human myoblast cell line  (Inserm Transfert)

Journal: bioRxiv

Article Title: Engineering novel AAV capsids by global de-targeting and subsequent muscle-specific tropism in mice and NHPs

doi: 10.1101/2025.05.19.654800

Figure Lengend Snippet: A–C. HEK293T, C2C12, Huh-7, HelaRC32, and CHO-K1 cell lines being transduced with AAV9-, AAV2-, AAV.Zero1-, AAV.Zero2-, or AAV.Zero3-CAG-Fluc-P2A-EGFP at MOI=1E5 after 120 hours. A. Representative images of each treatment. Scale bar: 100 μm. B. Quantification of the integrated gray value of the EGFP fluorescence strength in HEK 293T and C2C12 cells (n=3). C. Quantification of relative virus genome copy numbers in HEK 293T and C2C12 cells (n=2). D–G. 8-week-old C57BL/6J mice were systemically injected with 1E12 vg per mouse (∼4E13 vg/kg) of AAV9-, AAV2-, AAV.Zero1-, AAV.Zero2-, or AAV.Zero3-CAG-Fluc-P2A-EGFP and data were collected 21 days post-injection. Representative whole body in vivo bioluminescence images (D). Quantification of firefly luciferase luminescence from liver (n=5) (E). Quantification of fold-difference in Fluc mRNA expression in various tissues (F) compared with normalized AAV9. The p values were calculated between AAV9 and each other groups by Student’s t -test (n=4–5), * p < 0.05, ** p < 0.01, *** p < 0.001 and **** p < 0.0001. Representative western blot images detecting luciferase and GAPDH in quadriceps, heart, and liver (G).

Article Snippet: The human embryonic kidney cell line HEK 293T (American Type Culture Collection (ATCC), CRL-11268), human myoblast cell line C2C12 (ATCC, CRL-1772), human cervical carcinoma cell line HelaRC32 (ATCC, CRL-2972), and human hepatoma HuH-7 cell line (National Collection of Authenticated Cell Cultures, SCSP-526) were cultured in Dulbecco’s Modified Eagle Medium (DMEM) (Thermo Fisher Scientific, C11995500BT) supplemented with 10% fetal bovine serum (FBS; Excell Bio, FSP500) and 1% penicillin-streptomycin (P/S; Biosharp, BL505A) and the hamster ovary cell line CHO-K1 (Cobioer Gene Technology Co., CBP60296) was maintained in F12K media (Sigma-Aldrich, N3520-10X1L) supplemented with 10% FBS and 1% P/S.

Techniques: Transduction, Fluorescence, Virus, Injection, In Vivo, Luciferase, Expressing, Western Blot

Journal: bioRxiv

Article Title: Engineering novel AAV capsids by global de-targeting and subsequent muscle-specific tropism in mice and NHPs

doi: 10.1101/2025.05.19.654800

Figure Lengend Snippet: A. Sequence alignments of the RGD-containing peptides 2A, 4E, and 4A inserted into AAV.Zero3 at R585. B. Representative images of in vitro transduction in HEK 293T and C2C12 cell lines were transduced at MOI=1E5 with AAV9-, AAV2-, MyoAAV 2A-, AAV.Zero3 2A-, MyoAAV 4E-, AAV.Zero3 4E-, MyoAAV 4A-, or AAV.eM-CAG-Fluc-P2A-EGFP 72 hours after transduction. Scale bar: 100 μm. C–E. 8-week-old C57BL/6J mice were systemically injected with 2E11 vg per mouse (∼8E12 vg/kg) AAV9-, AAV2-, MyoAAV 2A-, AAV.Zero3 2A-, MyoAAV 4E-, AAV.Zero3 4E-, MyoAAV 4A-, or AAV.eM-CAG-Fluc-P2A-EGFP. Representative whole body in vivo bioluminescence images 21 days post-injection (C). Viral genome copy number per diploid from mice tissues, including quadriceps, abdomen, and heart (left) and liver (right) detected by qPCR with standard curve (D). The p value was calculated between AAV9 and each other capsid by unpaired Student’s t -test (n=6), * p < 0.05, ** p < 0.01, *** p < 0.001. Quantification of the Fluc mRNA in mice tissues, including quadriceps, abdomen, and heart (left) and liver (right) 21 days post-injection (E). The p value was calculated between AAV9 and each other capsid by unpaired Student’s t -test (n=11–17), * p < 0.05, ** p < 0.01, *** p < 0.001, *** p < 0.001. F–G. 8-week-old C57BL/6J mice were systemically injected with 2E11 vg per mouse (∼8E12 vg/kg) AAV9-, AAV2-, MyoAAV 4A-, or AAV.eM-CAG-Fluc-P2A-EGFP. Representative western blot images detecting luciferase and GAPDH of quadriceps and liver tissues 21 days post-injection (F). Representative cross-section fluorescent images of quadriceps and liver tissues 21 days post-injection (G). Green: EGFP, Blue: DAPI. Scale bar: 25 μm.

Article Snippet: The human embryonic kidney cell line HEK 293T (American Type Culture Collection (ATCC), CRL-11268), human myoblast cell line C2C12 (ATCC, CRL-1772), human cervical carcinoma cell line HelaRC32 (ATCC, CRL-2972), and human hepatoma HuH-7 cell line (National Collection of Authenticated Cell Cultures, SCSP-526) were cultured in Dulbecco’s Modified Eagle Medium (DMEM) (Thermo Fisher Scientific, C11995500BT) supplemented with 10% fetal bovine serum (FBS; Excell Bio, FSP500) and 1% penicillin-streptomycin (P/S; Biosharp, BL505A) and the hamster ovary cell line CHO-K1 (Cobioer Gene Technology Co., CBP60296) was maintained in F12K media (Sigma-Aldrich, N3520-10X1L) supplemented with 10% FBS and 1% P/S.

Techniques: Sequencing, In Vitro, Transduction, Injection, In Vivo, Western Blot, Luciferase

Journal: bioRxiv

Article Title: Engineering novel AAV capsids by global de-targeting and subsequent muscle-specific tropism in mice and NHPs

doi: 10.1101/2025.05.19.654800

Figure Lengend Snippet: A. Representative images Huh-7, HelaRC32, and CHO-K1 cell lines 72 hours after being transfected with AAV9-, AAV2-, MyoAAV 2A-, AAV.Zero3 2A-, MyoAAV 4E-, AAV.Zero3 4E-, MyoAAV 4A-, or AAV.eM-CAG-Fluc-P2A-EGFP at MOI=1E5. Scale bar: 100 μm. B. Quantification of relative virus genome copy numbers in HEK 293T and C2C12 cells transduced with AAV9-, AAV2-, MyoAAV 2A-, AAV.Zero3 2A-, MyoAAV 4E-, AAV.Zero3 4E-, MyoAAV 4A-, or AAV.eM-CAG-Fluc-P2A-EGFP at MOI=1E5 (n=2). C. Different dilutions of AAV2-antibody containing serum were incubated with constant amounts of virus at MOI=1E6 and tested for neutralization of transduction using an in vitro assay (n=3–4). D. Different dilutions of AAV9-antibody containing serum were incubated with constant amounts of virus at MOI=1E6 and tested for neutralization of transduction using an in vitro assay (n=3–6). E–F. 8-week-old C57BL/6J mice were systemically injected with 2E11 vg per mouse (∼8E12 vg/kg) of AAV9-, AAV2-, MyoAAV 2A-, AAV.Zero3 2A-, MyoAAV 4E-, AAV.Zero3 4E-, MyoAAV 4A-, or AAV.eM-CAG-Fluc-P2A-EGFP- and data were collected 21 days post-injection. Quantification of the firefly luciferase luminescence intensity from hindlimbs (n=6) (E). Representative western blot images detecting luciferase and GAPDH in quadriceps, heart, abdomen, biceps, and liver (F).

Article Snippet: The human embryonic kidney cell line HEK 293T (American Type Culture Collection (ATCC), CRL-11268), human myoblast cell line C2C12 (ATCC, CRL-1772), human cervical carcinoma cell line HelaRC32 (ATCC, CRL-2972), and human hepatoma HuH-7 cell line (National Collection of Authenticated Cell Cultures, SCSP-526) were cultured in Dulbecco’s Modified Eagle Medium (DMEM) (Thermo Fisher Scientific, C11995500BT) supplemented with 10% fetal bovine serum (FBS; Excell Bio, FSP500) and 1% penicillin-streptomycin (P/S; Biosharp, BL505A) and the hamster ovary cell line CHO-K1 (Cobioer Gene Technology Co., CBP60296) was maintained in F12K media (Sigma-Aldrich, N3520-10X1L) supplemented with 10% FBS and 1% P/S.

Techniques: Transfection, Virus, Transduction, Incubation, Neutralization, In Vitro, Injection, Luciferase, Western Blot

Journal: The EMBO Journal

Article Title: CHC22 clathrin recruitment to the early secretory pathway requires two-site interaction with SNX5 and p115

doi: 10.1038/s44318-024-00198-y

Figure Lengend Snippet: ( A , B ) Representative immunoblot ( n = 4–6) of immunoprecipitates (IP) of CHC22, from control HeLa cells ( A ) and differentiated human skeletal muscle cell line AB1190 (Myotubes) ( B ). Samples were immunoblotted for CHC22, SNX5, SNX6, and tubulin. Lanes show Input (5%), bead-only (no antibody) control (Bead) and CHC22 immunoprecipitate (22 IP). The migration positions of MW markers are indicated at the left in kilodaltons (kD). ( C ) The structure of a single clathrin triskelion formed from three clathrin heavy chains with the Hub region indicated by red dotted triangle and the trimerization domain (TxD), magenta), proximal leg region (dark blue), knee and distal leg regions (grey) and terminal domain (light blue), based on PDB 31YV (Fotin et al, ). ( D ) AlphaFold-generated model of the interaction between CHC22 Hub (blue proximal leg residues 1278–1519 and magenta TxD residues 1520–1640) and the BAR domain of SNX5 (residues 202–404, orange). Two angles of view are displayed. ( E ) Representative immunoblot ( n = 3–5) of in vitro binding of full-length GST-SNX5 (right lanes) or GST alone (left lanes) to CHC22 Hub (22 Hub) or CHC17 Hub (17 Hub) immobilized on Ni-NTA beads. Purified protein input (2%) and bead-only (no Hub immobilized) control (Bead) are shown for each prey. Samples were immunoblotted for SNX5, His-tag or GST with detected proteins indicated at the right. The position of MW markers is indicated at the left in kD. (F) Representative immunoblot of in vitro binding of full-length GST-SNX5 (right lanes) or GST alone (left lanes) to CHC22 Hub or CHC22 TxD immobilized on Ni-NTA beads ( n = 3). Purified protein input (2%) and beads without CHC22 fragment added (Bead) are shown for each prey. Samples were immunoblotted for SNX5, His-tag or GST with detected proteins indicated at the right. The position of MW markers is indicated at the left in kD. .

Article Snippet: The human myoblast cell line AB1190 was grown in complete Skeletal Muscle Cell Growth Medium (Promocell) with provided supplemental cocktail and 10% FBS (Gibco) to reach a 15% final supplement concentration (volume/volume) (Camus et al, ).

Techniques: Western Blot, Control, Migration, Generated, In Vitro, Binding Assay, Purification

Journal: bioRxiv

Article Title: Effects of HMGCR deficiency on skeletal muscle development

doi: 10.1101/2024.05.06.591934

Figure Lengend Snippet: shRNA mediated knockdown of Hmgcr in C2C12 myoblasts. (A) qPCR was performed in triplicate for each sample with n=5 experiments. Transcript levels were normalized against Gapdh with fold change calculated as Hmgcr knockdown versus scrambled control. Unpaired t-test results are shown. ***, p < 0.0002. (B) shRNA mediated knockdown of Hmgcr in C2C12 myoblasts. Successful shRNA transfection was confirmed by detecting GFP positive cells.

Article Snippet: C2C12 myoblasts were transfected with either a cocktail of three different shRNAs targeting mouse- Hmgcr or scrambled shRNA control ( GeneCopoeia ) using Lipofectamine 3000 ( Invitrogen ).

Techniques: shRNA, Knockdown, Control, Transfection

Journal: bioRxiv

Article Title: Effects of HMGCR deficiency on skeletal muscle development

doi: 10.1101/2024.05.06.591934

Figure Lengend Snippet: The oxygen consumption rate (OCR) of Hmgcr shRNA versus scrambled shRNA C2C12 myoblasts was determined using the RESIPHER system at (A) 24 hours, (B), 48 hours, and (C) 72 hours. The OCR of the Hmgcr knockdown cells was increased compared to scrambled controls at 24 and 48 hours. (D) Rotenone treatment at 72 hours confirms that the increased oxygen consumption occurred via mitochondrial respiration. (E) A A 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay at 72 hours shows that the total cellular metabolic activity is higher in Hmgcr knockdown cells compared to scrambled controls. Paired t-tests were performed. *, p <0.05. Error bars show standard error of the mean (SEM).

Article Snippet: C2C12 myoblasts were transfected with either a cocktail of three different shRNAs targeting mouse- Hmgcr or scrambled shRNA control ( GeneCopoeia ) using Lipofectamine 3000 ( Invitrogen ).

Techniques: shRNA, Knockdown, MTT Assay, Activity Assay