Hsf 1 antibody

The NCI/RES-ADR cells were incubated in medium containing various drug formulations for 12 h. After irradiated with laser for 1 min at 1 W cm⁻² (if needed), cells were further cultured for 12 h, washed twice with 1 × PBS at 37 °C, detached using trypsin/ethylenediaminetetraacetic acid (EDTA), and fixed with 4% paraformaldehyde for 20 min at room temperature. After washing, the fixed cells were incubated in 3% BSA and 0.1% TritonX-100 in 1 × PBS at room temperature for 1 h to block potential nonspecific binding and permeabilize the cell plasma membrane, respectively. Following that, the fixed and permeabilized cells were incubated overnight at 4 °C with HSP90 antibody (Cell Signaling, 4874S), HSP70 antibody (Cell Signaling, 4872S), mutant p53 antibody (Abcam, ab32049), HSF-1 antibody (ThermoFisher, PA3–017), and P-gp antibody (Sigma, P7965) at the dilution ratio of 1:200. Unbounded antibody was washed away with 1 × PBS for 3 times. Cells were then incubated with secondary antibody (ThermoFisher) at the dilution ratio of 1:200 in 1 × PBS with 1% BSA at room temperature for 1 h. After washing with 1 × PBS twice, the cells were analyzed using a BD (Franklin Lakes, NJ, USA) LSR-II flow cytometer and Diva software.

7–10 × 10⁶ NF or CAF at subconfluency were fixed in 1% formaldehyde and sonicated using the Bioruptor Pico sonication device (Diagenode; B01060001) using 15 cycles (30 s on; 30 s off) at maximum intensity. Purified chromatin was then separated for: (i) immunoprecipitation using Dynabeads Protein G (Life Technologies: 10003D) coated with 8 µg and 4 µg of HSF1 antibody (ThermoFisher: RT-405-P, and Cell Signalling: 4356 S, respectively) per ChIP experiment; (ii) non-immunoprecipitated chromatin, used as Input control; and (iii) assessment of sonication efficiencies using a 1% agarose gel. For the quantitative PCR briefly, reactions were carried out in 10 μL volume containing 5 μL of Sybergreen mix (Applied Biosystems; 4472918), 0.5 μL of primer (5 μM final concentration), 2.5 μL of genomic DNA and 2 μL of DNAse/RNAse-free water. A three-step cycle programme and a melting analysis were applied. The cycling steps were as follows: 10 s at 95 °C, 30 s at 60 °C and 30 s at 72 °C, for 40 cycles. Enrichment of the immunoprecipitated sample was confirmed using positive and negative controls. The exact loci of the primers are as follows: negative control (gene desert): chr6:116908976–116909177; Dkk3 Promoter (P): chr7:112159485–112159638; Dkk3 Enhancer (E): chr7:112183116–112183344; Rilpl, within the gene (positive control): chr5:124510011–124510190.