Fig. 4

Cryab overexpression increases clonogenic survival and transwell migration in vitro. a Proliferation of E1A/Ras-transformed MEFs Cryab^Wt (Wt) and Cryab^Tg (Tg) measured using IncuCyte (Mean ± SEM, an average of 2 biological repeats in duplicate and 2 independent experiments Student’s t-test, ****P < 0.0001), b Representative image (left) and quantification (right) of indicated genotypes of E1A-Ras-transformed MEFs for clonogenic survival (colony formation) assay. Each cell line was seeded at densities of 500 in each well incubated for 14 days, the cells were then processed using the crystal violet stain to visualize the colonies. Quantification performed by analysis of all images c Representative image (upper) and quantification (lower) of indicated genotypes of E1ARas-transformed MEFs for clonogenic survival (colony formation) assay as aforementioned above after initial 1 h of heat stress (43ºC). d Representative image (upper) and quantification (lower) of wells for indicated genotypes of E1ARas-transformed MEFs for transwell migration assay after an initial 1 h of heat stress (43ºC), (scale bars, 400 µm) Data in (b-d) presented as Mean ± SEM, an average of 3 biological repeats and 3 independent experiments Student’s t-test, ****P < 0.0001). e Immunoblot analysis was performed on E1A/Ras-transformed MEFs after exposure to heat stress at 43 °C for 1 h. f Quantification of apoptosis by Annexin V/PI flow cytometry in MEF cells of Cryab^Wt (Wt) and Cryab^Tg (Tg) MEFs after exposure to heat stress at 43 °C for 1 h. Upper panel is representative contour diagram of FACS profile via Annexin V-FITC/PI staining. Live cells which exclude PI and lack Annexin V-FITC binding are confined to left lower quadrant (Q4) of each panel. Dead cells (apoptosis) appear first in the right lower quadrant(Q3) detected with Annexin V-FITC and then in the upper right quadrant (Q2; late apoptotic cells) as a result of the loss of cell membrane integrity and are positive for both Annexin V-FITC binding and for PI uptake. Lower panel, Bar graph demonstrates the percent of early and late apoptotic cells in Cryab^Wt (Wt) and Cryab^Tg (Tg) MEFs after exposure to heat stress at 43 °C for 1 h. Data presented as Mean ± SD, an average of 3 biological repeats and 2 independent experiments Student’s t-test, **P < 0.01)