Mouse anti cytochrome c

Cells were treated with hyperthermia in combination with the indicated chemotherapy. After two hours, medium was refreshed and 1:1,000 of the apoptosis inhibitor Q-VD-OPh (20 μM, MP Biomedicals) was added for 24 hours, after which the cells were fixed with 4% PFA for 15 minutes. Fixed cells were subsequently labelled with mouse-anti-Cytochrome C (1:100, BD Pharmingen) and secondary antibody Alexa Fluor goat-anti-mouse 488 (ThermoFisher Scientific).

Protein measurements were carried out using the Pierce BCA Protein Assay Kit (Thermo Scientific) in accordance with the manufacturer’s instructions. Samples were mixed with Laemmli’s loading buffer, boiled for 5 min, and subjected to SDS-PAGE (12%) followed by blotting onto nitrocellulose membranes for 30 min at 25 V using the Mini Trans-Blot Cell (Bio-Rad). Membranes were blocked for 1 hr with 5% non-fat milk in TBS at room temperature and subsequently probed overnight at 4°C with the primary antibody (1:1000). The following primary antibodies were used: rabbit anti-vinculin, rabbit anti-hexokinase-II, rabbit anti-Na⁺/K⁺-ATPase, rabbit anti-cleaved caspase-3 (all from Cell Signaling), mouse anti-caspase-2, mouse anti-caspase-3, mouse anti-PARP1, mouse anti-cytochrome c (all from BD Transduction Lab), mouse anti-lamin B, rabbit anti-Endonuclease G, mouse anti-AIF (all from Santa Cruz Biotech), mouse anti-caspase-8 (Enzo Life Science), rabbit anti-GAPDH (Trevigen), and rabbit anti-ERp29 (kindly provided by Dr. S. Mkrtchian, Karolinska Institutet). After four times washes in TBST (0.05% Tween-20 in TBS), membranes were incubated with appropriate horseradish peroxidase-conjugated secondary antibodies purchased from Cell Signaling (1:4000) for 1 hr at room temperature. Blots were developed using ECL (Amersham Biosciences) and documented using Chemi-Doc (Bio-Rad).

One hour after the last rTMS treatment, anesthetized rats were sacrificed by decapitation. Pn were isolated, homogenized, and treated as previously described [13 (link)]. Samples were incubated with the following primary antibodies: rabbit anti-GFAP (1:2500; Dako, Denmark), rabbit anti-Iba-1 (1:500; Wako, Japan), and mouse anti-cytochrome-c (1:1000; BD Pharmingen, UK). Densities of protein bands in the Western blots were measured, and mean ratios between proteins and β-actin were reported as percentage of control values. The relative levels of immunoreactivity were determined by densitometry using the free software ImageJ (National Institutes of Health, Bethesda, MD, USA). Further details are reported in Additional file 2.

Cells plated on coverslips in 12-well plates and fixed with 4% paraformaldehyde for 15 min, permeabilized with 0.1% v/v Triton X-100/1xPBS (10 min), and blocked with 10% normal goat serum prior to incubation with primary antibodies overnight. Rabbit anti-ki67 (Millipore, 1:200), rabbit anti-Tom20 (Thermofisher, 1:1000), mouse anti-cytochrome C (BD, 1:500), rabbit anti-APP (Cell sig, 1:200), mouse anti-P62 (abcam, 1:500) and mouse anti-Tom20 (Santa Cruz, 1:100). Cells were then labeled with appropriate secondary antibody raised in goat (Invitrogen). Images were taken with the LSM800 (Zeiss) confocal microscope by using the 40×/0.5 EC Plan-Neofluar objective or the 63×/1.4 Oil Plan-Apochromat objective. Excitation and acquisition parameters were constrained across all paired comparisons. For fluorescent quantification, morphometric measurements of images were performed using Image-Pro Plus software (MediaCybernetics). Mitochondrial morphology quantification was conducted by the Mito-Morphology Macro^{44 (link)} through ImageJ software (National Institutes of Health) as previous described^{43 (link)}. In brief, images of 30 cells from random view field of each group were first processed with a median filter to obtain isolated and equalized fluorescence, then individual mitochondria were analyzed for the lengths of major axes.

mouse anti-cytochrome c (2 h, RT, 1:1000, clone 7HB8.2C12; BD Pharmingen, Franklin Lakes, NJ, USA), rabbit anti-TOM20 (1:200; Santa Cruz, Dallas, TX, USA), mouse β-tubulin (2 h, RT, 1:1000 in immunocytochemistry (IHC) and in western blots, clone TUB2.1, Sigma Aldrich), mouse anti-DRP1 (2 h, RT, 1:1000 in IHC and western blot; BD Bioscience, Franklin Lakes, NJ, USA) for Figure 3a/c and Supplementary Figures S3A–C, mouse anti-DRP1 (2 h, RT, 1:800; BD Bioscience) for Figure 3d, rabbit anti-p-DRP1 (1:500; Cell Signalling, Danvers, MA, USA), mouse anti-actin (1:1000; MP Biomedicals, Santa Ana, CA, USA), mouse anti-KDEL (o/n, 4 °C, 1:500; Abcam, Cambridge, UK), mouse anti-PDI (o/n, 4 °C, 1:1000; Enzo Life Sciences, Lörrach, Germany), rabbit anti-GM130 (o/n, 4 °C, 1:250; Abcam), mouse anti-giantin (o/n, 4 °C, 1:1000; Enzo Life Sciences). Generation of rabbit anti-COXII (1:1000) has been described before.^{22 (link)} Alexa Fluor 488-conjugated phalloidin (1:500; Invitrogen) was used to visualize F-actin.