Anti beclin1

The thymol (purity > 98.5%), LPS (Escherichia coli 055:B5, L2880), and dimethyl sulfoxide (DMSO, D4540) were obtained from Sigma-Aldrich (St. Louis, MO, USA). The mouse monoclonal anti-β-actin (AA128), HRP-labeled goat anti-rabbit IgG (H + L), and HRP-labeled goat anti-mouse IgG (H + L) antibodies (A0208, A0216) were purchased from Beyotime (Shanghai, China). The following antibodies were used in this work: anti-IκB α (4814, Cell Signaling Technology, Danvers, MA, USA), anti-phospho-NF-κB p65 (Ser536, 3033S, Cell Signaling Technology), anti-NLRP3 (AF2155, Beyotime), anti-IL-1β (AF7209, Beyotime), anti-beclin1 (ab231341, Abcam), anti-ATG7 (AA820, Beyotime), anti-LC3B (ab229327, Abcam, Cambridge, UK), anti-phospho-AMPK-α (Thr172, Cell Signaling Technology), anti-phospho-mTOR (Ser2448, D9C2, Cell Signaling Technology), anti-caspase-3 (WL02117, Wanleibio, Shenyang, China), and anti-cleaved caspase-9 (WL01838, Wanleibio).

Forty-eight hours after transfection, cells were fixed with Cytofix/Cytoperm™ (BD Biosciences) and incubated with anti-Beclin 1 (1:200; Abcam, Cambridge, UK) or anti-Fibulin 5 (1:200; Biorbyt, Cambridge, UK) antibodies for 40 min. Next, the cells were incubated with secondary antibodies, as appropriate, for 30 min. The transfected cells expressing GFP protein encoded by the pSELECT-GFPzeo backbone and protein of interest were analyzed with a BD FACS Canto II Cytometer and BD FACS Diva Software v. 6.1.3 (BD Biosciences, San Jose, CA, USA).

The total protein of lung tissues and cells was collected with RIPA buffer (Servicebio, Wuhan, China) with cocktail and phosphatase inhibitors (Thermo Fisher Scientific, Wuhan, China), followed by electrophoresed through 8–12% sodium dodecyl sulfate–polyacrylamide gel and then transferred to 0.45 μm PVDF membrane (Merck Millipore, USA). A total of 30 ug of protein was used for western blot experiments. After blocked with 5% milk, the membrane was immunoblotted with following primary antibodies(Except for labeled, all from Cell Signaling Technology, MA, USA) at 4 °C overnight: anti-ERRα (1:1000), anti-ZO-1 (1:1000; Abcam), anti-VE-cadherin (1:1000), anti-Occludin(1:1000), anti-JAM-A(1:1000; Abcam), anti-Sirt3 (1:1000), anti-Bcl-2 (1:1000), anti-Bax (1:1000), anti-Smac (1:1000), anti-Cytochrome c (1:1000), anti-caspase 3 (1:1000), anti-cleaved caspase 3 (1:500), anti-caspase 9 (1:1000), anti-cleaved caspase 9 (1:500), anti-LC3A/B(1:1000), anti-Beclin1 (1:1000), anti-p62 (1:1000), or anti-GAPDH (1:10000, Abcam, internal control). After incubated with the HRP-conjugated secondary antibodies at 25℃ for 1 h, the immunoblots were visualized using the ECL system. The gray value was analyzed using the Image J and calculate the relative protein level based on the density ratio of the target protein to GAPDH (internal control).

The total cell lysate was prepared using RIPA buffer as described [49 (link)] and Western blotting was carried out as detailed [50 (link)]. The following antibodies were used: rabbit polyclonal anti-Beclin1 (1:2000, Abcam), Anti-Bcl2 (1:500, Abcam), Anti-CD9 antibody (1:250 Abcam) and anti-rabbit or anti-mouse secondary antibodies conjugated to horse-radish-peroxidase (1:20,000, Abcam) and β-Actin (1:5000, Santacruz Inc.,). Following TBST washes, protein bands were visualized using ECL and ChemiDoc Imaging System and quantified with Image-J.

As previously mentioned^{48 (link)}, proteins were isolated and electroblotted. The membranes were blocked for 1 h with 5% BSA (ZSGB-BIO, Beijing, China) and incubated overnight at 4℃ with the primary antibodies (anti-Beclin-1, 1:1000dilution; anti-LC3, 1:2000 dilution; anti-RUNX2, 1:1000 dilution; anti-P62/SQSTM1, 1:5000 dilution, all from Abcam, MA, USA). After washing, membranes were incubated for 1 h with the secondary horseradish peroxidase antibody (Solarbio, Beijing, China). Finally, the intensity of the bands was determined using the Bio-Rad VersaDoc imaging system and the ECL kit (Solarbio, Beijing, China).

The antibodies used in this study were as follows: anti-P2X7 (Abcam, Cambridge, UK; cat. no. ab109054), anti-collagen II (Abcam; cat. no. ab34712), anti-MMP13 (Abcam; cat. no. ab39012), anti-AMPKα1 (Abcam; cat. no. ab32047), anti-mTOR (Abcam; cat. no. ab109268), anti-NLRP3 (Proteintech; cat. no. 19771-1-AP), anti-caspase-1 (Proteintech; cat. no. 22915-1-AP), anti-LC3B (Abcam; cat. no. ab192890), anti-Beclin-1 (Abcam; cat. no. ab62557), anti-glyceraldehyde 3-phosphate dehydrogenase (GAPDH; Proteintech; cat. no. 10494-1-AP), and horseradish peroxidase (HRP)-labeled IgG (Beyotime; cat. no. A0208). The reagents used in the experiment were as follows: MIA (Sigma, St. Louis, MO, USA; cat. no. I2512), the P2X7 receptor agonist BzATP (Sigma; cat. no. B6396), the mTOR activator MHY1485 (Sigma; cat. no. SML0810), the mTOR inhibitor rapamycin (Sigma; cat. no. V900930), the NLRP3 inhibitor CY-09 (Sigma; cat. no. SML2465), the AMPK activator A-769662 (Sigma; cat. no. SML2578), and the AMPK inhibitor compound C (Sigma; cat. no. P5499). The concentrations, dosages, and preparation of the reagents were described previously [43 (link), 45 (link)].

Icariin (ICA, purity >98.61%) was purchased from Cheng Du Purechem-Standard Co., Ltd. (Chengdu, China). Haematoxylin, eosin, Nissl staining solution and ECL chemiluminescence detection kit were purchased from Servicebio (Wuhan, China), RIPA buffer and BCA protein assay kit were purchased from Applygen (Beijing, China), PVDF was purchased from Millipore (MA, USA). Anti-LC3B (#83506), anti-p-ULK1 (ser757) (14202S), anti-p-mTOR (#5536), anti-p-AMPK (#2535) were purchased from Cell Signalling Technology (MA, USA). Anti-LC3 (14600-1-AP) was purchased from Proteintech (Wuhan, China). Anti-p62 (ab56416) and anti-Beclin1 were purchased from Abcam (Cambridge, UK). Anti-Atg5 (NB110-53818SS) was purchased from Novus Biologicals (CO, USA). Anti-NeuN (#2967854) was purchased from Millipore (MA, USA). Alexa Fluor 488 Donkey anti-Mouse IgG (H + L) (138499) and Alexa Fluor 594 Donkey anti-Rabbit IgG (H + L) (140019) were purchased from Jackson ImmunoResearch (PA, USA).