Na931

The following antibodies were used in this study: primary—mouse anti-E-Cadherin (1:200 610181, BD Transduction Lab), mouse anti-β-actin (1:200, A5441, Sigma), goat anti-Ctr (OBT0978, AbD Serotec), mouse anti-HSP60 (1:5000, ALX-804-701, Alexis), rabbit anti-Ki67 (9027, Cell Signaling), rabbit anti-pSTAT1 (1:1000, 9167, Cell Signaling), rabbit anti-pSTAT3 (Tyr705) (1:1000 9145, Cell Signaling), cleaved caspase-3 (1:1000, 9664, Cell Signaling), goat anti-LIF (1:500, AF-250-NA, R&D Systems); secondary - sheep anti-mouse-HRP (1:3000, NA931, Amersham), donkey anti-rabbit-HRP (1:3000, NA934, Amersham), donkey anti-goat-HRP (1:3000, 800073, Biomol), donkey anti-mouse-Alexa488 (1:300, 715-546-140, Dianova), donkey anti-goat-Cy3 (1:300, 705-165-003, Dianova), donkey anti-rabbit-Alexa488 (1:300, 711-546-152, Dianova), donkey anti-mouse-Dylight 647 (1:300, 715-605-150, Dianova), CD326 (EpCAM)-FITC antibody (1:50, 130-080-301, Miltenyi), mouse anti-human CD24-BV711 antibody (1:200, 563371, BD Biosciences), mouse anti IgG1-APC (1:100, 130-098-846, Miltenyi), and mouse anti-human CD133/1 (AC133)­APC (1:100, 130­098­829, Miltenyi). As a DNA-labeling agent, Draq5 (5 µM, 62252, Thermo Scientific) was used.

Whole‐cell extracts were prepared in radioimmune precipitation assay (RIPA) buffer (50 mmol/L Tris‐HCl [pH 7.5], 150 mmol/L NaCl, 1% NP‐40, 0.1% SDS, 0.5% sodium deoxycholate, 0.5 mmol/L DTT, 0.5 mmol/L PMSF and 2.5 mmol/L Roche protease inhibitor cocktail). The extracts were subjected to Western blot analysis as described previously.¹¹ Briefly, 20 to 40 μg of total protein were separated using 7‐12% SDS‐PAGE and transferred to PVDF membrane. The membrane containing transferred proteins was blocked by incubating with 5% BSA containing 0.05% Tween‐20 and incubated overnight at 4°C with primary antibody to detect CYCLIN D1 (Abcam, ab134175), c‐MYC (Abcam, ab62928), SURVIVIN (Abcam, ab76424), α‐TUBULIN (Abcam, ab4074), DKK1 (Abcam, ab109416), DKK3 (Abcam, ab186409), β‐ACTIN (Cell Signaling Technology, 4970), CYCLIN D3 (Cell Signaling Technology, DCS22) and PRMT5 (Thermo Fisher, MA1‐25470). After incubation with primary antibody, the membrane was treated with HRP‐conjugated goat anti‐mouse (Amersham Biosciences, NA931) or anti‐rabbit (Amersham Biosciences, NA934V) secondary antibody. Next, proteins were visualized using the ECL detection kit (Amersham, RPN2209) in a Western blot imager (Flurochem E system, proteinsimple).

To evaluate the isolated mitochondria and other homogenate fractions, samples were analyzed by SDS page using tris-glycine gels and transferred onto PVDF for subsequent blotting procedures (Biorad, 4561094 and 1704156). Antibodies used for blotting procedures included an antibody against MTP18 (Abcam, ab198217), Complex IV subunit I (MTCO1; Abcam, ab14705), and GAPDH (Cell Signaling Technology, 2118 S). An MTP18 positive control protein lysate was used in blots to confirm the detection of MTP18 (Santa Cruz, sc-125655). Prestained protein ladders were loaded in all gels and were used to identify the molecular weight of proteins (Biorad, 1610375, and Thermofisher Scientific, 26617). All blotting procedures with primary antibodies were carried out overnight, at 4 °C in 5% milk in a T(0.1%)-TBS solution. All secondary antibodies HRP conjugated were incubated for 4 hr (1:5000) at room temp, prior to ECL activation and imaging procedures (General Electric, NA931, NA9340, Amersham Imager 680). Loading normalization and band quantification was conducted using the ImageJ gel analyzer tool.