Journal: Frontiers in Pharmacology

Article Title: Celastrol directly binds with VAMP7 and RAB7 to inhibit autophagy and induce apoptosis in preadipocytes

doi: 10.3389/fphar.2023.1094584

Figure Lengend Snippet: Celastrol bond with Vamp7 and Rab7 to inhibit autophagy and subsequently induce apoptosis (A–D) , 3T3-L1 preadipocytes were transfected with blank, Vamp7, Rab7 and Vti1b plasmids for 48 h, and then treated with 0, 1 and 2 μM celastrol for 24 h and subjected to phase contrast light microscope imaging (A) ( n = 4), flow cytometry analysis (B, C) ( n = 4), and Western blotting of P62 and LC3 I/II (D) ( n = 4) (E–H) , Surface Plasmon Resonance studies of VAMP7 with celastrol (E) and hesperidin (F) , RAB7 with celastrol (G) and hesperidin (H) were shown. (I) RAB7-GTP pull-down assay was performed in preadipocytes after 1 and 2 μM celastrol treatment for 16 h and shown by Western blotting of RAB7-GTP and total RAB7 ( n = 3). Protein expression was calculated relative to β -actin and depicted at the top of each blot. Error bars represent SEM; ns, no significance; *** p < 0.001. Veh, vehicle; CON, control; Cela, C, celastrol; 1C, 1 μM celastrol; 2C, 2 μM celastrol; NC, negative control.

Article Snippet: The SPR assays were performed to analyze the interactions between the compounds and VAMP7, RAB7 proteins (Sino Biological, China) by using a Biacore T100 machine with Sensor Chip CM5 (GE Healthcare, United States) at 25°C.

Techniques: Transfection, Light Microscopy, Imaging, Flow Cytometry, Western Blot, SPR Assay, Pull Down Assay, Expressing, Negative Control

Journal: Animal Nutrition

Article Title: Retinoic acid alleviates rotavirus-induced intestinal damage by regulating redox homeostasis and autophagic flux in piglets

doi: 10.1016/j.aninu.2023.12.007

Figure Lengend Snippet: Effects of retinoic acid (RA) administration on autophagy activation in jejunum from control and RA-treated groups. (A and B) Western blotting analysis of autophagy-related proteins including autophagy induction and regulation (pBeclin/Beclin, pULK1/ULK1, ATG7, ATG16 and ATG14), autophagy cargo recognition and degradation (p62, ATG5, ATG3), and autophagy maturation and lysosomal fusion (syntaxin, SNAP29, Rab7A and LC3I/II) ( n = 3). (C to G) Gray value analysis of the above protein expressions ( n = 3). KD = kiloDalton; Beclin = coiled-coil, moesin-like BCL2-interacting protein; ULK1 = Unc51-like kinase 1; ATG = autophagy-related gene; p62 = sequestosome 1; SNAP29 = synaptosome associated protein 29; RAB7A = RAS-related protein Rab-7a; LC3 = microtubule-associated protein light chain 3. ∗ P < 0.05, ∗∗ P < 0.01 and ∗∗∗ P < 0.001.

Article Snippet: Western blotting was conducted as previously described ( ) using primary antibodies against zonula occludens-1 (ZO-1) (13663, Cell Signaling Technology, Danvers, MA, USA), occludin (911131, Cell Signaling Technology, Danvers, MA, USA), claudin-1 (13255, Cell Signaling Technology, Danvers, MA, USA), pNRF2 (ab76026, Abcam, Cambridge, UK), NRF2 (16396-1-AP, Proteintech, Wuhan, China), heme oxygenase 1 (HO-1) (10701-1-AP, Proteintech, Wuhan, China), sequestosome 1 (p62) (ab56416, Abcam, Cambridge, UK), glutathione peroxidase 4 (GPX4) (ab125066, Abcam, Cambridge, UK), Unc51-like kinase 1 (ULK1) (8054, Cell Signaling Technology, Danvers, MA, USA), pULK1 (14202, Cell Signaling Technology, Danvers, MA, USA), autophagy-related gene (ATG) 7 (8558, Cell Signaling Technology, Danvers, MA, USA), ATG16 (8089, Cell Signaling Technology, Danvers, MA, USA), ATG14 (96752, Cell Signaling Technology, Danvers, MA, USA), coiled-coil, moesin-like BCL2-interacting protein (Beclin) (3495, Cell Signaling Technology, Danvers, MA, USA), pBeclin (14717, Cell Signaling Technology, Danvers, MA, USA), ATG5 (9980, Cell Signaling Technology, Danvers, MA, USA), ATG3 (3415, Cell Signaling Technology, Danvers, MA, USA), syntaxin (2869, Cell Signaling Technology, Danvers, MA, USA), synaptosome associated protein 29 (SNAP29) (sc-390602, Santa Cruz, Dallas, Texas, USA), RAS-related protein Rab-7a (RAB7A) (9367, Cell Signaling Technology, Danvers, MA, USA), LC3I/II (4108, Cell Signaling Technology, Danvers, MA, USA), and β-actin (3700, Cell Signaling Technology, Danvers, MA, USA).

Techniques: Activation Assay, Western Blot

Journal: Animal Nutrition

Article Title: Retinoic acid alleviates rotavirus-induced intestinal damage by regulating redox homeostasis and autophagic flux in piglets

doi: 10.1016/j.aninu.2023.12.007

Figure Lengend Snippet: Effects of retinoic acid (RA) administration on regulating autophagic pathways in jejunum from control, rotavirus (RV) and RV + RA groups. (A) Fluorescence density of RV in all six treatment groups in jejunum sample (red fluorescence indicates RV protein detection). Scale bar = 200 μm. (B and C) Western blotting analysis of autophagy-related proteins including autophagy induction and regulation (pBeclin/Beclin, pULK1/ULK1, ATG7, ATG16 and ATG14), autophagy cargo recognition and degradation (p62, ATG5 and ATG3), and autophagy maturation and lysosomal fusion (syntaxin, SNAP29, RAB7A and LC3I/II) ( n = 3). (D to I) Gray value analysis of the above protein expressions ( n = 3). KD = kiloDalton; Beclin = coiled-coil, moesin-like BCL2-interacting protein; ULK1 = Unc51-like kinase 1; ATG = autophagy-related gene; p62 = sequestosome 1; SNAP29 = synaptosome associated protein 29; RAB7A = RAS-related protein Rab-7a; LC3 = microtubule-associated protein light chain 3. ∗ P < 0.05, and ∗∗ P < 0.01.

Article Snippet: Western blotting was conducted as previously described ( ) using primary antibodies against zonula occludens-1 (ZO-1) (13663, Cell Signaling Technology, Danvers, MA, USA), occludin (911131, Cell Signaling Technology, Danvers, MA, USA), claudin-1 (13255, Cell Signaling Technology, Danvers, MA, USA), pNRF2 (ab76026, Abcam, Cambridge, UK), NRF2 (16396-1-AP, Proteintech, Wuhan, China), heme oxygenase 1 (HO-1) (10701-1-AP, Proteintech, Wuhan, China), sequestosome 1 (p62) (ab56416, Abcam, Cambridge, UK), glutathione peroxidase 4 (GPX4) (ab125066, Abcam, Cambridge, UK), Unc51-like kinase 1 (ULK1) (8054, Cell Signaling Technology, Danvers, MA, USA), pULK1 (14202, Cell Signaling Technology, Danvers, MA, USA), autophagy-related gene (ATG) 7 (8558, Cell Signaling Technology, Danvers, MA, USA), ATG16 (8089, Cell Signaling Technology, Danvers, MA, USA), ATG14 (96752, Cell Signaling Technology, Danvers, MA, USA), coiled-coil, moesin-like BCL2-interacting protein (Beclin) (3495, Cell Signaling Technology, Danvers, MA, USA), pBeclin (14717, Cell Signaling Technology, Danvers, MA, USA), ATG5 (9980, Cell Signaling Technology, Danvers, MA, USA), ATG3 (3415, Cell Signaling Technology, Danvers, MA, USA), syntaxin (2869, Cell Signaling Technology, Danvers, MA, USA), synaptosome associated protein 29 (SNAP29) (sc-390602, Santa Cruz, Dallas, Texas, USA), RAS-related protein Rab-7a (RAB7A) (9367, Cell Signaling Technology, Danvers, MA, USA), LC3I/II (4108, Cell Signaling Technology, Danvers, MA, USA), and β-actin (3700, Cell Signaling Technology, Danvers, MA, USA).

Techniques: Fluorescence, Western Blot