Skimmed milk

The cultures used in the study, namely Lactobacillus rhamnosus K4E and Lactobacillus helveticus K14 (NCBI GenBank Accession No. KX950834 and KU644578), were isolated from the various ethnic fermented foods of Meghalaya, India, and deposited in Animal Science Laboratory, Department of Rural Development and Agricultural Production, North-Eastern Hill University, Tura Campus, Meghalaya, India (14 (link)). These isolates were propagated in De Man, Rogosa and Sharpe (MRS) agar; HiMedia, Mumbai, India) for 24 h at 37 °C. Their probiotic potential was studied earlier and they were selected for this study due to their maximum probiotic characteristics (3 (link)). Small, smooth yellow seeds of a local variety of soybeans were obtained from a local market in Tura town (West Garo Hills, Meghalaya). The pure lactic cultures (L. rhamnosus K4 and L. helveticus K14) grown in MRS were transferred into sterile rehydrated skimmed milk and glycerol (both HiMedia), and 1-mL aliquots were placed in cryovials and stored at −20 °C. After two successive transfers of the test organisms in MRS broth (HiMedia) and incubation at 37 °C for 24 h, each activated culture was inoculated into skimmed milk and incubated at 37 °C for another 16 h. These cultures were then transferred into soy milk to check their activity (8 (link)).

Immunoblotting was performed following protocols as described previously [20 (link)]. Cells were lysed in modified RIPA buffer (Sigma-Aldrich) and protein content was measured using Bradford reagent (Thermo Scientific). The cellular protein lysates were run in denaturing polyacrylamide gels and thereafter transferred to PVDF membrane (Millipore) for blocking with 5% skimmed milk (HiMedia). The blots were then probed or re-probed with specific primary antibodies and detected using enhanced chemi-luminescence (ECL) detection system following the manufacturer's protocol. The primary antibodies used were as follows: anti-p38, anti-JNK, anti-ERK1/2, anti-PARP-1, anti-ATG and anti-LC3B-II. The secondary antibodies were horseradish peroxide-conjugated goat anti-rabbit IgG. Expression was quantitated using ImageJ software.

Cell culture media components such as DMEM, RPMI-1640 medium, Ham’s F12 K medium, penicillin, streptomycin, Hi-Sep Lymphocyte separation medium, HiKaryo XL RPMI medium, MTT, acridine orange, BSA, skimmed milk, prestained protein marker and tween 20 were purchased from HiMedia Laboratories, Mumbai, India. DMSO, trypan blue, Hoechst 33258, DCFH-DA, Rhodamine-123, Tris base, TRI reagent, Oligo(dT)-18 mer primers, Protease/phosphatase Inhibitor Cocktail and KBr were purchased from Sigma-Aldrich, USA. Fetal bovine serum, Dulbecco’s Phosphate Buffered Saline (DPBS), Trypsin–EDTA, Alexa Fluor 488 Annexin V/Dead Cell Apoptosis Kit, Fluo-3 AM, propidium iodide and Geltrex LDEV-Free Reduced Growth Factor Basement Membrane Matrix were purchased from Thermo Fisher Scientific, USA. M-MuLV reverse transcriptase enzyme, RNase A, dNTP mix and agarose were purchased from Genei Laboratories Pvt. Ltd, Bangalore, India. Triton X-100, EDTA, glycine, ethidium bromide, BCIP, NBT, silica gel for column chromatography (60–120 meshes), vanillin, sulphuric acid and solvents such as petroleum ether, chloroform, acetone, methanol (all HPLC grade) were procured from Sisco Research Laboratories Pvt. Ltd., Mumbai, India. Antibodies were procured from Cell Signaling Technology, Inc., USA and Sigma-Aldrich, USA. Primers sets were purchased from Eurofins Genomics India, Bangalore, India.

Western blotting was performed following methods described elsewhere [18 (link)]. Briefly, RIPA buffer (Sigma-Aldrich) was used for the extraction of proteins, and Bradford reagent for estimation. Proteins were loaded in lab-made denaturing polyacrylamide gels and transferred to polyvinylidene fluoride membranes (PVDF, Bio-Rad). Skimmed milk (HiMedia) was used for blocking. The blots were probed and re-probed with specific primary antibodies (dilution 1:1000) and detected using enhanced chemiluminescence (ECL; Thermo Fisher Scientific) on ChemiDoc (Bio-Rad). GAPDH (dilution 1:2000) was used as a loading control. Wherever required, the blots were cut to probe with different antibodies against proteins of different molecular weights. The make of antibodies is mentioned in the 'chemical and reagents' section.

Cells and tissue were homogenized in ice-cold lysis buffer with 1X protease inhibitory cocktail (Sigma Aldrich, USA). Total protein content was quantified using Bradford reagent (Bio-Rad, USA). Equal amounts of protein from each sample were loaded on 10% gel for SDS-PAGE. Protein was further transferred to PVDF membrane (Bio-Rad, USA) and blocked with 5% skimmed milk (HiMedia) following overnight incubation in primary antibody (SIRT-1, 1:500; PGC-1α, 1:700) prepared in 3% BSA. Secondary antibody (anti-rabbit, 1:1000; anti-mouse, 1:5000) treatment was done for 1 h, and blots were developed using ECL reagent. The membrane was stripped using stripping buffer and was re-probed with β-actin (1:1000).