Regulation of L-type Voltage Gated Calcium Channel CACNA1S in Macrophages upon Mycobacterium tuberculosis Infection
Fig 9
Cross regulation of CACNA1S, ROS and pCREB during Rv2463 stimulation or M. tb infection.
For Panel A, J774 cells were stimulated with 25 μg/ml Rv2463 or infected with 2 MOI M. tb H37Rv for 48h in the presence (bold lines) or absence (dotted lines) of indicated inhibitors to ROS. CACNA1S levels were monitored by flow cytometry. For Panel B, J774 cells were transfected with siRNAs to CACNA1S for 36h followed by stimulation with 25 μg/ml Rv2463 for 48h. ROS levels were monitored by FACS. Grey line represents Rv2463 stimulated cells transfected with control siRNAs, while bold line represents Rv2463 stimulated cells transfected with siRNAs specific to CACNA1S. Dotted line represents unstimulated cells transfected with control siRNAs. For Panel C, J774 cells were stimulated with 25 μg/ml Rv2463 or infected with 2 MOI M. tb H37Rv for indicated times in the presence or absence of indicated inhibitors to ROS and nuclear extracts were probed for pCREB levels. Numbers below the blots indicate relative intensities of the bands. Arrow shows the specific band. Data from one of three experiments is shown. In Panel A, P<0.024 for Rv2463 v/s DPI+Rv2463; P<0.013 for M. tb v/s DPI+M. tb. In Panel B, P<0.006 for siControl+Rv2463 v/s siCACNA1S+Rv2463. In Panel C, at 120 minutes, P<0.024, for M. tb v/s DPI+M. tb. Two-tailed Student’s t-test was employed for P values.