Chemidoc xr

Overnight cultures were grown in liquid YPD at 30 ^oC, vortexed, and diluted to an OD₆₀₀ of 0.04 in fresh liquid YPD in a final volume of 1 mL. From this, 70 μLs was plated onto YPD solid agar medium and spread using glass beads. Plates were allowed to dry at room temperature for 1 hour and a 25 mM fluconazole disc was then placed in the center of the plate. Plates were incubated for 48 hours at 30 °C before imaging using a ChemiDoc XRS+ molecular imager (Bio-Rad, Hercules, CA). Plate images were analyzed using DiskImageR^{85 (link)}, and the fraction of growth (FoG20) and resistance (RAD20) scores used to determine tolerance and resistance, respectively.

RT-PCR was performed as previously described elsewhere to quantify the endogenous mRNA expression levels. The total RNA of cells was isolated using TRIsure reagent (#BIO-38033; BIOLINE, Memphis, TN, USA) according to the manufacturer’s instructions. A total of 5 μg of RNA was used to synthesize cDNA using a SensiFAST cDNA Synthesis kit (#BIO-65054; BIOLINE). PCR was performed using 2X MyTaqRedMix (#BIO-25043; BIOLINE) under the conditions of an annealing temperature of 60°C and 30 cycles. Oligonucleotide primer sequences used were as follows: CXCR4 forward, 5′-AATCTTCCTGCCCACCATCT-3′ and reverse, 5′-GACGCCAACATAGACCACCT-3′; SOX2 forward, 5′-TGGACAGTTACGCGCACAT-3′ and reverse, 5′-CGAGTAGGACATGCTGTAGGT-3′; GAPDH forward, 5′-AGGTGAAGGTCGGAGTCAAC-3′ and reverse, 5′-TTCCCGTTCTCAGCCTTGAC-3′. The PCR samples underwent analysis on a 2% agarose gel, were stained with ethidium bromide (Mbiotech, Inc., Hanam, Korea), and were observed under UV light using an image analyzer (ChemiDoc XRS; Bio-Rad Laboratories, Inc., Hercules, CA, USA). To measure band density, ImageJ (NIH, USA) software was used.

Murine PSCA expression was assessed by protein western blot in parental and transfected cells used in animal studies. Cell culture extracts from TRAMP-C2, Luc-MC38, Luc-MC38-EV and Luc-MC38-mPSCA were quantified for protein content with the Pierce BCA Assay Kit (23225, Thermo). Protein lysates (25 µg protein) were loaded onto pre-made MiniProtean TGX 7.5% PA gels (456–1023, Bio-Rad) and run by electrophoresis. Semi-dry transfer onto a Polyvinylidene fluoride or polyvinylidene difluoride (PVDF) membrane was then performed on a Transblot SD unit (170-3940, Bio-Rad). Membranes were blocked and then incubated overnight at 1:1,000 with anti-mouse PSCA (17171-1-AP, Proteintech) or 1:10,000 anti-vinculin (AB129002, Abcam) as loading control in TBS-T (170–6435, Bio-Rad). After three rounds of washing, membranes were incubated with 1:2,000 secondary HRP antibody (AB205718, Abcam). After a short incubation with SuperSignal West Pico PLUS Chemiluminescent substrate (34577, Thermo), membranes were imaged with a Chemidoc XRS⁺ (Bio-Rad). Images were initially processed with Image Lab (Bio-Rad) and later prepared for publication with ImageJ (Fiji).

Briefly, protein lysates were extracted with RIPA and quantified using BCA kits. Equal protein amounts were separated by 6–12% SDS-PAGE and transferred to PVDF membranes. After blocking, membranes were incubated overnight with primary antibodies (P4HB, CSB-PA00254A0Rb; GAPDH, AF1186) at 4 °C and then with secondary antibodies for 2 h at room temperature. Bands were visualized using the Bio-Rad ChemiDoc XRS.

C20 cells were seeded in complete culture medium in a p100 Petri dish with a density of 1.5 × 10⁴ cells/cm². After treatments, cells were collected by scraping, centrifuged at 2500xg and lysed by adding RIPA buffer (0.5% sodium deoxycholate, PBS pH 7.4, 1% Igepal, 0.1% SDS; and protease inhibitors—4 µg/mL apronitin, 1 µM ortovanoate, and 0.1 mg/mL PMSF). For the evaluation of p65 nuclear translocation, cells were gently lysed with a subcellular fractionation buffer (Sucrose 250 mM, HEPES 20 mM, KCl 10 mM, MgCl2 1.5 mM, EDTA 1 mM, EGTA 1 mM, DTT 1 mM, pH = 7.4) added with protease inhibitor cocktail. The suspension was then vortexed and centrifuged at 600xg for nuclei precipitation. This step was repeated al least three times. The nuclear pellet was then separated from the cytosolic pellet and RIPA buffer with protease inhibitors was added to both. For the analysis of phosphorylated Smad2/3, NaF (Sigma) was added to the RIPA buffer. The cellular suspension was sonicated in ice and maintained on a rotating wheel at 4 °C for 2 h. Cell lysate was then centrifuged at 13000xg for 20 min and total proteins were quantified by using the BioRad DC Protein Assay. 30–50 µg for each cell protein extract was mixed with the Laemmli solution and resolved by electrophoresis SDS-PAGE in precast stain-free gels (Bio-Rad, Hercules, California) with a 4–20% gradient of polyacrylamide. Gel was then activated by using ChemiDoc™ XRS· + (Bio-Rad, California) instrument; then proteins were transferred to a PVDF membranes. Total proteins for each lane were imaged for normalization of the obtained band [114 (link)]. The PVDF membrane was incubated with blocking solution for 2 h (5% non-fat dry milk in Tris-buffered saline (TBS)/Tween20 or 5% BSA/0.1% Tween 20) and then incubated overnight at 4 ℃ with the primary antibody. The day after, the membrane was washed with TBS/0.05% Tween20 (Sigma-Aldrich) and incubated for two hours at room temperature with anti-rabbit or anti-mouse IgG light chains conjugated to peroxidase. After 3 washes with TBS-Tween, and one last wash in TBS, the peroxidase was detected by adding the chemo-luminescent substrate ECL (Bio-Rad, California) and by using ChemiDoc. Antibodies and dilutions used are listed below: anti-p65 mouse pAb (Santa-Cruz, sc-8008) 1:200; Anti-TSPO Rabbit mAb (Abcam, ab109497), 1:5000; anti-CYP11A1 Rabbit mAb (Cell Signalling Technology, D8F4F), 1:500; anti p-Smad2/3 (Cell Signalling, #8685) 1:10000; Anti-Rabbit IgG (A6154 Sigma Aldrich) 1:10000; Anti-Mouse IgG (31430, Invitrogen) 1:10000.