Antibodies
Primary antibodies were as follows: The anti-HRS/HGS antibodies (ab155539; WB 1:1,000, IF 1:100,); -MEF2A (ab32866; IF 1:500), and -GSK3β (ab32391 WB 1:1,000 and IF 1:100) were from Abcam. The anti-TSG101 (sc-7964; WB 1:500) were from Santa Cruz Biotechnology. The anti-EEA1 (CST3288; IF 1:100); -phospho-ERK1/2 (Thr202, Tyr204) (CST3179; WB 1:2,000); -total-ERK1/2 rabbit (CST9102; WB 1:1,000); -LC3 (CST2775; WB 1:1,000 and IF 1:100), -GAPDH (CST2118; WB 1:10,000), -total-AKT2 (CST2964; WB1:1,000), -phospho-AKT2-Ser474 (CST85995; WB 1:1,000); -FOXO1 (CST2880; WB 1:1,000 and IF 1:100); -Ubiquitin (CST14049; WB 1:1,000); -IRS1 (CST2382; IF 1:100); Histone H3 (CST4499S; WB 1:5,000); Rab7 (CST95746; IF 1:100), Rab5 (CST46449; IF 1:100), and phosphor-EGFR-Tyr1068 (D7A5, CST3777S; WB 1:000; IF1:100) were from Cell Signaling Technology. The anti-SQSTM1/p62 (GP62-C; WB 1:4,000 and IF 1:200) was from Progen and the anti-myogenin (F5D; WB 1:200, IF1:100) and the anti-myosin heavy chain (MHC) antibody (MF20 hybridoma cell culture supernatant) were from Developmental Studies Hybridoma Bank (DSHB). The anti-clathrin heavy chain (610499; IF 1:100) and the anti-pY216GSK3β (612312; WB 1:1000) were from BD Biosciences. The anti-KDEL (NBP1-97469; IF 1:100) were from Novus Biologicals. The anti-MyoD (MA1-41017; WB 1:1000 and IF 1:100), -EGFR (PA1-1110; WB 1:1,000), -PTPN23 (PA5-76478; WB 1:1000) were from Thermo Fisher Scientific. The anti-GM130/GOLGA2 (NBP2-53420; IF 1:200) was from Bio-techne.
Secondary antibodies are as follows: For Western blotting, the anti-mouse (NA931V) and anti-rabbit (NA934V) labeled with peroxidase (GE Healthcare UK) were used at 1:10,000.
For immunofluorescence, the goat anti-mouse dylight-488 (115-486-072), goat anti-mouse dylight-549 (115-506-072), the donkey anti-mouse Alexa-Fluor 647 (715-606-151), and the donkey anti-rabbit rhodamine (TRITC) (711-025-152) were purchased from Jackson Lab. The donkey anti-rabbit Alexa-Fluor 488 (A21206) was purchased from Molecular probe. All these secondary antibodies were used at 1:1000. Antibodies for flow cytometry are as follows: CD34-FITC (#11-0341-82); Ly-6A/E (Sca-1)-PE (#12-5981-82); CD45-PE (#12-0451-82); and CD31 (PECAM-1)-PE (#12-0311-82) were purchased from Thermo Fisher Scientific and were used as previously described by the manufacturer.
Drugs and compounds
The inhibitor of MEK1/2-ERK1/2 U0126 (U120) and the GSK3β inhibitor 1-azakenpaullone (A3734) were used at 3 μM in DMSO and were purchased from Sigma-Aldrich. The cycloheximide (ref 239763-1GM) from Millipore was used at 10 μg/mL in DMSO.
Constructs
The psPAX2 plasmid is a 2nd generation lentiviral packaging construct encoding the HIV-1 Gag, GagPol, Tat, and Rev accessory proteins, and the pMDG2 plasmid encoding the vesicular stomatitis envelope glycoprotein (VSVg) was kindly provided by Didier Trono (EPFL). The pLKO1 constructs encoding small hairpin RNAs (shRNA) directed against murine Hrs (Hrs#1 TRCN0000313945; Hrs#2 TRCN0000314016 and Hrs#3TRCN0000088688), Tsg101 (Tsg101#1 TRCN0000054603; Tsg101#2TRCN0000054604, Tsg101#3 TRCN0000054607) were from Sigma-Aldrich (Mission shRNA plasmid DNA).
The HIV-psi-LVRU6GP encoding the shCT (CSHCTR001-1) or shPtpn23/HD-PTP#1 et #2 (MSH025913-1 and MSH025913-2 respectively) were kindly provided by Giampetro Schiavo and were previously described in [88].
The negative pLKO1 shRNA control (shCT) as well as the human HRS-shRNAs constructs (huHRS#1TRCN0000037894; huHRS#2 TRCN0000037895 and huHRS#3 TRCN0000037896) were kindly provided by Clotilde Thery and were previously described in [89].
The HIV-1 pLv-hTERT-CDK4 for human primary myoblast immortalization bearing the expression cassettes encoding the HTert and CDK4 proteins and the puromycin resistance was from CloneSpace LLC (SKU: CS1031).
The pCDNA3.1-Myc-Empty and pCDNA3.1-Myc-murine-Hrs were kindly provided by Mark von Zastrow [19]. The pEGFPC1 encoding the GFP-human-HRS construct was kindly provided by Dr daSilva and was previously described in [90].
Cell lines and primary muscle cells
The C2C12 skeletal myoblast cell line (ATCC CRL-1772) was kindly provided by S.Rome (IGFL, ENS-Lyon, France) and was previously described in [91]. C2C12 were grown in the growth medium (GM) Dulbecco’s modified Eagle’s medium containing glutamax/pyruvate/glucose (DMEM Gibco) supplemented with 10% (v/v) of fetal bovine serum (FBS, Gibco) and 1% penicillin streptomycin (P/S) and maintained at 37 °C under 5% CO2. Myoblast to myotube cell differentiation was induced by shifting the cells into the differentiation medium (Diff; DMEM supplemented with 2% of horse serum HS, Gibco and 1% P/S).
Primary muscle cells were isolated as previously described [92]. Briefly, mice were sacrificed and hind limb muscles were recovered and enzymatically dissociated with collagenase B (11 088 831 001, Roche) and Dispase (04 942 078 001, Roche) for 30 min at 37 °C. Digestion was stopped by the addition of FBS and the mixture was filtered through a 70-μm cell strainer (15-1070 BioLogix) and mildly centrifuged at 350×g (5804 r, Eppendorf) for 7 min at 4 °C. The cell pellet was resuspended in phosphate buffer (1× PBS) containing 2% FBS and incubated with appropriate antibodies (α7 integrin-647, CD34-FITC, CD45-PE, CD31-PE, Sca1-PE) at 4 °C during 45 min protected from the light. Cells were centrifuged at 350×g for 5 min at 4 °C and resuspended in phosphate buffer containing 2% FBS and passed through 30 μm CellTrics strainer. Cells were FACS sorted with FACSAria (ANIRA, Gerland, Lyon, France). The primary muscle cells are the CD31/CD45/Sca1− CD34/α7integrin+ population. Primary muscle cells were grown into DMEM F/12 supplemented with 20% of HS, 1% PS, and 2.5 μg/mL of FGF and dishes coated with Matrigel (#354234, Corning). Differentiation of primary muscle cells was induced by shifting the growth medium to DMEM F/12 with 2% of horse serum (HS) and 1% P/S. The human embryonic kidney cell line (HEK293T) was obtained from Généthon (Evry, France) and were cultured in DMEM-Glutamax supplemented with 10% (v/v) FBS and P/S.
The human Y711i immortalized myoblast cell line was obtained as follows. Quadriceps muscle biopsy from a 64-year-old male was obtained from the CBC (Centre de Biotechnologie Cellulaire, Groupement Hospitalier Est, Bron France). Muscle stem cells were extracted, sorted, and tested for their myogenicity as previously described [93]. Cells were then expended in proliferating KMEM medium (1 volume of M199, 4 volumes of Dulbecco’s modified Eagle’s medium (DMEM), 20% fetal bovine serum (v/v), 25 μg/mL Fetuin, 0.5 ng/mL β-FGF, 5 ng/mL EGF, 5 μg/mL Insulin from Gibco BRL). The Y711 primary cell immortalization was carried out by transducing cells with the HIV-1 lentivector encoding the HTert and CDK4 overnight in KMEM culture medium. One day after transduction, cells were passaged and cultured in presence of puromycin (1 μg/mL) for selection for 3–4 days until the death of non-transduced primary myoblast control cells was completed. Once puromycin was selected, transduced myoblasts were cultured in KMEM-D medium (KMEM medium with dexamethasone 0.2 μg/mL) in absence of puromycin. After 1 week of culture in KMEM-D medium, immortalized myoblasts can be cultured for a long term and were named Y711i (i for immortalized) myoblast. Differentiation of Y711i myoblasts into Y711i myotubes was carried out in free DMEM or free DMEM+insulin (10 μg/mL) for 4–5 days.
RNA interference and lentivector vector production
The lentiviral vector particles were produced by transient transfection of the packaging construction plasmid HIV-1 psPAX2, a minimal genome (HIV-1 pLKO1) bearing the expression cassette encoding the shCT control (shCT) or against HRS/Hrs and Tsg101 components or the HIV-psi-LVRU6GP encoding the shCT and sh-Ptpn23/HD-PTP (#1) and the plasmid encoding the VSV-G-envelope expressing construct pMDG2 (DNA ratio 8:8:4 μg of the respective plasmids) into HEK293T cells (4 × 106 cells plated 1 day before transfection) by the calcium phosphate method [94]. At 48 h post-transfection, virus-containing media was collected, centrifugated at 3000×g for 5 min (Multifuge X1; Thermo Fisher Scientific) and filtrated (0.45 μm filter, Millipore). Viral particles were normalized using the qPCR Lentivirus Titration Kit from abm (LV900) or by radioactive Reverse Transcriptase RT test and titrated on C2C12, primary muscle cell, or human primary Y711 cells for the lentivector encoding the hTERT-CDK4 cassette. Transduction of C2C12, primary muscle cells, or Y711 human myoblasts were carried with different MOI in 6-well plates for C2C12 (80,000 cells/well) or 100-mm dishes for primary muscle cells (200,000 cells/dish) or in 12-well plates (80,000 cells plated 1 day before) in presence of 6 μg/mL of polybrene overnight.
One day after transduction, cells were passaged and cultured in presence of 1 to 4 μg/mL puromycin (P8833, Sigma-Aldrich) for 2 to 3 days. Efficiency of the knockdown was monitored by Western blotting using the anti-HRS and anti-TSG101.
Alternatively, to control Irs1 antibody specificity, Irs1 was downregulated using small RNA siIrs1 ON-TARGET plus Mouse Irs1(L-040503-02-0005) or ON-TARGET plus non-targeting pool (D-001810-10-05) as negative control from Dharmacon (Horizon). siRNAs were transfected using the Lipofectamine RNAi max reagent (Thermo Fisher Scientific) according to the procedure depicted by the manufacturer.
Transfection of C2C12 cells
C2C12 myoblasts were transfected with 2–4 μg of DNA and with 10 μL of Lipofectamine 2000 reagent (Invitrogen) in OptiMEM according to the manufacturer protocol and differentiated 7 h or 1 day later. Cells were recovered 4–6 days after transfection for further analysis.
Rescue experiment
Rescue experiments were carried out using the Hrs#3TRCN0000088688 shRNA that targets the 3′ untranslated region of the murine Hgs mRNA. For this purpose, C2C12 shHrs#3 selected cells were transfected using the non-targeted shHrs pcDNA3.1-Myc-murine-Hrs encoding construct or the pcDNA3.1-Myc-Empty as negative control. Transfected cells were then placed in Diff for 96 h and differentiation of myoblasts into myotubes was assessed by confocal immunofluorescence imaging.
Counting cells
Cells were first counted and plated. Six, 24, and 72 h after plating, cells were trypsinized and counted using a cell counting Malassez chamber.
RNA extraction and quantitative RT-PCR
Total RNA was isolated from cultured cells grown either in 100 or in 35-mm dishes using Tri-reagent (Sigma-Aldrich) or RNeasy Mini kit (74104, Qiagen). The RNA quality was verified by agarose gel. Reverse transcriptase was performed on mRNA using 5× reaction buffer (Thermo Fisher Scientific), RevertAid H Minus Reverse Transcriptase (EP0451, Thermo Fisher Scientific), and random hexamer (Eurogentec) and Biometra thermocycler. RT-qPCR reaction using SYBR Green Mastermix (Qiagen) in the CFX-connect system (Bio-Rad) was performed on the cDNA using the oligonucleotides (sens/antisens) myogenin (CAATGCACTGGAGTTCGGTC /ACAATCTCAGTTGGGCATGG), hrs (GACAAGCTGGCACAGATACG/CTCTGCACCTCCAGGTACTC), mef2A (CAGCATTCCAGGGGAAGTAA/AATCAAAGGATAAGC), and cyclosporine B (AACTTTGCCGAAAACCACAT/GATGGCACAGGAGGAAAGAG).
The relative levels of mRNAs were calculated with the second derivative method (2−(ΔΔCt) equation) and normalized to cyclosporine B expression with the Bio-Rad CFX software.
Immunofluorescence and confocal microscopy imaging
Naïve and shCT, -Hrs, -Tsg101 C2C12, and Y711i and primary myoblasts/myotubes were grown in 6-well plates, 35-mm dishes, or on 12-mm diameter coverslips (Thermo Fisher Scientific) and fixed with 4% paraformaldehyde (PFA) in 1× PBS at room temperature for 10 min or alternatively with methanol at − 20 °C for 3 to 5 min. Cells were permeabilized with 0.2% or 0.3% Triton X-100 (Sigma-Aldrich) for 5 min at room temperature and blocked 1 h with 1× PBS containing 5% normal goat serum (NGS, Gibco). Muscle fibers were fixed with 4% PFA at room temperature for 20 min. Fibers were permeabilized with 1% Triton X-100 for 15 min and blocked for 1 h with 1× PBS containing 0.5% BSA, 2% NGS, and 0.1% Triton X-100. Cells and fibers were labeled overnight with the indicated primary antibody in 1× PBS containing 2% of BSA for cells or in blocking buffer for fibers at 4 °C. After extensive washing steps with 1× PBS 0.02% Triton X-100, cells and fibers were stained for 1 h at room temperature with secondary antibodies coupled with Alexa-Fluor 488 or 555. After washings, nuclear DNA was counterstained with DAPI (D9542; Sigma-Aldrich) for 5 min at room temperature and washed 3 times.
Images were acquired using the confocal microscope Zeiss LSM-880 (CIQLE platform from the Faculty of Medicine of Lyon, France) and analyzed using the ImageJ software (version 1.8).
Pulse-chase experiments and colocalization analysis
ShCT, shTsg101, or shHrs cells in proliferation were preincubated with cycloheximide (10 μg/mL) for 60 min before EGF stimulation, as well as during the course of the experiment to prevent EGFR synthesis as previously described [49]. Cells were pulse-stimulated during 5 min with 50 ng/mL of EGF-488 (E13345, Thermo Fisher Scientific) and then washed with warm DMEM containing cycloheximide. After chase times (15, 30, and 45 min) were indicated, cells were fixed with 4% PFA for 10 min and immunostained as described in the “Immunofluorescence and confocal microscopy imaging” section. Colocalization between EGF-488 and EEA1 was quantified with the ImageJ software (version 1.8). To quantify colocalization, a binary mask corresponding to the overlap between the green (EGF-488) and the red (EEA1) channels was generated according to [95]. This mask corresponded to the surface of colocalization between EGF-488 and EEA1 on one confocal plane. A ratio was calculated between the surface of colocalization and the total green surface (EGF-488). Results were given as percentage of colocalization of EGF-488 with EEA1. The same procedure was used to quantify colocalization of HRS around nuclei with CHC, Rab5a, Rab7, GM130, and KDEL. In this case, only the ratio of overlapping between HRS and the target proteins was used.
C2C12 myoblast and primary muscle cell differentiation analysis
To determine the rate of myoblast differentiation, the myogenic index was used as a morphological parameter of muscle differentiation. The number of nuclei in cells positive for the MHC staining and the total number of nuclei were counted in > 6 randomly selected fields per well. Using ImageJ software (version 1.8), the myogenic index (in %) was then calculated as: \( \left(\frac{\mathrm{number}\ \mathrm{of}\ \mathrm{nuclei}\ \mathrm{in}\ \mathrm{positive}\ \mathrm{MHC}\ \mathrm{staining}}{\mathrm{total}\ \mathrm{number}\ \mathrm{of}\ \mathrm{nuclei}\ \mathrm{in}\ \mathrm{counted}\ \mathrm{fields}}\right)\times 100 \).
Immunoblotting and fractionation
Whole cell lysates were prepared using radio-immunoprecipitation assay (RIPA) lysis buffer (50 mM Tris HCl, pH 8, 150 mM NaCl, 1 mM EDTA, 1% NP-40, 0.5% Na-deoxycholate, 0.1% SDS) supplemented with protease inhibitor cocktail (Complete; Sigma-Aldrich) and phosphatase inhibitor (PhosphoStop; Roche) and centrifugated at 10,000×g, for 10 min at 4 °C or directly lysed in 1× sample buffer (60 mM TrisHCl buffer at pH 6.8, 2% SDS, 10% glycerol, 5% β-mercapto-ethanol, 0.025% bromophenol blue). Protein concentration was determined with the Bradford reagent (Bio-Rad Laboratories), and 5–30 μg of proteins was loaded and separated by SDS-PAGE electrophoresis gels in presence of 2,2,2-trichloroethanol (TCE, T54801, Sigma-Aldrich) for stain free gel analyzes. Gels were transferred to polyvinylidene difluoride (PVDF) membranes (Millipore, IPVH0001 0.45 μm, Millipore) by semi-dry electrotransfer apparatus (TransBlot® TurboTM, Bio-Rad Laboratories) in TOWBIN buffer (25 mM Tris, 192 mM glycine, 20% methanol) or wet electrotransfer (25 mM Tris, 192 mM glycine, 20% ethanol), and rate of transfer was controlled by stain free analyzes using the ChemiDocTM Touch imaging system (Bio-Rad Laboratories) analyzer. Membranes were incubated with primary antibody in 5% bovine serum albumin (BSA) in Tris-Buffered Saline (TBS buffer; UP74004B, Interchim) overnight at 4 °C. Membranes were successively washed 3 × 5 min in TBS 0.1% Tween-20 and were next probed with horseradish peroxidase-conjugated secondary antibodies for 1 h at room temperature. After three washing steps, immunodetection was carried out using the ECLTM reagent (Amersham). Western blotting revelation was carried with a ChemiDocTM Touch imaging system and quantification of immunoblots was done using the image lab software (Bio-Rad Laboratories).
For nuclear and cytosol fractionation experiments, freshly cells were recovered in Buffer A (20 mM Tris HCl, pH 8, 1 mM EDTA, 5 mM DTT, protease inhibitors, and anti-phosphatase) for 15 min at 4 °C and then lyzed using a dounce tissue grinder. Cell lysate was centrifuged at 2400×g for 5 min and the resulting supernatant was recovered as the cytosolic fraction, whereas pellet corresponding to nuclei fraction was lyzed in Buffer B (20 mM Tris HCl, pH 8, 20% glycerol, 420 mM NaCl, 1.5 mM MgCl2, 0.2 mM EDTA, 0.5 mM DTT, protease inhibitors, and anti-phosphatase) for 20 min and sonicated. The lysate was centrifuged at 9600×g for 15 min, and the resulting supernatant was recovered as the nuclei fraction. Cytosolic and nuclear fractions were analyzed by Western blotting as described above.
EGFR degradation
shCT, shHrs, and shTsg101 cells were preincubated for 1 h in growth medium in presence of 10 μg/mL of cycloheximide. Cells were then cultured in growth medium in presence of cycloheximide (10 μg/mL) and with EGF Recombinant Human protein (50 ng/mL; Gibco) for 15, 60, and 120 min. After these time points, cells were recovered and analyzed by Western blotting.
Quantification and statistical analysis
All experiments were performed at least three independent times. All images analysis has been quantified with the ImageJ software (version 1.8). The number of individual experiments and the number of cells or different organelles analyzed are indicated in the figure legends. We tested our datasets for normal distribution and chose an appropriate test accordingly using Prism 6.0 (GraphPad Software). Data are given as mean ± SEM. The nonparametric two-sided U test (Mann–Whitney U test) was used to test two samples with unequal variance. For more than two samples, we used nonparametric Kruskal–Wallis test. For a multiple factorial analysis of variance, two-way ANOVA was applied. P values < 0.05 were considered statistically significant (shown as a single asterisk in figures, P values < 5%); P values < 0.01 were considered highly statistically significant (shown as two asterisks in figures, P values < 1%); P values < 0.001 were considered very highly statistically significant (shown as three asterisks in figures, P values < 0.1%) and P values < 0.0001 were considered extremely highly statistically significant (shown as four asterisks in figures, P values < 0.01%). Samples were not randomized for the experiments. No samples were excluded from the analysis.
Mice
Control (C57BL/10) and mdx (C57BL/10 ScSn-Dmdmdx/J) mice were provided by Jackson Laboratory. The generation of control (mTORflox/flox) and muscle-specific mTOR knockout (HSA-Cre+; mTORflox/flox herein called mTORmKO) mice on F6; C57BL/6 background has been previously described in [60].
Animals were provided with mouse chow and water ad libitum in a restricted-access, specific pathogen-free animal care facility at AniRA PBES, Lyon, France. All procedures were performed in accordance with national and European legislation on animal experimentation.
Protein extract from tibialis anterior muscle of 11-week-old mdx and 16-week-old mTORmKO male mice were prepared as described in [60].