had proceeded normally. Twenty-four h later, recipient cells were assessed for Hgs, ALIX, P44, and GAPDH levels and the A. phagocytophilum load (Progeny). Statistically significant (**, P < 0.01; ***, P < 0.001) are indicated. Data shown are representative of three independent experiments.
Fig 3: EGFR is accumulated in Hrs-depleted cells and its activation partially correlates with induction of the MEK1/2/ERK1/2-pathway. a Left panel: representative immunofluorescence images of pulse-chase experiments: shCT and shHrs#3 C2C12 were stimulated for 5 min with 50 ng/mL of EGF-488 (green) and after removing unbound ligand-chased for the indicated amount of time. Cells were probed with the anti-EEA1 to visualize EE and trafficking of EGF-488/EGFR was followed through the EE pathway. White arrows indicate colocalization of EGF-488 (in green) with the EEA1 EE marker (red) and DAPI (blue). Note the accumulation of EGF-488 in EEA1-positive compartments (yellow merge) in shHrs#3-depleted cells. Right panel: mask of colocalization between EGF-488- and EEA1-positive compartments. Scale bars, 20 µm. b Quantifications of colocalization of EGF-488 in EEA1 compartments. Analysis shows the percentage of EGF-488 overlapping with EEA1 and establishes the endocytic trafficking of EGF ligand through the EE pathway (EEA1 compartment). Open circles correspond to shCT and black squares to shHrs#3 conditions. The data represent mean +/- SEM. Analyses were done on 5 fields per condition on n = 3 experiments. Significance was assessed using a Mann–Whitney U test; ***p < 0.001; **p < 0.005. ns not significant. c Degradation of EGFR. shCT and shHrs#3 cells were stimulated with 50 ng/mL of EGF and with 10 µg/mL of cycloheximide to inhibit de novo synthesis of EGFR. Cells were recovered 15 (lanes 1,4), 60 (lanes 2,5), and 120 (lanes 3,6) min after stimulation and cells extracts were analyzed by Western blotting using anti-EGFR, -pY1068-EGFR, -ERK1/2, and -pT202/Y204ERK1/2. GAPDH was used as a loading control. d Quantifications of EGFR (left panel) and pY1068-EGFR (right panel) protein levels. Data are presented as the ratio of EGFR/GAPDH and pY1068-EGFR/GAPDH. Data are mean +/- SEM, n = 3–4 experiments. Significance was assessed using a two-way ANOVA test; **p < 0.01, ****p < 0.0001; ns not significant. e Representative Western blotting of protein extracts from shCT and shHrs#3 C2C12 collected in Pro (lanes 1 and 7) and at 7, 24, 48, 72, and 96 h of differentiation (lanes 2–6 and 8–12) and probed with anti-MHC, -EGFR, -pY1068-EGFR, and -GAPDH. f Representative immunofluorescence images of shCT and shHrs#3 C2C12 at 24 h of differentiation and probed with anti-pY1068-EGFR (green), -Rab5a (red) for EE and DAPI (blue). White arrows showed the pY1068-EGFR signal associated with Rab5a vesicles. Scale bar, 10 µm
Fig 4: Hrs is dynamically expressed and distributed during myogenesis in C2C12 and primary muscle cells. a Representative Western blotting of myoblast C2C12 cell extracts in Pro status (lane 1) and at 7, 24, 48, 72, and 96 h of differentiation (lanes 2–6) and probed with anti-MHC, -Myogenin as makers of differentiation, and anti-HRS antibody. b Quantification of the Hrs protein level from experiments as presented in a. Data are presented as ratio of Hrs/GAPDH and normalized to the Pro status starting point. Data represent mean +/- SEM. n = 8 experiments. Significance was assessed using a Kruskal–Wallis test; **p < 0.01; ***p < 0.001. c qRT-PCR of Hrs-mRNA from C2C12 cells collected in Pro and at 7, 24, 48, 72, and 96 h of differentiation. Data represent mean +/- SEM, n = 3 experiments. Significance was assessed using a Kruskal–Wallis test; ns not significant. d Representative Western blotting of C2C12 in Pro or at 16 h of differentiation treated with DMSO or 200 nM MG132 proteasome inhibitor. Membrane was probed with anti-HRS, -ubiquitin. GAPDH was used as loading control
Fig 5: Hrs silencing inhibits myoblast to myotube differentiation. a Representative Western blotting of C2C12 transduced with HIV-1 lentivectors shCT (lane 1) or shHrs-mRNA shHrs#1 (lane 2), #2 (lane 3), #3 (lane 4) and probed with the anti-HRS and anti-GAPDH. b Quantification of shCT and shHRS#3 C2C12 cell number at 6, 24, or 72 h. Data represent mean +/- SEM, n = 3 experiments. Significance was assessed using two-tailed Mann–Whitney U test. ns not significant. c Representative immunofluorescence images of shCT and shHrs#3 C2C12 myoblasts and probed with anti-Lamp1 (green) for LE/LYS or anti-EEA1 (red) for EE and DAPI (blue). White dotted squares represent a focus of the selected regions (see insets). Scale bar, 20 µm. d Representative immunofluorescence images of shCT and shHrs#1-3 C2C12 at 96 h of differentiation and probed with the anti-MHC (green) as a marker of myotubes and DAPI (red) for nuclear staining. Scale bar, 200 µm. e Representative Western blotting of shCT and shHrs#3 C2C12 myoblasts at the Pro status (lanes 1,7) or during the differentiation 7–96 h (lanes 2–6 and 8–12) and probed with the anti-HRS and -MHC. GAPDH was used as a loading control. f Quantification of the MHC protein level present in experiments like in e. Data are presented as ratio of MHC/GAPDH and normalized to the Pro starting point condition. Data represent mean +/- SEM, n = 3 experiments. Significance was assessed using a two-way ANOVA test; *p < 0.05, **p < 0.01. g Quantification of the myogenic index corresponding to the number of nuclei present in MHC-positive cells as shown in d. Data represent mean +/- SEM, each dot represents one image, n = 24 images from two independent experiments. h Representative Western blotting of primary muscle cells transduced with the lentivector shCT (lane 1) or shHrs#3 (lane 2) and probed with the anti-HRS. GAPDH was used as a loading control. i Representative immunofluorescence images of shCT and shHrs#3 primary muscle cells at 48 h of differentiation and probed with the anti-MHC (green) and DAPI (red). Scale bar, 200 µm. j Quantification of the myogenic index corresponding to the number of nuclei present in MHC-positive cells as shown in i. Data represent mean +/- SEM, each dot represents one image, n = 16 images from two independent experiments
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