Image lab version 6

For performing western analysis to estimate Edc3-mCherry, Dhh1-GFP levels in wild-type and Δsbp1 under unstressed, stressed, and recovery conditions, an aliquot of cells were collected by centrifuging at 3234 g for 1 min at room temperature for each condition while doing microscopy experiments. Cells were then lysed in 100 µl lysis buffer containing 50 mM Tris–Cl pH 7.5, 50 mM NaCl, 2 mM MgCl₂, 0.1% Triton-X100, 1 mM β-Mercaptoethanol, 1× cOmplete mini-EDTA-free Protease Inhibitor Cocktail (Roche, catalog no. 04693132001) and lysed by vortexing at 4 °C in bead-beater with glass beads. Unbroken cells and debris were removed by centrifugation at 2800 g for 5 min at 4 °C, followed by a 1-min spin at 15,800 g to remove any protein aggregates. 100 µg of total protein was loaded onto SDS-PAGE gels. Western analysis was performed using anti-mCherry antibody (Abcam, cat. ab167453), anti-GFP (Santa Cruz, catalog no. sc-9996; 1:1000 dilution), and anti-PGK1 (Abcam, catalog no. ab113687; dilution 1:1000). Western data analysis was done using Biorad ImageLab version 6.0.1.

U1 and U937 cells (seeding density: 10 × 10⁶ cells/well) were either left untreated for 48 h or treated with DON (12.5 µM for 48 h), prostratin (untreated for 24 h followed by 6 µM prostratin for 24 h), or DON + Prostratin (12.5 µM DON for 48 h and 6 µM prostratin for last 24 h). At 48 h, activation of HIV was measured by intracellular staining of HIV-1 core antigen-FITC, KC57 as mentioned earlier. Cells were harvested, washed in PBS, and centrifuged. Cell pellets were processed for mitochondrial extraction using Mitochondria Isolation Kit for Cultured cell (Thermo Scientific) using a reagent-based method as per manufacturers guidelines, followed by measuring OXPHOS protein levels using the total OXPHOS Human WB antibody cocktail (Abcam) with mitochondrial loading control VDAC using the antibody VDAC clone B-6 (Santa Cruz Biotechnology). Relative protein quantification was performed using ImageLab version 6.0.1 (Bio-Rad Laboratories Inc), results analyzed using Mann–Whitney U-test or unpaired t-test and visualized using GraphPad Prism 8.4.3 (significance p < 0.05). All laboratory experiments were performed in three independent replicates. Analysis was performed using unpaired t-test or Mann–Whitney U-test and visualized using GraphPad Prism 8.4.3 (significance p < 0.05). Uncropped and unedited blot images are included as Fig. S10.

To analyse protein levels, cells were harvested in SDS (sodium dodecyl sulfate) lysis buffer and sonicated. Protein concentrations were determined using the Compat-Able Protein Assay Preparation Reagent Set (Thermo Fisher Scientific, #23215, Vienna, Austria) and the Pierce BCA Protein Assay Kit (Thermo Fisher Scientific, #23227, Vienna, Austria) according to the manufacturer´s instructions. 20 µg of total protein per sample was separated by SDS polyacrylamide gel electrophoresis and blotted onto PVDF membranes, which were probed with anti-PPARγ (#23215, Cell Signaling), anti-FABP4 (#10004944, Cayman) or anti-DPP4 antibodies (#ab28340, Abcam). Goat anti-rabbit IgG-HRP (#W4011, Promega) and goat anti-mouse IgG-HRP (#W4021, Promega) served as secondary antibodies. Signals were detected using a chemoluminescence detection system, and membranes were stained with Ponceau S for normalization to total protein. Densitometric analysis was done using ImageJ 1.51n (NIH, USA) and Image Lab version 6.0.1 (Bio-Rad, Feldkirchen, Germany).

Amounts of liver extracts containing 50 µg of proteins were separated by SDS-PAGE and electrotransferred to PVDF membranes (GE10600023 Amersham™ Hybond^®® P Western blotting membranes, PVDF; Merck KGaA, Darmstadt, Germany), as previously described [33 (link)]. Membranes were then incubated with primary antibodies (diluted 1:1000, according to producer instruction), listed in Supplementary Table S1, diluted in TBS containing 2% nonfat dry milk and 0.05% Tween, followed by incubation with appropriated horseradish peroxidase (HRP)-conjugated secondary antibodies (7074S, Anti-rabbit IgG, HRP-linked Antibody, 7076S Anti-rabbit IgG, HRP-linked Antibody, Cell Signaling Technology, Inc., Denver, MA, USA) diluted in TBS containing 2% nonfat dry milk and 0.05% Tween (antibody dilution 1:5000 or 1:10,000). Chemiluminescence images of protein bands were detected using the Clarity Western ECL substrate (Bio-Rad) and the ChemiDoc™ Imaging System (Bio-Rad Laboratories, Hercules, CA, USA) and quantified by densitometric image analysis software (Image Lab Version 6.0.1; Bio-Rad). Results were normalized to the densitometric values of internal loading controls, including GAPDH bands for total and cytosolic extracts, and histone H3 bands for nuclear extracts, and then expressed as fold of standard-diet-fed mice values.

Cultured cells were lysed in RIPA buffer containing 50 mM Tris-HCl, 150 mM NaCl, 2 mM EDTA, 2 mM EGTA, 0.5% sodium deoxycholate, 1% Triton X-100, 0.1% SDS, 50 mM NaF, 0.2 mM Na₃VO_4, and 0.2% protease inhibitor cocktail (Sigma). Samples were prepared as described previously^{67 (link)}. Equal amounts of protein (15 μg) were separated in duplicate on an Any-kD TGX polyacrylamide gel (BioRad). Protein was transferred to a PVDF membrane (TransBlot Turbo, BioRad) and blocked for 24 hours at 4 °C in 5% blotting-grade blocker (BioRad). Blots were incubated in primary OXPHOS antibody (1:1000, 110413, Abcam) and then HRP-conjugated goat anti-mouse antibody (1:5000, 31430, Invitrogen), each for 2 hours at room temperature. Bands of interest were compared against positive control (Rat Heart Mitochondria, Abcam) and all targets were probed on the same membrane, but required different exposure times using signal accumulation mode (ChemiDoc, BioRad). Volume intensity analysis of matched exposure duration for each target was completed using Image Lab Version 6.0.1 (BioRad). Total protein volume intensity obtained by UV activated Stain-Free imaging was used for data normalization.