PMC

PR-M structure. A, Schematic diagram of the functional domains, exons, and amino acid number for nuclear PR-B, PR-A, and PR-M. B, Northern analysis of poly-A RNA from breast cancer and breast epithelial cell lines, hybridized with a 43-bp [³²P]ATP end-labeled probe directed to the 5′ UTR, shows predominant bands at 6.1 and 2.5 kb. C, A repeat, clearer, Northern blot for T47D cells is shown. See also Supplemental Table 1.

Change in extracellular oxygen levels in Tet-On HeLa cells expressing PR-M. A, A decrease in extracellular oxygen levels (reflected by an increase in probe emission) was seen in Tet-On HeLa cells expressing PR-M treated with 10⁻⁸M R5020 (PRMR). Using repeated-measures analysis adjusting for time effect, PRMR was significantly different from cells expressing PR-M treated with vehicle (PRMC), control transfected cells treated with R5020 (PTRER), and control transfected cells treated with vehicle (PTREC). Results are from duplicate experiments (mean ± SD). B, Tet-On HeLa cells expressing a truncated form of PR-M lacking the initial 30 amino acids (PR-M-30aa) showed no evidence of a change in oxygen levels with 10⁻⁸M R5020 (PR-M-30aaR) treatment or vehicle (PR-M-30aaC) in duplicate experiments (mean ± SD). Positive controls included treatment of pTRE transfected cells with sodium pyruvate (Pyru) (4mM) plus ADP (10⁻⁵M) or sodium pyruvate (4mM) alone, whereas negative controls included treatments with rotenone (10μM) or dimethylsulfoxide (DMSO).

Identification of PR-M in mitochondria by mass spectrometry. A, Reaction with the C19 antibody in the left lane shows endogenous expression of a 38-kDa protein consistent with PR-M in T47D-Y breast cancer cells, known to lack expression of nPR. The next lane identifies a HIS-tagged rPR-M (44 kDa). rPR-M was generated in a cell-free E. coli system followed by a denaturing purification with urea on a nickel column. The control (Ctl) reaction lacked rPR-M template and showed no evidence of a band at 44 kDa. The right lane is from a different blot showing the 44-kDa rPR-M compared with the 38-kDa PR-M in T47D breast cancer cells. The far right blot shows the rPR-M also identified with the MAB 462 antibody. B, SYPRO Ruby-stained gel of rPR-M and mitochondrial membrane protein from human myometrial tissue before immunoprecipitation (pre-IP) and after immunoprecipitation (IP) with MAB 462 antibody, showing a similar protein banding pattern. The band at 38 kDa in the mitochondrial immunoprecipitation preparation submitted for mass spectrometry analysis showed a PR-specific peptide. Western blot analysis with the C19 antibody of the same mitochondrial immunoprecipitation preparation also identifies the 38-kDa protein.

Change in ψm after RNAi in T47D breast cancer cells. A, Transfection with siRNA duplexes directed to exons 1, 2, and 4 of PR-M corresponding to exons 4, 5, and 7 of nPR decreased PR-M expression (dark bars), whereas a decrease in nPR transcript levels did not reach statistical significance (light bars, duplicate assays). B, Transfection with siRNA duplexes directed to exons 1 and 2 of nPR significantly decreased nPR expression (duplicate assays). C, Transfection with siRNA duplexes directed to exons 1 and 2 of nPR significantly decreased S100P transcript levels after 18 hours treatment with 10⁻⁸M R5020 (duplicate assays). D, Transfection with siRNA to PR-M inhibited the increase in ψm with 30 minutes R5020 treatment (dark bars). Control siRNA transfected cells showed an increase in ψm (light bars). E, Transfection with siRNA to nPR showed an increase in ψm with 30 minutes R5020 treatment (dark bars), also seen with control siRNA transfected cells (light bars). ADP (10⁻⁵M) served as a positive control. All values are expressed as mean ± SEM. See also Supplemental Figures 6 and 9. Abbreviations: Ctl, control; #Exps, number of experiments performed on different days; #Obs, total number of wells assayed.

Localization of PR-M to the outer mitochondrial membrane. A, Western blot with MAB 462 of protein from human heart mitochondria (HM) and fractions enriched in proteins of the outer membrane (OM) and the inner membrane and matrix (mitoplast [MP]) showed the 38-kDa PR-M primarily in the OM. Enrichment of fractions was shown by reactions with antibodies to mitochondrial porin and to complex III subunit core I (Cx III), respectively. See also Supplemental Figures 3 and 4. B, Transmission electron microscopy of myometrial tissue with C19 antibody directed to the HBD. Note that the lack of tissue fixation in osmium (which would reduce or eliminate antigenicity) prevents the membranes from showing up as dark tracks. Panel a shows a myocyte without nuclear (n) staining. Mitochondrial staining predominantly of the outer membrane (arrows) is seen along with sparse cytosolic staining. Panel b shows a myocyte with nuclear staining and mitochondrial staining (arrows). Panel c shows a cell devoid of all staining. The morphology of this cell with sparse mitochondria suggests it is not a myocyte. Panel d shows a myocyte reacted with antigen-preabsorbed antibody lacking staining. Also see Supplemental Figure 5.

Change in mitochondrial activity in Tet-On HeLa cells expressing PR-M. A, Ethidium bromide-stained gel of RT-PCR product for PR-M in Tet-On HeLa cells after doxycycline (DOX) induction (Ut, untransfected; pTRE, control plasmid; PR-M, pTRE plasmid with PR-M cDNA). B, An increase in ψm in Tet-On HeLa cells expressing PR-M (dark bars) was seen after 30 minutes treatment with R5020. No significant change in ψm was seen in control transfected cells (light bars). Treatment with ADP (10⁻⁵M) served as a positive control. C, The increase in ψm in Tet-On HeLa cells expressing PR-M seen after 30 minutes treatment with R5020 was inhibited by cotreatment with the PR antagonist RTI-6413-049b. D, No significant change was seen in ψm with MPA (left) or progesterone (right) treatment for 60 minutes in Tet-On HeLa cells expressing PR-M (dark bars) or control transfected cells (light bars). A positive linear trend was seen in the percent difference of MPA to ethanol (F = 5.17, P = .038) and in the percent difference of P to ethanol (F = 4.76, P = .05). In control transfected Tet-on HeLa cells, there was no linear trend in the percent difference of MPA to ethanol (F = 0.059, P = .811) or in the percent difference of progesterone to ethanol (F = 0.3, P = .59) (Supplemental Figure 7). All values are expressed as mean ± SEM. Abbreviations: DMSO, dimethylsulfoxide; EtOH, ethanol; #Exps, number of experiments performed on different days; #Obs, total number of wells assayed; P, progesterone.

Mitochondrial localization of PR-M. A, Cos-1 cells transfected with a carboxy GFP-tagged PR-M (PR-M-GFP), control transfected GFP, a carboxy GFP-tagged truncated PR-M lacking the initial 16 amino acids (PR-M-16aa-GFP), or GFP containing the initial 30 amino acids of PR-M on the N terminus (+30-GFP). Mitochondria (Mito) are stained with Mitofluor or Mitotracker. Confocal imaging showed colocalization of stained mitochondria with PR-M-GFP and +30-GFP as shown by the yellow color. See also Supplemental Figure 1. B, Western blot analyses of purified human heart mitochondrial protein performed with a mouse antibody (MAB 462) and a rabbit antibody (C19) directed to the HBD of PR showed a predominant band at 38 kDa (PR-M). No specific binding was seen with replacement of the primary antibody with mouse or rabbit IgG. See also Supplemental Figure 2. C, Western blot analyses performed with antibodies to histone deacetylase (HDAC) and syntaxin 6 showed relative purity of the preparation. Prohibitin was used as a positive control. D, Western blot analysis with a mouse antibody (AB 52) directed to the N terminus of nPR showed no evidence of the 38-kDa PR-M protein. LDM, low density microsome.