Lc3a b 1 2

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LC3A/B-I/II is a protein that is involved in the process of autophagy, which is a cellular mechanism for recycling and degrading damaged or unwanted cellular components. This protein exists in two forms, LC3A/B-I and LC3A/B-II, which can be detected and quantified using appropriate laboratory techniques. The primary function of this protein is to play a role in the formation and maturation of autophagosomes, which are vesicles that engulf the cellular components targeted for degradation.

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6 protocols using lc3a b 1 2

Western Blot Analysis of Liver Proteins

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The weighed liver samples were homogenized in RIPA lysis buffer (Beyotime Biotechnology, Shanghai, China) and centrifuged to obtain the supernatant, and the protein concentration was measured using a protein analysis kit (Beyotime Biotechnology, Shanghai, China). Protein preparations (20 μg) were separated by electrophoresis using 6%, 8%, and 12% SDS-polyacrylamide gels (SDS-PAGE) and then transferred onto polyvinylidene fluoride (PVDF) membranes. The membranes were blocked with 5% skim milk in 0.1% Tween-20 (TBST) buffer for 1 h and further incubated with specific primary antibodies GAPDH (#5174, Cell signaling, Danvers, MA, USA), SIRT1 (A17307, Abclonal, Wuhan, China), PGC-1α (A12348, Abclonal, Wuhan, China), P62 (A11247, Abclonal, Wuhan, China), and LC3A/B-I/II (4108S, Cell signaling, Danvers, MA, USA). The membranes were then incubated with the secondary antibody Goat Anti-Rabbit IgG H&L (HRP) (1:5000, ab205718; Abcam), and each membrane was ultimately scanned using a CCD camera (Chemidoc MP Imaging System, BioRad) to evaluate the intensity of bands.

Wu L., Chen Q., Dong B., Geng H., Wang Y., Han D., Zhu X., Liu H., Zhang Z., Yang Y., Xie S, & Jin J. (2023). Resveratrol alleviates lipopolysaccharide-induced liver injury by inducing SIRT1/P62-mediated mitophagy in gibel carp (Carassius gibelio). Frontiers in Immunology, 14, 1177140.

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Western Blot Analysis of Autophagy Markers

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Total protein was extracted from samples with RIPA lysis buffer, separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred onto 0.45 μm PVDF membranes (Millipore). The following primary antibodies were used in the assays: antibodies for cleaved caspase-3 (no. 9654; Cell Signaling Technology, Danvers, MA, USA), GRP78 (no. 3183; Cell Signaling Technology), CHOP (no. 2895; Cell Signaling Technology), LC3A/B II/I (no. 12741; Cell Signaling Technology), p62 (no. 5114; Cell Signaling Technology), ATG-5 (no. 12994; Cell Signaling Technology), ATG-7 (no. 8558; Cell Signaling Technology), mTOR (no. 2983; Cell Signaling Technology), p-mTOR (Ser2448) (no. 5536; Cell Signaling Technology), p38 MAPK (no. 9212; Cell Signaling Technology), and p-p38 MAPK (no. 4511; Cell Signaling Technology), and β-actin (no. 3700; Cell Signaling Technology) was chosen as loading control.

Wu H, & Chang Q. (2018). The cryoprotectant trehalose could inhibit ERS-induced apoptosis by activating autophagy in cryoprotected rat valves. PLoS ONE, 13(3), e0194078.

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Autophagy Modulation Enhances Osteogenic Differentiation

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BMSCs at a density of 5 × 10⁴ cells/well were seeded in six-well plates. For autophagic inhibition, BMSCs were treated with 2 mM 3-methyladenine (3-MA, Sigma, United States) with or without leonurine (10 μM). First, 3-MA was dissolved in dimethyl sulfoxide (DMSO) prior to addition to the culture medium, with the final concentration of DMSO below 0.1% of the medium. Alizarin red staining was performed for 14 days and ALP staining was performed for 6 days as described above.
Additionally, WB and qPCR were performed with the no treatment (control) group, leonurine treatment group, 3-MA treatment group, and 3-MA with leonurine treatment group. The antibodies LC3AB I/II (1:1,000) and ATG7 (1:500) used in WB analyses were purchased from Cell Signaling Technology (CST, Beverly, MA, United States); P62 (1:1,000) was purchased from Abcam (Cambridge, MA, United States). The primers used in the qPCR analysis are listed in Table 1.

Zhao B., Peng Q., Poon E.H., Chen F., Zhou R., Shang G., Wang D., Xu Y., Wang R, & Qi S. (2021). Leonurine Promotes the Osteoblast Differentiation of Rat BMSCs by Activation of Autophagy via the PI3K/Akt/mTOR Pathway. Frontiers in Bioengineering and Biotechnology, 9, 615191.

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Western Blot Analysis of Autophagy and ER Stress Markers

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Sample preparation and western blotting were performed as previously described [51 (link)]. Anti-Atg5-Atg12 complex (#4180), -Atg16L1 (#8089), -Bak (#12105), -Beclin1 (#3495), -Bim (#2933), -Calnexin (#2679), -CHOP (#2895), -cleaved PARP (#9541), -cytochrome c (#4280), -Ero1-Lα (#3264), -ERp44 (#3798), -ERp72 (#5033), -GRP94 (#2104), -LC3A/B-I/II (#12741), -Mcl-1 (#5453), -PDI (#3501) and -VDAC (#4661) antibodies were from Cell Signaling Technology (Beverly, MA, USA). Anti-β-actin (ab8226), -Bcl-2 (ab692), -cleaved caspase-3 (ab2302), -GAPDH (ab9485), -GRP78 (ab21685) and -p62 (ab56416) antibodies were from Abcam (Cambridge, MA, USA). Anti-XBP1s (sc-7160) antibody was from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Anti-cleaved ATF6 (NBP1-40256) antibody was from Novus Biologicals (Littleton, CO). Horseradish peroxidase-conjugated goat anti-mouse and anti-rabbit immunoglobulin G secondary antibodies were from GenDEPOT (Barker, TX, USA). The blots were developed using a chemiluminescent detection kit (Ab Frontier, Seoul, Republic of Korea). Densitometric quantification of western blot bands was performed using Image J software, version 1.49 (http://rsb.info.nih.gov/ij/index.html).

Lee S., Kim S., Hwang S., Cherrington N.J, & Ryu D.Y. (2017). Dysregulated expression of proteins associated with ER stress, autophagy and apoptosis in tissues from nonalcoholic fatty liver disease. Oncotarget, 8(38), 63370-63381.

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Apoptosis and Cell Cycle Regulation Assays

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Phosphate-buffered saline (PBS), fetal bovine serum (FBS), RPMI-1640, and trypsin were obtained from Gibco (Grand Island, NY, USA). The A549 cell line was obtained from the American Type Culture Collection (Manassas, VA, USA) and was cultured in RPMI-1640 medium containing 10% FBS and 1% penicillin-streptomycin at 37 °C in an atmosphere of 5% CO₂. Cytochrome c, Bax, Bcl-2, PARP, cleaved PARP, caspase-3, cleaved caspase-3, caspase-9, cleaved caspase-9, p53, p21, Cyclin A2, Cyclin E1, Cyclin B1, CDK2, Erk, p-Erk, JNK, p-JNK, p38, p-p38, mTOR, p-mTOR, Akt, p-Akt, FAK, p-FAK, PI3K, p-PI3K, p62, LC3A/B- I/II, β-actin, GAPDH and all secondary antibodies were acquired from Cell Signaling Technology (Beverly, MA, USA). Necrostatin-1 (Nec-1, an inhibitor of RIP1K), Z-VAD-FMK (an inhibitor of pan caspase), Z-DEVD-FMK (an inhibitor of caspase-3), N-Acetyl-L-cysteine (NAC), mitochondrial membrane potential assay kit for JC-1, reactive oxygen species (ROS) assay kit, cell cycle and apoptosis analysis kit, and DAPI were obtained from Beyotime Institute of Biotechnology (Shanghai, China). Smp24 and FITC-labeled Smp24 were synthesized as reported in our previous study [11 (link)].

Guo R., Chen X., Nguyen T., Chai J., Gao Y., Wu J., Li J., Abdel-Rahman M.A., Chen X, & Xu X. (2022). The Strong Anti-Tumor Effect of Smp24 in Lung Adenocarcinoma A549 Cells Depends on Its Induction of Mitochondrial Dysfunctions and ROS Accumulation. Toxins, 14(9), 590.

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Western Blot Analysis of Cav1, Autophagy, and Mitophagy

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Cells were washed three times with ice-cold 1× PBS (Gibco), and then lysed with RIPA Buffer including Protease and Phosphatase Inhibitors (Roche Lifesciences). Protein concentration was measured using the Bradford reagent (BioRad) and equal amounts of proteins (20 µg per lane) were resolved on SDS-PAGE (BioRad). Proteins were electro-transferred onto polyvinylidene-difluoride (PVDF) membrane. After blocking with 5% nonfat milk, blots were incubated with α-Cav1 (Cell Signaling, Cat# 3267S, 1:1000 dilution), GAPDH (Cell Signaling, Cat# 2118, 1:1000 dilution); autophagy marker LC3A/B-I/II (Cell Signaling, Cat# 12741, 1:1000 dilution) or mitophagy markers Parkin and PINK1 (Cell Signaling, Cat# 4211 and 6946, respectively, both at 1:1000 dilution). Uncropped original immunoblot scans are provided in Supplementary Fig. 9.

Vykoukal J., Fahrmann J.F., Gregg J.R., Tang Z., Basourakos S., Irajizad E., Park S., Yang G., Creighton C.J., Fleury A., Mayo J., Paulucci-Holthauzen A., Dennison J.B., Murage E., Peterson C.B., Davis J.W., Kim J., Hanash S, & Thompson T.C. (2020). Caveolin-1-mediated sphingolipid oncometabolism underlies a metabolic vulnerability of prostate cancer. Nature Communications, 11, 4279.

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