Fig 1.
Human REV3L contains a mitochondrial localization signal (MLS) and localizes to mitochondria.
(A) Schematic drawings of the MLS and NLS in the long and short forms of human REV3L. Amino acid numbers and sequences of MLS and NLS are indicated. (B) Fluorescence microscopic pictures showing the localization of Rev3MLS attached GFP protein in mitochondria of NIH3T3 cells. (C) Western blot analysis showing mitochondrial localization of Rev3MLS attached GFP protein and mitochondria-encoded COX II protein using protein extract from proteinase K-treated mitochondria. (D) Western blot analyses of cytoplasmic (cyto), nuclear (nuc), and mitochondrial (mito) protein fractions from mouse heart and human breast epithelial cell line MDA-MB-231 showing the localization of long and short isoforms of REV3 protein in different cellular compartments. (E) Fluorescence microscopic pictures showing the localization of Rev3NLS attached GFP protein in the nucleus of NIH3T3 cells.
Fig 2.
Rev3 inactivation affects mitochondrial functions.
(A) Western blot analysis of representative subunits of mitochondrial respiratory complexes I-V in Rev3+/+ and Rev3-/- mouse embryonic fibroblasts. The bar graph represents fold change in COX II protein expression (mean ± s.d.) from at least three independent experiments. *P<0.05 versus Rev3+/+; Student's t-test. (B) RT PCR analyses of mitochondrial-encoded gene Cox II in Rev3+/+ and Rev 3-/- cells. The bar graph represents fold change in mRNA expression (mean ± s.d.) from at least three independent experiments. *P<0.05 versus Rev3+/+; Student's t-test. (C) Enzymatic activity of mitochondrial respiratory complex IV in Rev3+/+ and Rev3-/- cells. Bars represent the means ± s.d. *P<0.05 versus Rev3+/+; Student's t-test. (D) Glucose consumption by Rev3+/+ and Rev3-/- cells. Bars represent the mean ± s.d. *P<0.05 versus Rev3+/+; Student's t-test. (E) Mitochondrial membrane potential of Rev3-/- cells relative to Rev3+/+ cells in the presence or absence of a mitochondrial membrane potential inhibitor CCCP treatment. Bars represent the mean ± s.d. *P<0.05 versus Rev3+/+ and **P<0.05 versus Rev3+/+ CCCP; Student's t-test. (F) ROS production in Rev3+/+ and Rev3-/- cells in the presence or absence of OXPHOS inhibitor Antimycin A treatment. Bars represent the mean ± s.d. *P<0.05 versus Rev3+/+ and **P<0.05 versus Rev3+/+ Antimycin A; Student's t-test.
Fig 3.
Inhibition of OXPHOS increases REV3 expression.
(A) RT PCR analysis of Rev3 gene expression in 143B parental and rho° cells. Treatment of 143B parental cells with mitochondrial OXPHOS inhibitors increases expression of Rev3. Black vertical lines indicate where intervening lanes have been removed for clarity. (B) Co-immunoprecipitation of POLG1 and POLG2 with REV3 in Rev3+/+ and 143B cells with or without UV treatment. Rev3-/- cells were used as negative control. (C-D) RT PCR (C) and Western blot (D) analyses showing increased mRNA and protein expression of REV3 in MCF7 cells containing tetracycline-inducible, dominant negative mutant POLG1 (MCF7 Polg1 DN) after 12 days of dox induction. The bar graphs represent fold change in mRNA or protein expression (mean ± s.d.) from at least three independent experiments. *P<0.05 versus MCF7 cells at day 0; Student's t-test.
Fig 4.
REV3 protects cells from mtDNA damage.
(A) RT PCR results showing a time-dependent increase in Rev3 gene expression in Rev3+/+ cells after UV exposure. The bar graph represents fold change in mRNA expression (mean ± s.d.) from at least three independent experiments. *P<0.05 versus UV-unexposed control; Student's t-test. (B) RT PCR analysis showing increased expression of Polg1 and Polg2 in Rev3-/- cells relative to Rev3+/+ cells. The bar graph represents fold change in mRNA expression (mean ± s.d.) from at least three independent experiments. *P<0.05 versus Rev3+/+; Student's t-test. (C) Representative ChIP results showing binding of REV3 to the mtDNA (D loop and COX II region) in Rev3+/+ cells. Experiment was carried out in triplicate. A further increase in REV3 binding to the D loop region is evident after UV exposure. (D) mtDNA damage analysis in UV-exposed (2.5 J/m2) HEK293 cells expressing full-length Rev3 and HEK293 cells expressing Rev3 without the MLS (Rev3-MLS) after 6 h of exposure. Bars represent the mean ± s.d. *P<0.05 versus full-length Rev3 expressing cells; Student's t-test. (E) mtDNA content of UV-exposed (2.5 and 5 J/m2) Rev3+/+ and Rev3-/- cells after 6 and 24 h of exposure. Bars represent the mean ± s.d. *P<0.05 versus respective UV-unexposed cells; Student's t-test.
Fig 5.
Mitochondrial REV3 contributes to tumorigenic properties.
(A-B) RT PCR analysis of Rev3 gene expression in the non-neoplastic breast epithelial cell line MCF-12A and in 10 neoplastic breast epithelial cell lines (A), and in a set of 10 primary breast tumors and matched normal breast tissues from the same patients (B). Breast tumor samples showing increased mRNA expression of Rev3 compared to respective matched normal breast tissue are denoted by asterisks (*) (B). (C-D) Levels of survival (C) and Matrigel invasion capacity (D) of full-length Rev3 and Rev3+MLS-NLS expressing HEK293 cells. Bars represent mean ± s.d. *P<0.05 versus vector-transfected cells; Student's t-test. (E) Matrigel invasion capacity of Rev3 shRNA-transfected HeLa cells (Rev3 shRNA-1 and Rev3 shRNA-2). Bars represent the mean ± s.d. *P<0.05 versus scrambled shRNA-transfected HeLa cells; Student's t-test. (F) Comparison of cell-culture migration capacity of Rev3 shRNA-transfected HeLa cells (Rev3 shRNA-1 and Rev3 shRNA-2) with scrambled shRNA-transfected HeLa cells.