Journal: MedComm

Article Title: Obestatin Treatment Counteracts Muscle Wasting by Reactivation of Autophagy in Duchenne Muscular Dystrophy

doi: 10.1002/mco2.70563

Figure Lengend Snippet: Dystrophic conditions set a different activation pattern for the obestatin/GPR39 system compared with normal human myotubes. (A) KM155C25 and DMD myotubes were treated with obestatin (10 nM) or Dexa (1 µM) in the presence or absence of bafilomycin treatment. LC3, p62, Murf1, pmTOR(S2448), mTOR, pS6(S235/236), S6, pAMPK(T172), and AMPK were analyzed by immunoblotting. Immunoblots are representative of the mean value. Data were expressed as the mean ± SEM obtained from intensity scans from at least three independent experiments ( *,#,&,⊄,$ p < 0.05). (B) Upper panel : Representative images from LAMP2 and p62 immunostaining of DMD myotubes treated with obestatin (10 nM) or vehicle (control) for 24 h. Lower panel : The changes in MFI of LAMP2 and p62 are shown. Pearson's coefficient ( r ) indicates the correlation of intensity values of green and red pixels in dual‐channel images. Data were expressed as arbitrary units (au) ( n = 5 per group; mean ± SEM; * p < 0.05). (C) Upper panel : Representative images from LAMP2 and ubiquitin immunostaining of DMD myotubes treated with obestatin (10 nM) or vehicle (control) for 24 h. Lower panel : The changes in MFI of LAMP2 and ubiquitin are shown. Pearson's coefficient ( r ) indicates the correlation of intensity values of green and red pixels in dual‐channel images. Data were expressed as au ( n = 5 per group; mean ± SEM; * p < 0.05). (D) Immunoblot analysis of pAMPK(T172), AMPK, pmTOR(S2448), mTOR, pS6(S235/236), S6, pULK1(S318), pULK1(S758), ULK1, pBeclin1(S15), and Beclin1 in full‐time course of DMD myotubes treated with obestatin (10 nM) or insulin (1.72 µM). Protein level was normalized to its nonphosphorylated control (AMPK, mTOR, S6, ULK1, or Beclin1, respectively) and expressed as fold of control (designated as time 0). Data were expressed as the mean ± SEM obtained from intensity scans from at least three independent experiments.

Article Snippet: To knockdown NEDD4, AMPKα or USP10 expression, siRNA specifically targeting human NEDD4‐L (sc‐75894; Santa Cruz Biotechnology, CA, USA), AMPKα (sc‐45312; Santa Cruz Biotechnology), or USP10 (sc‐76811; Santa Cruz Biotechnology) was used. siRNA duplexes targeting preproghrelin or c‐Src were selected from ON‐TARGET plus SMARTpool siRNA from Thermo Fisher Scientific (Dharmacon, CO, USA). shRNA‐mediated gene silencing was used for ULK1 knockdown (sc‐44182‐SH; Santa Cruz Biotechnology) in DMD cells.

Techniques: Activation Assay, Western Blot, Immunostaining, Control, Ubiquitin Proteomics

Journal: MedComm

Article Title: Obestatin Treatment Counteracts Muscle Wasting by Reactivation of Autophagy in Duchenne Muscular Dystrophy

doi: 10.1002/mco2.70563

Figure Lengend Snippet: Obestatin stimulates autophagy in an NEDD4‐L‐dependent manner in human DMD myotubes. (A) Immunoblot analysis of pAMPK(T172), AMPK, pBeclin1(S15), Beclin1, VPS34, pULK1(S555), ULK1, LC3, and p62 in DMD myotubes transfected with sicontrol or siAMPKα1/2 and treated with obestatin (10 nM). Data were expressed as the mean ± SEM ( n = 3 per group; *,# p < 0.05). (B) sicontrol and siNEDD4‐L DMD cells differentiated for 3 days were treated with obestatin in the presence or absence of MG132. Protein extracts were analyzed by immunoblot to detect NEDD4‐L, pAMPK(T172), AMPK, pmTOR(S2448), mTOR, pULK1(S318), pULK1(S758), ULK1, VPS34, pBeclin1(S15), Beclin1, LC3, and p62. Immunoblots are representative of the mean value. Data were expressed as mean ± SEM ( n = 3 per group; *,# p < 0.05). (C) Immunoblot analysis of VPS34 and NEDD4‐L in extracts of DMD and KM155C25 cells transfected with control or NEDD4‐L siRNAs after obestatin treatment (10 nM). Data were expressed as mean ± SEM ( n = 3 per group; *,# p < 0.05). (D) Coimmunoprecipitation and immunoblot analysis of extracts of DMD cells transfected with control or NEDD4‐L siRNAs and treated with obestatin (10 nM) by using anti‐VPS34 antibody and immunoblotted with Beclin1, ATG14, UVRAG, VPS34, or NEDD4‐L antibody. Data were expressed as mean ± SEM ( n = 3 per group; *,# p < 0.05).

Article Snippet: To knockdown NEDD4, AMPKα or USP10 expression, siRNA specifically targeting human NEDD4‐L (sc‐75894; Santa Cruz Biotechnology, CA, USA), AMPKα (sc‐45312; Santa Cruz Biotechnology), or USP10 (sc‐76811; Santa Cruz Biotechnology) was used. siRNA duplexes targeting preproghrelin or c‐Src were selected from ON‐TARGET plus SMARTpool siRNA from Thermo Fisher Scientific (Dharmacon, CO, USA). shRNA‐mediated gene silencing was used for ULK1 knockdown (sc‐44182‐SH; Santa Cruz Biotechnology) in DMD cells.

Techniques: Western Blot, Transfection, Control

Journal: MedComm

Article Title: Obestatin Treatment Counteracts Muscle Wasting by Reactivation of Autophagy in Duchenne Muscular Dystrophy

doi: 10.1002/mco2.70563

Figure Lengend Snippet: USP10 is required for deubiquitination of VPS34 and AMPKα in human DMD myotubes. (A) Immunoblot analysis of pAMPK(T172), AMPK, pULK1(S318), ULK1, NEDD4‐L, VPS34, Beclin1, LC3, and p62 in DMD myotubes transfected with sicontrol or siUSP10 and treated with obestatin (10 nM, 3 h). Data were expressed as the mean ± SEM ( n = 3 per group; *,# p < 0.05). (B) Immunoblot analysis of Beclin1, VPS34, pAMPK(T172), and AMPK in DMD cells transfected with HA‐USP10 or HA‐USP10 CA and treated with obestatin (10 nM, 3 h). Data were expressed as the mean ± SEM obtained from intensity scans ( n = 3; *,# p < 0.05). (C) Immunoprecipitation analysis of the AMPKα ubiquitination in DMD cells after obestatin (10 nM, 20 min) treatment and in the presence of MG132. Analysis of ubiquitin K48 and K63 linkage was developed by immunoblot. Data were expressed as mean ± SEM ( n = 3 per group; * p < 0.05). (D) Coimmunoprecipitation analysis of DMD cells treated with obestatin (10 nM, 20 min) by using anti‐USP10 antibody and immunoblotted with anti‐pAMPK(T172), anti‐AMPKα, anti‐NEDD4‐L, or anti‐USP10 antibody. Data were expressed as mean ± SEM ( n = 3 per group; * p < 0.05). (E) Coimmunoprecipitation analysis of DMD cells treated with obestatin (10 nM, 20 min) by using anti‐AMPKα antibody and immunoblotted with anti‐USP10, anti‐NEDD4‐L, anti‐pAMPK(T172), or anti‐AMPKα antibody. Data were expressed as mean ± SEM ( n = 3 per group; *,# p < 0.05). In panels (A)–(E), immunoblots are representative of the mean value.

Article Snippet: To knockdown NEDD4, AMPKα or USP10 expression, siRNA specifically targeting human NEDD4‐L (sc‐75894; Santa Cruz Biotechnology, CA, USA), AMPKα (sc‐45312; Santa Cruz Biotechnology), or USP10 (sc‐76811; Santa Cruz Biotechnology) was used. siRNA duplexes targeting preproghrelin or c‐Src were selected from ON‐TARGET plus SMARTpool siRNA from Thermo Fisher Scientific (Dharmacon, CO, USA). shRNA‐mediated gene silencing was used for ULK1 knockdown (sc‐44182‐SH; Santa Cruz Biotechnology) in DMD cells.

Techniques: Western Blot, Transfection, Immunoprecipitation, Ubiquitin Proteomics

Journal: MedComm

Article Title: Obestatin Treatment Counteracts Muscle Wasting by Reactivation of Autophagy in Duchenne Muscular Dystrophy

doi: 10.1002/mco2.70563

Figure Lengend Snippet: Tyrosine switch on NEDD4‐L facilitates activation of autophagy through the USP10 recruitment and the VPS34 stabilization in human DMD myotubes. (A) Immunoblot analysis of pc‐Src(Y416), c‐Src, pAMPK(T172), or AMPK in DMD myotubes transfected with sicontrol or si‐c‐Src and treated with obestatin (10 nM, 3 h). Data were expressed as the mean ± SEM ( n = 3 per group; * p < 0.05). (B) Coimmunoprecipitation analysis of DMD cells treated with obestatin (10 nM, 20 min) by using anti‐NEDD4‐L antibody and immunoblotted with ubiquitin, pY, pc‐Src(Y416), USP10, VPS34, or NEDD4‐L antibody. Data were expressed as mean ± SEM ( n = 3 per group; * p < 0.05). In panels (A) and (B), immunoblots are representative of the mean value. (C) Proposed model by which NEDD4‐L regulates autophagy via obestatin signaling under dystrophic conditions. Tyrosine switch on NEDD4‐L activates autoubiquitination that serves as a scaffold to recruit the deubiquitinase enzyme USP10 to form a deubiquitination complex, which stabilizes VPS34 to promote autophagy through the removal of the ubiquitin chains on VPS34 and activation of Beclin1 complex. In parallel, NEDD4‐L favors AMPK exposure to CaMKKß and consequent activation of ULK1. Under DMD conditions, AMPK does not inhibit mTORC1, but sustains ULK1/Beclin1 activity and autophagy. In nonpathological conditions, lack of action of non‐RTK on NEDD4‐L favors interaction between c‐Src and NEDD4‐L, thus impairing the VPS34 stabilization and AMPK activation.

Article Snippet: To knockdown NEDD4, AMPKα or USP10 expression, siRNA specifically targeting human NEDD4‐L (sc‐75894; Santa Cruz Biotechnology, CA, USA), AMPKα (sc‐45312; Santa Cruz Biotechnology), or USP10 (sc‐76811; Santa Cruz Biotechnology) was used. siRNA duplexes targeting preproghrelin or c‐Src were selected from ON‐TARGET plus SMARTpool siRNA from Thermo Fisher Scientific (Dharmacon, CO, USA). shRNA‐mediated gene silencing was used for ULK1 knockdown (sc‐44182‐SH; Santa Cruz Biotechnology) in DMD cells.

Techniques: Activation Assay, Western Blot, Transfection, Ubiquitin Proteomics, Activity Assay

Journal: Autophagy

Article Title: Pycard deficiency inhibits microRNA maturation and prevents neointima formation by promoting chaperone-mediated autophagic degradation of AGO2/argonaute 2 in adipose tissue.

doi: 10.1080/15548627.2023.2277610

Figure Lengend Snippet: Figure 3. AGO2 is degraded through chaperone-mediated autophagy pathway. Stromal vascular fraction isolated from WT mice were differentiated into adipocytes and used for the following experiments. (A-B) immunoblotting analysis of AGO2 expression in the cells treated with 10 µM lactacystin for indicated time. (C-D) cells were treated with NH4Cl combined with leupeptin for indicated time, and AGO2 level was measured. (E-G) cells were transfected with control siRNA (siCtrl) or cathepsin B siRNA (siCtsb) or cathepsin D siRNA (siCtsd), and AGO2 level was analyzed. (H-I) immunoblotting analysis of AGO2 expression in the cells transfected with control siRNA (siCtrl) or Ulk1 siRNA (siUlk1). (J-K) cells were treated with 6-AN or starvation for indicated time, and AGO2 level was measured. (L-N) cells were transfected with control siRNA (siCtrl) or Hspa8 siRNA (siHspa8) or Lamp2 siRNA (siLamp2), and AGO2 expression was analyzed. (O) interaction of AGO2 and HSPA8 in adipose tissue were detected by immunoprecipitation and immunoblotting analysis. (P) co-staining of AGO2 and lysosomal HSPA8 in abdominal adipose tissue. Red: AGO2; green: HSPA8; blue: DAPI. (Q) the interaction of AGO2 and LAMP2 in adipose tissue was detected by immunoprecipitation and immunoblotting analysis. (R) co-staining of AGO2 and LAMP2 in abdominal adipose tissue. Red: AGO2; green: LAMP2; blue: DAPI. Data are presented as mean ± SD. * p < 0.05, ** p < 0.01, and n. S. non-significant, derived from Student’s t tests.

Article Snippet: For transfections of Cathepsin B siRNA (Santa Cruz Biotechnology, sc -29,933), Cathepsin D siRNA (Santa Cruz Biotechnology, sc -29,934), Hspa8 siRNA (Santa Cruz Biotechnology, sc -35,593), Lamp2 siRNA (Santa Cruz Biotechnology, sc -35,791), Ulk1 siRNA (Santa Cruz Biotechnology, sc -44,849), Prmt8 siRNA (Santa Cruz Biotechnology, sc -152,473), and Mir106b mimic in adipocytes, the cells that had been differentiated for 6 days were electroporated using SE Primary Cell 4D-Nucleofector X Kit (Lonza, V4XC– 1012) according to the manufacturer’s instructions.

Techniques: Isolation, Western Blot, Expressing, Transfection, Control, Immunoprecipitation, Staining, Derivative Assay

Journal: Autophagy

Article Title: Pycard deficiency inhibits microRNA maturation and prevents neointima formation by promoting chaperone-mediated autophagic degradation of AGO2/argonaute 2 in adipose tissue.

doi: 10.1080/15548627.2023.2277610

Figure Lengend Snippet: Figure 4. Pycard deficiency promotes CMA-mediated AGO2 degradation in adipocytes. Stromal vascular fraction isolated from WT and pycard−/− mice were differentiated into adipocytes and used for the following experiments. (A-B) cells were treated with NH4Cl (N) combined with leupeptin (L) for 24 h. AGO2 level was detected by immunoblotting analysis. The ratio of N/L to veh treatment after normalization by endogenous control. (C-D) cells were transfected with control siRNA (siCtrl) or Hspa8 siRNA (siHspa8), AGO2 protein level was measured by immunoblotting analysis. (E-F) cells were transfected with control siRNA (siCtrl) or Lamp2 siRNA (siLamp2), and western blot was performed to measure AGO2 protein expression. (G-H) cells were transfected with control siRNA (siCtrl) or Ulk1 siRNA (siUlk1), andAGO2 protein expression was analyzed by immunoblotting analysis. (I) the interaction of AGO2 and HSPA8 in differentiated adipocytes was determined by immunoprecipitation and immunoblotting analysis. (J) the interaction of AGO2 and LAMP2 was analyzed in differentiated adipocytes. Data are presented as mean ± SD. * p < 0.05, ** p < 0.01, and n.S. non-significant, derived from Student’s t tests.

Article Snippet: For transfections of Cathepsin B siRNA (Santa Cruz Biotechnology, sc -29,933), Cathepsin D siRNA (Santa Cruz Biotechnology, sc -29,934), Hspa8 siRNA (Santa Cruz Biotechnology, sc -35,593), Lamp2 siRNA (Santa Cruz Biotechnology, sc -35,791), Ulk1 siRNA (Santa Cruz Biotechnology, sc -44,849), Prmt8 siRNA (Santa Cruz Biotechnology, sc -152,473), and Mir106b mimic in adipocytes, the cells that had been differentiated for 6 days were electroporated using SE Primary Cell 4D-Nucleofector X Kit (Lonza, V4XC– 1012) according to the manufacturer’s instructions.

Techniques: Isolation, Western Blot, Control, Transfection, Expressing, Immunoprecipitation, Derivative Assay

Journal: Autophagy

Article Title: Pycard deficiency inhibits microRNA maturation and prevents neointima formation by promoting chaperone-mediated autophagic degradation of AGO2/argonaute 2 in adipose tissue.

doi: 10.1080/15548627.2023.2277610

Figure Lengend Snippet: Figure 5. Pycard deficiency promotes PRMT8-mediated AGO2 methylation and increases AGO2 binding to HSPA8. (A) the differentiated WT and pycard−/− adipocytes were treated with NH4Cl combined with leupeptin for 24 h. Acetylated AGO2 level was determined by immunoprecipitation (IP) of acetylate lysin (ac-lysin) and followed by immunoblotting analysis of AGO2. (B) the differentiated WT and pycard−/− adipocytes were treated with NH4Cl combined with leupeptin for 24 h. Methylated AGO2 level was determined by IP of AGO2 and followed by immunoblotting analysis of either SYM10 or ASYM24. (C) Messenger RNA levels of PRMTs in the differentiated WT and pycard−/− adipocytes were analyzed by qRT-PCR. ** p < 0.01. (D) immunoblotting analysis of PRMT8 in epididymal adipose tissue (EpiAdi) or perivascular adipose tissue (PVAT) collected from WT or pycard−/− mice. (E) the differentiated WT or pycard−/− adipocytes were transfected with control siRNA (siCtrl) or Prmt8 siRNA (siPrmt8), protein levels of AGO2 and PRMT8 were detected by immunoblotting analysis. (F) primary mature adipocytes were treated with PRMT8 inhibitor (PRMT8i, GSK3368715, 2 nM) or vehicle (veh) for 48 h. AGO2 methylation level was determined by IP and immunoblotting analysis. (G) primary mature adipocytes were treated with PRMT8 inhibitor (PRMT8i, GSK3368715, 2 nM) or vehicle (veh) for 48 h. The interaction between AGO2 and HSPA8 was determined by IP and immunoblotting analysis. Data are presented as mean ± SD. * p < 0.05, ** p < 0.01, and n.S. non-significant, derived from Student’s t tests.

Article Snippet: For transfections of Cathepsin B siRNA (Santa Cruz Biotechnology, sc -29,933), Cathepsin D siRNA (Santa Cruz Biotechnology, sc -29,934), Hspa8 siRNA (Santa Cruz Biotechnology, sc -35,593), Lamp2 siRNA (Santa Cruz Biotechnology, sc -35,791), Ulk1 siRNA (Santa Cruz Biotechnology, sc -44,849), Prmt8 siRNA (Santa Cruz Biotechnology, sc -152,473), and Mir106b mimic in adipocytes, the cells that had been differentiated for 6 days were electroporated using SE Primary Cell 4D-Nucleofector X Kit (Lonza, V4XC– 1012) according to the manufacturer’s instructions.

Techniques: Methylation, Binding Assay, Immunoprecipitation, Western Blot, Quantitative RT-PCR, Transfection, Control, Derivative Assay