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. 2018 Apr 4;13(4):e0195050. doi: 10.1371/journal.pone.0195050

Fig 6. Revisiting the sealed heating method–V158411 decreases Chk1 nuclear immunofluorescence in HT29 cells heated in a low well volume.

Fig 6

(A) HT29 cells were treated with 0–20 μM V158411 for 10 minutes in 25 μL media before being heated at the indicated temperature for 3 minutes followed by room temperature for 5 minutes. Nuclear Chk1 intensity was determined by immunofluorescent staining using the EP691Y antibody and high content imaging. Values are the mean of three wells ± SD. (B) HT29 cells were treated with 0–20 μM V158411 for 10 minutes in 25 μL media before being heated to 49°C. For 37°C, mean of n = 4 ± SD; for 49°C, dose response curves of 3 independent experiments are shown (mean of 3 wells ± SD). (C) U2OS cells were treated with 0–20 μM V158411 for 10 minutes in 25 μL media before being heated to 49°C. Mean of 3 determinations ± SD. (D) HT29 cells were treated with 0–20 μM V158411 for 10 or 30 minutes in 50 μL media before being heated to 51°C. Mean of 3 wells ± SD. (E) HT29 or U2OS cells were treated with 0–10 μM V158411 under identical conditions of cell number to media volume as (B) above for 10 minutes. Protein expression levels were determined by immunoblotting and quantified by densitometry.