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. 2010 Sep 7;30(21):5123–5134. doi: 10.1128/MCB.00619-10

FIG. 7.

FIG. 7.

Overexpression of RNase H1 in mitochondria impairs growth and results in mtDNA loss. (A) FT293-CAT, FT293-M27, FT293-M1-M27I, and FT293-M1 cells were grown in the media with (filled squares) or without (open squares) 1 μg/ml doxycycline. The FT293-CAT cell line has the CAT gene inserted at the flip-in site. Likewise, the other FT293 cell lines have the cDNA for the Rnaseh1 mRNA, starting at M1 (FT293-M1), M27 (FT293-M27), or M1 with the M27 AUG changed to AUC (FT293-M1-M27I). The numbers of cells were counted with a hemocytometer. (B) RNase H1-HA-NESi is expressed, activated, and localized to mitochondria in 293 cells. RNase H1 mRNA levels were assayed by RT-qPCR at different time points and dosages of doxycycline. The initial black bars are the parental FT293 cell line lacking RNase H1-HA-NESi. Subsequent bars from left to right in each set are cells with RNase H-HA-NESi expressed at 0 (red), 1 (blue), 3 (yellow), and 10 (green) ng/ml doxycycline. Protein contents of cell lysates were analyzed by Coomassie brilliant blue (CBB) stain, Western blotting for the HA epitope, and the RNase H1 gel activity assay with poly(rA)/poly(dT) as the substrate. Equal amounts of total protein (as assessed from the CBB staining) were loaded in each lane. For clarity, activity is shown as a negative image. Expression of RNase H1-HA-NESi was induced for 6 days using 0, 3, 30, and 60 ng/ml doxycycline. At the bottom and to the right is shown confocal microscopy of FT293-RNase H1-HA-NESi. After 24 h of induction using 100 ng/ml doxycycline, the entire signal detected with the anti-HA antibody (green) colocalized with mitochondria (MitoTracker orange). mtDNA copy number decreases as the dosage of doxycycline and days of incubation increase. For each day, the first bar (black) is FT293 without the cDNA for human RNase H1 treated with 10 ng/ml doxycycline. Subsequent bars from left to right in each set contain 0 (violet), 0.1 (red), 1 (blue), 3 (yellow), or 10 (green) ng/ml doxycycline. At day 6, the values for 1, 3, and 10 ng/ml doxycycline are 0.036, 0.032, and 0.027, respectively. At day 9, the values for 1, 3, and 10 ng/ml doxycycline are 0.018, 0.011, and 0.010, respectively.