Journal: EBioMedicine
Article Title: Prolactin protects retinal pigment epithelium by inhibiting sirtuin 2-dependent cell death
doi: 10.1016/j.ebiom.2016.03.048
Figure Lengend Snippet: Endogenous PRL is a trophic factor for RPE cells. (a) RT-PCR of PRL receptor (PRLR, 200 bp) in ARPE-19 cell extracts. GAPDH was used as a positive control. bp, DNA ladder. RT-PCR was performed in RNA extracted from three independent cell cultures (N = 3). (b) Confocal stack image of ARPE-19 cells showing F-actin (red), nucleus (blue), and PRL receptor (green) immunofluorescence in x-y axis. Corresponding x-y axis images and z-axis projection were included in Supplemental Fig. 3a and b, respectively. PRL receptor was present in a non-uniform, punctuate distribution along the ARPE-19 cell border (left panel). The secondary antibody control was carried out by omitting the anti-PRL receptor antibody (right panel). Images were captured in three different regions of the culture plate (n = 3), three independent cultures were analyzed (N = 3). (c) Western blot analysis of PRL in conditioned media (CM) from 3-day ARPE-19 cells. A standard of 23-kDa PRL (Std) was included, and equal concentrations of total proteins were loaded. Also, a series of standard PRL dilutions was included at indicated concentrations. CM from five 3-day ARPE-19 cell cultures concentrated by a ~ 2-fold factor was analyzed and this same experiment was repeated three times (N = 3). (d) Quantification of PRL in the CM of 3-day ARPE-19 cells by the Nb2-bioassay. 10% FBS medium was included. Note that CM was not concentrated. Three samples from CM from five 3-day ARPE-19 cell cultures were analyzed and this same experiment was repeated three times (N = 3). (e) Effect on survival of ARPE-19 cells treated with 10% FBS, polyclonal α-PRL antibody (α-PRL Ab) or preimmune serum (ctl Ab), pure PRL receptor antagonist Del1-9-G129R-hPRL at 0.1 and 1 μM or untreated (ctl) for 48 h was measured by MTT assay (n = 8; N = 3 independent experiments). BLK, averaged blank. (f) ARPE-19 proliferation after treatment with 100 pM hPRL, 1 μM Del1-9-G129R-hPRL or no treatment was measured by incorporation of [ 3 H]thymidine for 24 h after the treatments. Scintillation signals were normalized to the untreated condition (n = 9; N = 3 independent replicates). All bar plots, mean plus S.E.M.; P values: ANOVA and Bonferroni post-hoc test. Of note, the control condition corresponds to cells that were pre-treated with 1% FBS medium during 12 h followed by a 48-h period in the same medium.
Article Snippet: Anti-human PRL rabbit polyclonal antibody IC5 and anti-TRPM2 (#ACC-043) were kind gifts from the National Hormone and Pituitary Program (UCLA Medical School, Torrance, CA) and Alomone Labs, respectively.
Techniques: Reverse Transcription Polymerase Chain Reaction, Positive Control, Immunofluorescence, Western Blot, MTT Assay
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![Endogenous <t>PRL</t> is a trophic factor for RPE cells. (a) RT-PCR of PRL receptor (PRLR, 200 bp) in ARPE-19 cell extracts. GAPDH was used as a positive control. bp, DNA ladder. RT-PCR was performed in RNA extracted from three independent cell cultures (N = 3). (b) Confocal stack image of ARPE-19 cells showing F-actin (red), nucleus (blue), and PRL receptor (green) immunofluorescence in x-y axis. Corresponding x-y axis images and z-axis projection were included in Supplemental Fig. 3a and b, respectively. PRL receptor was present in a non-uniform, punctuate distribution along the ARPE-19 cell border (left panel). The secondary antibody control was carried out by omitting the anti-PRL receptor antibody (right panel). Images were captured in three different regions of the culture plate (n = 3), three independent cultures were analyzed (N = 3). (c) Western blot analysis of PRL in conditioned media (CM) from 3-day ARPE-19 cells. A standard of 23-kDa PRL (Std) was included, and equal concentrations of total proteins were loaded. Also, a series of standard PRL dilutions was included at indicated concentrations. CM from five 3-day ARPE-19 cell cultures concentrated by a ~ 2-fold factor was analyzed and this same experiment was repeated three times (N = 3). (d) Quantification of PRL in the CM of 3-day ARPE-19 cells by the Nb2-bioassay. 10% FBS medium was included. Note that CM was not concentrated. Three samples from CM from five 3-day ARPE-19 cell cultures were analyzed and this same experiment was repeated three times (N = 3). (e) Effect on survival of ARPE-19 cells treated with 10% FBS, <t>polyclonal</t> α-PRL antibody (α-PRL Ab) or preimmune serum (ctl Ab), pure PRL receptor antagonist Del1-9-G129R-hPRL at 0.1 and 1 μM or untreated (ctl) for 48 h was measured by MTT assay (n = 8; N = 3 independent experiments). BLK, averaged blank. (f) ARPE-19 proliferation after treatment with 100 pM hPRL, 1 μM Del1-9-G129R-hPRL or no treatment was measured by incorporation of [ 3 H]thymidine for 24 h after the treatments. Scintillation signals were normalized to the untreated condition (n = 9; N = 3 independent replicates). All bar plots, mean plus S.E.M.; P values: ANOVA and Bonferroni post-hoc test. Of note, the control condition corresponds to cells that were pre-treated with 1% FBS medium during 12 h followed by a 48-h period in the same medium.](https://pub-med-central-images-cdn.bioz.com/pub_med_central_ids_ending_with_9382/pmc04909382/pmc04909382__gr2.jpg)