Ab52757

A PCR-based method was used to generate all plasmid constructs used in this study. Sequence encoding full-length human HSF1 CDS (NM_005526), and a series of deletion derivatives from SELENOF gene 5′-flanking region sequences were amplified by PCR using human gDNA extracted from HEK293T as templates. HSF1 was inserted into the pcDNA3.1-Myc for overexpression in eukaryotic cells. The 5′-flanking region (from −2055 to +909) deletion derivatives were inserted into the pGL4.10-Luc2 luciferase vector for Dual-Glo Luciferase assay. All constructs were verified by sequencing.
Primary antibody for anti-HSF1 was purchased from abcam (ab52757), anti-myc-tag was purchased from Cell Signaling Technology (2276), anti-HA-tag was purchased from TransGen biotech (HT301-01), and anti- glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was purchased from Abways (AB0036). Goat Anti-Rabbit IgG-HRP secondary antibodies were purchased from Abmart (M21002).

Human CRC cell lines SW480, SW620, DLD1, RKO were obtained from the American Type Culture Collection (ATCC). All cells were routinely cultured in RPMI 1640 (Invitrogen; 11875–093) or DMEM (Invitrogen; 11965–092) supplemented with 10% fetal bovine serum. All cells were incubated at 37 °C with 5% CO2 and 95% humidity. The following antibodies were used for western blotting and IHC: HSF1 (12972S, Cell Signaling Technology, CST; ab52757, Abcam); β-catenin (8480S, CST); β-actin (4970 L, CST); FLAG (F1804–1, Sigma); cyclinD1 (ab134175, Abcam); Cleaved PARP1 (9541-s, CST); METTL3 (a8370, Abclonal); GLS1 (ap8809b, Abgent). Pyrvinium (P0027), LiCl (793620), cycloheximide (R750107), chloroquine (C6628), MG132 (474790) and PD150606 (D5946) were purchased from Sigma-Aldrich.

The frozen myocardial tissue from Days 1, 7, 14, and 28 after radiation was weighed at 20 mg, added to 200 μL of tissue lysis solution (1:100 with protease inhibitor), homogenized in 60 Hz 10 times (45 s each time, interval 15 s), and centrifugated at 12,000 r in 4 °C for 15 min. The supernatant was collected as total protein solution. The concentration of protein solution was measured using the BCA method, and the loading buffer and protein solution (4:1) were mixed and then denatured at 95 °C for 10 min. After the gel preparation, electrophoresis, membrane transfer, and closure, the anti-JNK (Rabbit, Abcam, ab179461, Cambridge, UK), anti-p-JNK (Rabbit, Abcam, ab219584, Cambridge, UK), anti-NFATc4 (Rabbit, Abcam, ab3447, Cambridge, UK), anti-HSF1(Rabbit, Abcam, ab52757, Cambridge, UK) (diluted to 1:1000 with TBST), and anti-GAPDH (Rabbit, Abcam, ab9485, Cambridge, UK) (diluted to 1:10,000 with TBST) were added to the PVDF membranes and incubated overnight at 4 °C with slow shaking. The next day, the PVDF membranes were washed 3 × 10 min with TBST, added to HRP-labeled secondary antibody IgG (Abclonal, Boston, USA) (diluted to 1:10,000 with TBST), shaken slowly at room temperature for 1 h, and washed with TBST for 3 × 10 min. The targeted proteins were detected by enhanced chemiluminescence (ECL), calculated by Image J.