Journal: Disease Models & Mechanisms

Article Title: Muscle-specific lack of Gfpt1 triggers ER stress to alleviate misfolded protein accumulation

doi: 10.1242/dmm.050768

Figure Lengend Snippet: Gfpt1 -KI activated the UPR, which subsequently promoted translational suppression, protein folding, early autophagy and apoptosis in skeletal muscles. (A) Quantitative RT-PCR of Hspa5 , Ddit3 and Xbp1-s in the GAS muscles in WT and Gfpt1 -KI mice ( n =4 mice each). (B,C) Representative immunoblots and quantification of Grp78, Grp94 and Chop in the GAS muscles in WT and Gfpt1 -KI mice ( n =6 mice each). (D-F) Representative immunoblots and quantification of p-eIF2α, eIF2α and Bax in the GAS muscles in WT and Gfpt1 -KI mice ( n =4 mice each). (G) Representative confocal images of cross-sections of myofibers stained with antibodies against KDEL (Grp78/Grp94) and p62 in the GAS muscles in WT and Gfpt1 -KI mice. Arrows indicate colocalized Grp78-p62. Scale bar: 50 μm. (H) Percentage of the myofibers with KDEL (Grp78)-p62 colocalized puncta in WT and Gfpt1 -KI GAS muscles ( n =4 mice each). (A,C,E,F,H) Mean and s.e.m. are indicated. * P <0.05; ** P <0.01; *** P <0.001; **** P <0.0001 (two-tailed unpaired Student's t -test).

Article Snippet: The membranes were subsequently incubated with the following primary antibodies in TBS-T at 4°C overnight: rabbit monoclonal anti-GFPT1 antibody (1:1000, ab125069, Abcam), mouse monoclonal anti-RL2 antibody (1:800, sc-59624, Santa Cruz Biotechnology), mouse monoclonal anti-KDEL antibody (1:1000, sc-58774, Santa Cruz Biotechnology), mouse monoclonal anti-Chop antibody (1:2000, 2895, Cell Signaling Technology), rabbit monoclonal anti-phospho-eIF2α antibody (1:1000, 9721, Cell Signaling Technology), rabbit monoclonal anti-eIF2α antibody (1:1000, 5324, Cell Signaling Technology), rabbit monoclonal anti-Bax antibody (1:2000, 2772, Cell Signaling Technology), rabbit polyclonal anti-p62 antibody (1:1000, PM045, MBL), mouse monoclonal anti-Hsp70 antibody (1:1000, ADI-SPA-810-D, Enzo Life Sciences), rabbit monoclonal anti-LC3B antibody (1:1000, ab192890, Abcam), mouse monoclonal anti-MuRF1 antibody (1:1000, sc-398608, Santa Cruz Biotechnology), rabbit monoclonal anti-p-AMPKα (Thr172) antibody (1:1000, 2535, Cell Signaling Technology), rabbit monoclonal anti-AMPKα antibody (1:1000, 5831, Cell Signaling Technology), rabbit monoclonal anti-p-AKT (Ser473) antibody (1:1000, 4060, Cell Signaling Technology), rabbit monoclonal anti-AKT antibody (1:1000, 4691, Cell Signaling Technology), rabbit polyclonal anti-p-mTORC1 (Ser2448) antibody (1:1000, 2971, Cell Signaling Technology), rabbit polyclonal anti-mTOR antibody (1:1000, 2972, Cell Signaling Technology), rabbit monoclonal anti-FoxO1 antibody (1:1000, 2880, Cell Signaling Technology), rabbit monoclonal anti-Xbp1 antibody (1:1000, ab220783, Abcam) and rabbit polyclonal anti-Gapdh antibody (1:2000, G9545, Sigma-Aldrich).

Techniques: Muscles, Quantitative RT-PCR, Western Blot, Staining, Two Tailed Test

Journal: Disease Models & Mechanisms

Article Title: Muscle-specific lack of Gfpt1 triggers ER stress to alleviate misfolded protein accumulation

doi: 10.1242/dmm.050768

Figure Lengend Snippet: Denervation failed to activate the hexosamine biosynthesis pathway, markedly enhanced protein folding and compromised autophagy-mediated protein degradation in Gfpt1 -KI mice. (A-C) Representative immunoblots and quantification of Grp78, Grp94 and Gfpt1 in the GAS muscles in WT and Gfpt1 -KI mice ( n =3 to 4 mice each). Sh, sham-operated; De, denervated. (D) Representative confocal images of cross-sections of myofibers stained with antibodies against KDEL (Grp78/Gpr94) and p62 in the GAS muscles in WT and Gfpt1 -KI mice. Scale bar: 50 μm. (E) Quantification of D showing the percentage of myofibers with Grp78-p62 colocalization in the GAS muscles in WT and Gfpt1 -KI mice ( n =3 mice each). (F,G) Representative immunoblots and quantification of LC3 and p62 in the GAS muscles in WT and Gfpt1 -KI mice ( n =4 mice each). (H) Quantitative RT-PCR of total Gfpt1 in GAS muscles in WT and Gfpt1 -KI mice ( n =5 mice each). (I,J) Representative immunoblots and quantification of O -GlcNAcylated proteins in GAS muscles in WT and Gfpt1 -KI mice ( n =3 mice each). (B,C,E,G,H,J) Mean and s.e.m. are indicated. ns, no significance; * P <0.05; ** P <0.01; *** P <0.001; **** P <0.0001 (one-way ANOVA followed by Tukey's post hoc test).

Article Snippet: The membranes were subsequently incubated with the following primary antibodies in TBS-T at 4°C overnight: rabbit monoclonal anti-GFPT1 antibody (1:1000, ab125069, Abcam), mouse monoclonal anti-RL2 antibody (1:800, sc-59624, Santa Cruz Biotechnology), mouse monoclonal anti-KDEL antibody (1:1000, sc-58774, Santa Cruz Biotechnology), mouse monoclonal anti-Chop antibody (1:2000, 2895, Cell Signaling Technology), rabbit monoclonal anti-phospho-eIF2α antibody (1:1000, 9721, Cell Signaling Technology), rabbit monoclonal anti-eIF2α antibody (1:1000, 5324, Cell Signaling Technology), rabbit monoclonal anti-Bax antibody (1:2000, 2772, Cell Signaling Technology), rabbit polyclonal anti-p62 antibody (1:1000, PM045, MBL), mouse monoclonal anti-Hsp70 antibody (1:1000, ADI-SPA-810-D, Enzo Life Sciences), rabbit monoclonal anti-LC3B antibody (1:1000, ab192890, Abcam), mouse monoclonal anti-MuRF1 antibody (1:1000, sc-398608, Santa Cruz Biotechnology), rabbit monoclonal anti-p-AMPKα (Thr172) antibody (1:1000, 2535, Cell Signaling Technology), rabbit monoclonal anti-AMPKα antibody (1:1000, 5831, Cell Signaling Technology), rabbit monoclonal anti-p-AKT (Ser473) antibody (1:1000, 4060, Cell Signaling Technology), rabbit monoclonal anti-AKT antibody (1:1000, 4691, Cell Signaling Technology), rabbit polyclonal anti-p-mTORC1 (Ser2448) antibody (1:1000, 2971, Cell Signaling Technology), rabbit polyclonal anti-mTOR antibody (1:1000, 2972, Cell Signaling Technology), rabbit monoclonal anti-FoxO1 antibody (1:1000, 2880, Cell Signaling Technology), rabbit monoclonal anti-Xbp1 antibody (1:1000, ab220783, Abcam) and rabbit polyclonal anti-Gapdh antibody (1:2000, G9545, Sigma-Aldrich).

Techniques: Western Blot, Muscles, Staining, Quantitative RT-PCR

Journal: Disease Models & Mechanisms

Article Title: Muscle-specific lack of Gfpt1 triggers ER stress to alleviate misfolded protein accumulation

doi: 10.1242/dmm.050768

Figure Lengend Snippet: Thapsigargin induced the UPR in C2C12 myotubes, and Gfpt1 deficiency switched the cell fate from the activation of autophagy to the induction of apoptosis. (A) Estimation of cell viability by CCK-8 of C2C12 myoblasts transfected with scrambled siRNA (siControl) or siGfpt1 for 48 h ( n =3 dishes each). (B) Representative images of C2C12 myotubes stained with antibodies against KDEL (Grp78/Grp94) and p62. Scale bar: 50 μm. (C) Quantification of B showing the ratio of Grp78-positive aggregates per myonucleus in C2C12 myotubes transfected with siControl or siGfpt1 ( n =3 dishes each). (D) Quantitative RT-PCR of Hspa5 , Ddit3 and Bax in C2C12 myotubes transfected with siControl or siGfpt1 ( n =3 dishes each). (E,F) Representative immunoblots and quantification of Gfpt1, Grp78, Grp94 and Bax in myotubes transfected with siControl or siGfpt1 ( n =3 dishes each). (G) Temporal profile of thapsigargin (TG)-mediated induction of UPR in C2C12 myotubes for H-N. Note that siRNA was added 4 days before harvesting cells for B-F. (H-N) Representative immunoblots and quantification of the indicated proteins and the LC3-II/LC3-I ratio in C2C12 myotubes transfected with siControl or siGfpt1 ( n =3 dishes each). (A,C,D,F,I-N) Mean and s.d. are indicated. ns, no significance; * P <0.05; ** P <0.01; *** P <0.001 (two-tailed unpaired Student's t -test for A,C; two-way ANOVA followed by Sidak's post hoc test for D,F,I-N).

Article Snippet: The membranes were subsequently incubated with the following primary antibodies in TBS-T at 4°C overnight: rabbit monoclonal anti-GFPT1 antibody (1:1000, ab125069, Abcam), mouse monoclonal anti-RL2 antibody (1:800, sc-59624, Santa Cruz Biotechnology), mouse monoclonal anti-KDEL antibody (1:1000, sc-58774, Santa Cruz Biotechnology), mouse monoclonal anti-Chop antibody (1:2000, 2895, Cell Signaling Technology), rabbit monoclonal anti-phospho-eIF2α antibody (1:1000, 9721, Cell Signaling Technology), rabbit monoclonal anti-eIF2α antibody (1:1000, 5324, Cell Signaling Technology), rabbit monoclonal anti-Bax antibody (1:2000, 2772, Cell Signaling Technology), rabbit polyclonal anti-p62 antibody (1:1000, PM045, MBL), mouse monoclonal anti-Hsp70 antibody (1:1000, ADI-SPA-810-D, Enzo Life Sciences), rabbit monoclonal anti-LC3B antibody (1:1000, ab192890, Abcam), mouse monoclonal anti-MuRF1 antibody (1:1000, sc-398608, Santa Cruz Biotechnology), rabbit monoclonal anti-p-AMPKα (Thr172) antibody (1:1000, 2535, Cell Signaling Technology), rabbit monoclonal anti-AMPKα antibody (1:1000, 5831, Cell Signaling Technology), rabbit monoclonal anti-p-AKT (Ser473) antibody (1:1000, 4060, Cell Signaling Technology), rabbit monoclonal anti-AKT antibody (1:1000, 4691, Cell Signaling Technology), rabbit polyclonal anti-p-mTORC1 (Ser2448) antibody (1:1000, 2971, Cell Signaling Technology), rabbit polyclonal anti-mTOR antibody (1:1000, 2972, Cell Signaling Technology), rabbit monoclonal anti-FoxO1 antibody (1:1000, 2880, Cell Signaling Technology), rabbit monoclonal anti-Xbp1 antibody (1:1000, ab220783, Abcam) and rabbit polyclonal anti-Gapdh antibody (1:2000, G9545, Sigma-Aldrich).

Techniques: Activation Assay, CCK-8 Assay, Transfection, Staining, Quantitative RT-PCR, Western Blot, Two Tailed Test