Figure 2

The ATF-6 signaling during CHIKV and SINV infection. A) HEK293 cells (1×10⁵ cells) were cultured on coverslips and either mock or CHIKV/SINV infected at an MOI-1. At indicated time points post infection cells were fixed and immunofluorescence microscopy was performed to probe the virus replication-using antibody against dsRNA (red). Uninfected cells were used as negative control and nuclear stain DAPI (blue) was used as background control. From 12h post infection onwards ~90% of cells were infected with CHIKV/SINV and stained positive for dsRNA antibody. B, C) HEK293 cells (1×10⁵ cells) were infected with MOI-1 of CHIKV/SINV and at indicated time points post infection cells were lysed using TNET lysis buffer. Lysed samples were run on 12% SDS PAGE followed by Western blotting. Antibodies against BIP, ATF-6, HSP-90 and p58IPK were used to probe the ATF-6 pathway component protein levels during CHIKV/SINV infection. Anti-actin antibody was used to probe loading control and uninfected cells (0h) were used as baseline protein level control. D, E) Under the similar experimental conditions and time points stated above, real time RT PCR analysis of BIP, HSP-90 and p58IPK transcripts was done on total RNA extracted from CHIKV/SINV infected cells using specific primers (Materials and Methods) against each of three genes. All three transcripts are presented as fold change over 0h (uninfected cells) after normalization with Actin and GAPDH mRNA. The graphs were plot using graph-pad prism software and are representative of three independent repeats. Any significant change over 0h was determined using student unpaired T test and considered significant (*) if p value was less than 0.05.