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. 2022 Apr 25;96(10):e00165-22. doi: 10.1128/jvi.00165-22

FIG 2.

FIG 2

Temperature and virus effects on cell proliferation, viability, and protein production. (A) Cell proliferation of uninfected or infected C6/36 cells at different temperatures (16°C to 36°C). Dyed cells were infected with ZIKV, DENV, or CHIKV as described in the legend to Fig. 1 and were incubated at one of the six temperatures for 4 days. Intracellular fluorescence was normalized to that of uninfected cells on day 0. Data are presented as the mean ± SEM from three independent experiments. (B) Cell viability of uninfected or infected C6/36 cells incubated at six temperatures (16°C to 36°C) for 6 days. Cell viability was normalized to that of uninfected cells maintained at 28°C, and data are presented as the mean ± SEM from three independent experiments. Statistical differences for cell proliferation and viability were assessed by a two-way ANOVA with Dunnett’s correction for multiple comparisons. For each temperature, uninfected cells were compared to those infected with each virus. (C) Cell viability of uninfected or ZIKV-infected Vero cells incubated at six temperatures (28°C to 41°C) for 4 days. Cell viability was normalized to that of uninfected cells maintained at 37°C, and data are presented as the mean ± SEM from three independent experiments. Statistical differences were determined by a two-way ANOVA, followed by Bonferroni’s test for multiple comparisons. (D) Protein production at each temperature (16°C to 36°C) on days 1 and 3 was assessed after transfection of C6/36 cells with Ac5-STABLE2-neo plasmid. The data shown, expressed as mean GFP fluorescence, are means ± SEM from three replicates. Statistically significant mean fluorescence was assessed by using a two-way ANOVA on log-transformed data, followed by Dunnett’s test for multiple comparisons. For days 1 and 3, 28°C was compared to all other temperatures. The color of the significance symbol corresponds to the color for each day. (E) Total protein lysate was prepared for uninfected C6/36 cells incubated at six temperatures (16°C to 36°C) for 4 days. Seventy-five micrograms of total protein was denatured and run on a TGX Stain-Free gel (Bio-Rad). The gel was activated with UV light and imaged with a ChemiDoc XRS digital imaging system (Bio-Rad). *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001.