Caspase 9

The potential apoptotic effect of GS 1 mg/mL were assessed by Western blot analysis of caspases 3 and 9 and Poly (ADP-ribose) polymerase (PARP). Samples were prepared as previously reported for cell cycle proteins analysis and PVDF membranes were incubated with caspase 3 (Cell Signaling, 1:1000), caspase 9 (Cell Signaling, 1:1000) and PARP (Cell signaling 1:1000) in 2.5% milk in TBS-T overnight at 4 °C.

Proteins were separated on 10% or 12% SDS-PAGE gel and transferred to PVDF membranes (Millipore, IPVH00010), which were blocked with 5% BSA (Genview, FA016). Next PVDF membranes were incubated with the following antibodies: SREBP1 (PA1-337, ThermoFisher), Bax (50599-2-AP, Proteintech), Bcl2 (12789-1-AP, Proteintech), Caspase 3 (19677-1-AP, Proteintech), Caspase 9 (#9502, Cell Signaling Technology), AKT (#4691, Cell Signaling Technology), p-AKT (#4060, Cell Signaling Technology), JNK (10023-1-AP, Proteintech), p-JNK (80024-1-RR, Proteintech) and β-Actin (AC004, Abconal). After washed 3 times with TBST buffer, PVDF membranes were then incubated with horseradish peroxidase-conjugated anti-rabbit or anti-mouse secondary antibodies (Thermo Fisher Scientific). Western blot band were visualized by digital gel image analysis system (TANON 5500) and Pro-Light chemiluminescence detection kit (TIANGEN, PA112-01).

For Western blotting, total protein extracts were obtained in cell lysis buffer containing 150 mM NaCl, 1 mM EDTA, 1% NP-40, 50 mM Tris (pH 8.0) as well as phosphatase and protease inhibitors. Protein concentrations were determined by BCA staining (#23225, Thermo Fisher Scientific, Hennigsdorf, Germany) as compared to BSA concentration standard. Following SDS polyacrylamide gel electrophoresis, proteins were blotted on nitrocellulose membranes, as described previously [55 (link)].
Several primary antibodies were derived from Cell Signaling Technology (Danvers, MA, USA): caspase-3 (9662, rabbit, 1:1000), caspase-8 (9746, mouse, 1:1000), caspase-9 (9502, rabbit, 1:1000), Mcl-1 (4572, rabbit, 1:1000), Bcl-w (2724, rabbit, 1:1000) and Bcl-2 (2872, rabbit, 1:1000). Other primary antibodies were derived from Santa Cruz Biotech (Dallas, TX, USA): Bcl-x_L (sc-8392, mouse, 1:1000) and β-actin (sc-47778, mouse, 1:1000). As secondary antibodies, peroxidase-labeled goat anti-rabbit and goat anti-mouse were used (Dako, Hamburg, Germany; 1:5000).

Dulbecco’s modified Eagle’s medium (DMEM), Hoechst 33,342, Fluoromount, 2′,7′-Dichlorodihydrofluorescein diacetate (DCFH-DA), and Rhodamine-123 and Fetal Bovine Serum (FBS) were purchased from Sigma (St. Louis, MO, USA). 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) and trypsin were procured from Hi-media Pvt. Limited, Mumbai (India). Rabbit monoclonal Bcl-xl, p-53, p-NF-κB, Caspase3, and Caspase9 antibodies and anti-rabbit- HRP secondary antibody were obtained from Cell Signaling Technology, Danvers, MA, USA. PVDF membrane (MDI, Ambala). The RT-PCR chemical kit was purchased from Bio-Rad, CA, USA. The BD Cycletest plus DNA Kit was from BD Biosciences, San Jose, CA, USA. Chemicals and reagents of analytical (AR) grade were used to perform the experiments.

Atorvastatin calcium, Mevalonic acid, GGPP, FPP, Propidium iodide (PI) and 2′,7′-dichlorofluorescein diacetate (DCFH-DA) were purchased from Sigma-Aldrich (St. Louis, MO, USA). 3-(4,5-Dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) was from Amresco (Solon, OH, USA). 5,5′,6,6′-tetrachloro-1, 1′,3,3′-tetraethylbenzimidazole-carbocyanide iodine (JC-1) and cell mitochondria isolation kit were obtained from Beyotime Biotechnology (Shanghai, China). Annexin V-FITC Apoptosis Detection Kit, as well as antibodies against p-pRb (S780) (1:1,000), p27 (1:1,000) and cyclinD1 (1:1,000) were from BD Biosciences Pharmingen (San Jose, CA, USA). Antibodies against pRb (1:1,000), cyclinB1 (1:2,000), cdc2 (1:1,000), caspase-3 (1:1,000), caspase-9 (1:1,000), poly (ADP-ribose) polymerase (PARP) (1:1,000), Cytochrome c (1:1,000), YAP (1:1,000), p-YAP (Ser127) (1:1,000), Rho A (1:1,000), β-actin (1:1,000), anti-mouse (1:2,000), anti-rabbit HRP-conjugated and anti-rabbit Alexa Fluor 488-conjugated secondary antibodies (1:2,000) were from Cell Signaling Technology (Danvers, MA, USA). Antibodies against Bcl-2 (1:1,000), Bax (1:1,000) and Lamin B (1:500) were from Santa Cruz Biotechnology (Santa Cruz, CA, USA).

Cells were lysed in RIPA buffer (Beyotime, Jiangsu, China) containing a protease inhibitor cocktail (Sigma, St. Louis, CA, USA). Total protein was assessed using a BCA Protein Assay Kit (Beyotime). Western blot analysis was performed as previously described [18 (link)]. Antibodies against the following were used in this study, PABPC1 (1:1000, Abcam, SF, USA), IFI27, HA tag, Flag tag, TSG101, CD63, CD9, PCNA, Alix, Ki67, caspase 3, CXCL10, CD34, cleaved-PARP, PARP, eIF4G, cleaved caspase 9, caspase 9, ERK, p-ERK, IFI27, STAT3, p-STAT3, STAT1, p-STAT1, NF-kB, p-NF-kB, β-actin and GAPDH (all 1:1000, Cell Signaling Technology, MA, USA), EXOSC2 and EXOSC4 (both 1:1000, Santa Cruz, MA, USA).

For immunoblotting and co-immunoprecipitation: Actin (Santa Cruz, ♯sc-47778; 1:5000), ATF6 (Cell Signaling Technology, ♯65880; 1:1000), BCL-2 (AbCam, ♯ab182858; 1:500), BCL-XL (Cell Signaling Technology, ♯2764; 1:1000), Cleaved-Caspase-8 (Cell Signaling Technology, ♯8592; 1:1000), Caspase-3 (Cell Signaling Technology, ♯9665; 1:1000), Caspase-9 (Cell Signaling Technology, ♯9508; 1:1000), CHOP (Cell Signaling Technology, ♯2895; 1:250), GFP (AbCam, ♯ab13970; 1:2000), GRP78 (Cell Signaling Technology, ♯3177; 1:1000), FLAG-HRP (Sigma Aldrich, ♯A8592; 1:4000), HA-HRP (Roche, ♯11867423001; 1:2000), IP₃R1 (Thermo Fischer, ♯PA1-901; 1:1000), IP₃R1 & IP₃R2 [previously described⁷⁷ ; 1:1000], IP₃R3 (BD Biosciences, ♯610312; 1:1000), KDEL (AbCam, ♯ab12223; 1:2000), MCL-1 (Cell Signaling Technology, ♯5453; 1:1000), Nicastrin (BD Biosciences, ♯612290; 1:1000), PARP (Cell Signaling Technology, ♯9542; 1:1000), SERCA2 (Cell Signaling Technology, ♯4388; 1:1000), STIM1 (Cell Signaling Technology, ♯5668; 1:1000).
For immunofluorescence: DAPI (Thermo Fischer, ♯D1306; 1 µg/ml), HA (Cell Signaling Technology, ♯3724; 1:500), BAP31 (Enzo Life Sciences, ♯ALX-804-601-C100; 1:250).
For proximity ligation assay: HA (Cell Signaling Technology, ♯3724; 1:500), HA (Enzo Life Sciences, ♯ENZ-ABS118-0200; 1:200), IP₃R1 (Thermo Fischer, ♯PA1-901; 1:200), IP₃R3 (BD Biosciences, ♯610312; 1:200).