Fig. 2
LEA proteins enhance cell metabolic activity and viability of sheep fibroblasts exposed to cold stress. (a) Left: cell viability of sheep fibroblasts transfected with single LEA protein exposed to 10 °C for up to 7 days. Viability of the cell with Trypan Blue was controlled after 1-, 2-, 3- and 7 days (2-Way Anova plus post hoc Tukey test for multiple LEA groups and control at different time of cold-exposure; *p < 0.05 for CTR vs. WCOR410 and RAB17 (2 days); ***p < 0.001 for CTR vs. WCOR410 (3 days); ****p < 0.0001 for CTR vs. RAB17 (3 days); for CTR vs. WCOR410 and RAB17 (7 days). Error bars indicate SD. Right: cell viability of sheep fibroblasts transfected with single LEA protein exposed to 4 °C for up to 7 days. Viability of the cell with Trypan Blue was controlled after 1-, 2-, 3- and 7 days (2-Way Anova plus post hoc Tukey test for multiple LEA groups and control at different time of cold-exposure; *p < 0.05 for CTR vs. RAB17 (1 day) and WCOR410 vs. RAB17 (1 day); **p < 0.01 for CTR vs. RAB17 (2 days) and WCOR410 vs. RAB17 (7 days); ***p < 0.001 for CTR vs. WCOR410 (7 days); ****p < 0.0001 for CTR vs. WCOR410 (1-, 2-, 3 days) and RAB17 (3 days) and for WCOR410 vs. RAB17 (3 days)). Error bars indicate SD. (b) Percentage of metabolic active LEA-transfected cells after 3- and 7 days of cold stress. Cellular metabolic activity was assessed with MTT assay after cells were put back in culture for 24 h (2-Way Anova plus post hoc Tukey test for multiple LEA groups and control at different time of cold-exposure; *p < 0.05 for CTR vs. RAB17 (7 days); **p < 0.01 for WCOR410 vs. RAB17 (3 days); ***p < 0.001 for CTR vs. WCOR410 (7 days); ****p < 0.0001 for CTR vs. WCOR410 and RAB17 (3 days)). Error bars indicate SD. (c) Percentage of LEA-positive cells per field remained attached to culture dish after 1-, 2-, 3- and 7 days of cold stress (one-Way Anova plus post hoc Tukey test for LEA group at different time of cold-exposure; same letter indicate statistical difference between groups; *p < 0.05 for 1 vs. 7 days and for 2 vs. 7 days). Error bars indicate SD. (d) Representative fluorescence image of WCOR410-RFP positive cells per field after 1 day (upper) and 7 days (lower). Scale bar, 20 μm.
