C. trachomatis infection down-regulates p53 in a miR-30c–dependent manner. (A) Immunoblot of HUVECs transfected with siRNA negative control, miR-30c mimic, or inhibitor for 36 h followed by C. trachomatis (C.tr) infection for 12 h. The blots were probed with antibodies against the miR-30c target p53, chlamydial outer membrane protein A (cOmpA), and β-actin. (B) Immunoblot of HeLa cells expressing miR-30c sponge after AHT induction. Blots were probed with antibodies against miR-30c target p53, cOmpA, and β-actin. Induced and noninduced cells were infected with C. trachomatis for 24 and 36 h. (C) Sponge-mediated miR-30c depletion in HeLa results in mitochondrial fragmentation and affects chlamydial growth. Bars, 10 µm. Yellow box and inset point out the damaged mitochondrial architecture. (D) Quantification of chlamydial inclusion in induced and noninduced samples infected with C. trachomatis for 24 h. The samples were stained against cHSP60 (Cy3, 570 nm), and MitoTracker Deep Red FM was used to label the mitochondria. The number of inclusions was quantified using Object Count; ∼600 cells were analyzed from a random selection of six regions of interest (ROIs)/sample; n = 3. (E and F) Graphs represent the analysis of mitochondrial fragment length distribution and number of individual mitochondrial fragments in noninduced and AHT-induced miR-30c sponge cells. n = 6; ∼20 cells were analyzed from a random selection of ∼10 ROIs in each sample. Immunoblot of chimeric GFP protein under the control of the wild-type p53 3′ UTR (G) and mutant p53 3′ UTR (H). Blots were probed with antibodies against GFP, cHSP60, dsRed, and β-actin. (I) Quantification of chimeric GFP expression normalized to the expression of the dsRed transfection control (n = 3). All data represent mean ± SD. Asterisks denote significance by Student’s t test: *, P < 0.05; **, P < 0.01; ***, P < 0.001. See also Fig. S1.