Figure 4.

Changes in the relative abundance of the SUMO variants triggered by three different types of stress in A549 and HEK293A cells. A549 cells and HEK293A cells were exposed to three different types of stress, namely IAV (Influenza A virus infection using viral strain A/PR/8/34 H1N1 at MOI = 10 for 12 h), cold-shock (27 °C for 24 h), and heat-shock (43 °C for 1 h), as described under Materials and Methods. Untreated cell cultures plated at equal cell densities and maintained for equal time frames were used as controls. After treatment, total RNA was purified and CNest for each SUMO variant was determined. Alternatively, cells were lysed in 4 × Sample Buffer and processed for SDS-PAGE and immunoblotting. (a) Changes in the relative abundance of each SUMO variant, calculated by dividing the CNest value obtained upon stress by the CNest in the control (non-stressed sample). All data are shown in Log2 scale and represent the average values obtained from triplicate measurements in three independent experiments. Statistical significance was established using an unpaired Student’s T-Test, applying a Bonferroni correction to account for the number of multiple comparisons within each treatment. *p < 0.008; **p < 0.004. (b) Changes in total SUMO CNest per 100 ng of RNA triggered by the different stresses used. (c) Heat map representation of the data presented in A. Fc: fold change. (d). Immunoblot data showing changes in global SUMOylation levels triggered by Influenza A Virus infection, heat-shock, and cold-shock. The membranes were immunoblotted for GAPDH, washed, immunoblotted for SUMO1, stripped using a heat denaturation method, and subsequently blotted for SUMO2/3. 1, Mock infected cells; 2, IAV infected cells; 3, heat-shock control; 4, heat-shock sample; 5, cold-shock control; 6, cold-shock sample. *GAPDH.