Prohibitin regulates the FSH signaling pathway in rat granulosa cell differentiation

Supplementary Data

  • Supplementary Table 1 - Effects of pretreatment of MEK inhibitor (PD98059) in rat undifferentiated granulosa cells (GCs). (PDF 209 KB)
  • Supplementary Figure 1 - Gonadotropin dependent up-regulation of PHB positively correlates with steroidogenesis in rat granulosa cells (GCs). GCs were treated either with or without follicle stimulating hormone (FSH, 100ng/ml) and testosterone (T, 30ng/ml), and FSH plus T for 48hrs. Parallel controls (C) GCs were maintained without any treatments. A. Live cell phase photographs were taken under an inverted microscope at 400x magnification at 48hrs post-treatment in absence (a) or presence of T (b), FSH (c) and FSH+T (d). B. Bar diagrams representing estradiol (E2) and progesterone (P4) secretions into the medium as measured by RIA. C. Representative Western blot (WB) analysis of protein levels prohibitin (PHB) in GCs induced by FSH and T alone or in combination. Equal amounts of protein (25 μg) were applied to each lane, and WB analyzed for PHB. Tubulin was used as an internal control for cytosol. All numerical values are represented as mean ± SEM of three individual experiments (n = 3). Significant differences (P≤0.05) vs control (without treatment) is represented with star (*) in the respective panels. D. Immunocolocalization of endogenous PHB with Alexa fluor 488 (green, a) labeled secondary antibody and cytochrome c detected with Alexa fluor 594 (red, b) labeled secondary antibody in GC treated with FSH and T for 48hrs detected. The area of colocalization is seen as yellow (c) punctated spots within the cytoplasm. Image of colocalization of PHB and cytochrome c presented at higher magnification in image d. Data represents three individual experiments (n = 3) that were performed for each individual group. (PDF 459 KB)
  • Supplementary Figure 2 - Granulosa cells (GCs) survival in prohibitin (PHB) knock-down rat GCs treated with gonadotropin. GCs were transiently infected with AdshPhb (MOI 5, 10 and 20) or Ad-scrambled (MOI 5, 10 and 20) for 2hr and maintained in culture for 24hr in serum free media and treated with testosterone (T, 30ng/ml) in presence or absence of follicle stimulating hormone (FSH, 100ng/ml) for 48hrs in serum free media. Uninfected parallel control GCs groups were maintained and treated with T in presence or absence of FSH for 48h. A. Live cell photographs were taken under an inverted epifluorescence microscope at ×400 magnification showing green fluorescence for the eGFP-shPhb or eGFP alone along with phase contrast pictures at 48h after treatment. B. The bar graph represents the percentage of cells displaying nuclear morphologic changes characteristic of apoptosis. At least 200 cells/treatment/group were counted and assessed randomly from selected fields and blinded slides to avoid experimental bias. C. The bar graph represents the caspase-3 activity as % of control groups in cytosolic protein extracts from GCs after completion of treatments measured using the spectrophotometric substrate DEVD-pNA(D). All bar graphs represent the mean ± SEM of results from three independent experiments (n=3). Significant differences (P≤0.05) are represented with star (*) compare to parallel control group. (PDF 636 KB)
  • Supplementary Results - Supplementary Results (PDF 83 KB)

This Article

  1. J Mol Endocrinol May 1, 2016 vol. 56 no. 4 325-336
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