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Working model for the regulation of the <t>p16/RB</t> pathway and senescence induction by PR55α. Black lines depict our current understanding of the respective roles of the p16/RB and p53/p21 pathways in the promotion of cellular senescence in response to genotoxic stressors [ , ]. The CDKN2A locus produces both the p16 (INK4a) and p14 (ARF) proteins using both separate promoters and alternative splicing. The p16 protein blocks the CDK4/6 kinases leading to RB activation, which is required for G1 cell cycle arrest and senescence induction. The p14 protein stabilizes p53 by inhibiting the MDM2 E3 ubiquitin ligase, resulting in p21 (a p53 target gene) induction and subsequent inhibition of CDK2 and CDK4/6, which also leads to RB activation that promotes G1 cell cycle arrest and senescence. We have previously reported that p53 negatively regulates PR55α protein stability . However, the p53 mutational status had no detectable impact on the effects of PR55α on the expression of p16 and induction of senescence by IR. Red lines depict novel findings presented in this report: (1) PR55α-controlled PP2A enhances IR-induced p53/p21 signaling and (2) PR55α inhibits p16 transcription independently of p53 function.

Prl Sv40 Luc, supplied by Promega, used in various techniques. Bioz Stars score: 90/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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prl-sv40-luc - by Bioz Stars, 2026-10

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1) Product Images from "PR55α-controlled protein phosphatase 2A inhibits p16 expression and blocks cellular senescence induction by γ-irradiation"

Article Title: PR55α-controlled protein phosphatase 2A inhibits p16 expression and blocks cellular senescence induction by γ-irradiation

Journal: Aging (Albany NY)

doi: 10.18632/aging.205619

Working model for the regulation of the p16/RB pathway and senescence induction by PR55α. Black lines depict our current understanding of the respective roles of the p16/RB and p53/p21 pathways in the promotion of cellular senescence in response to genotoxic stressors [ , ]. The CDKN2A locus produces both the p16 (INK4a) and p14 (ARF) proteins using both separate promoters and alternative splicing. The p16 protein blocks the CDK4/6 kinases leading to RB activation, which is required for G1 cell cycle arrest and senescence induction. The p14 protein stabilizes p53 by inhibiting the MDM2 E3 ubiquitin ligase, resulting in p21 (a p53 target gene) induction and subsequent inhibition of CDK2 and CDK4/6, which also leads to RB activation that promotes G1 cell cycle arrest and senescence. We have previously reported that p53 negatively regulates PR55α protein stability . However, the p53 mutational status had no detectable impact on the effects of PR55α on the expression of p16 and induction of senescence by IR. Red lines depict novel findings presented in this report: (1) PR55α-controlled PP2A enhances IR-induced p53/p21 signaling and (2) PR55α inhibits p16 transcription independently of p53 function.

Figure Legend Snippet: Working model for the regulation of the p16/RB pathway and senescence induction by PR55α. Black lines depict our current understanding of the respective roles of the p16/RB and p53/p21 pathways in the promotion of cellular senescence in response to genotoxic stressors [ , ]. The CDKN2A locus produces both the p16 (INK4a) and p14 (ARF) proteins using both separate promoters and alternative splicing. The p16 protein blocks the CDK4/6 kinases leading to RB activation, which is required for G1 cell cycle arrest and senescence induction. The p14 protein stabilizes p53 by inhibiting the MDM2 E3 ubiquitin ligase, resulting in p21 (a p53 target gene) induction and subsequent inhibition of CDK2 and CDK4/6, which also leads to RB activation that promotes G1 cell cycle arrest and senescence. We have previously reported that p53 negatively regulates PR55α protein stability . However, the p53 mutational status had no detectable impact on the effects of PR55α on the expression of p16 and induction of senescence by IR. Red lines depict novel findings presented in this report: (1) PR55α-controlled PP2A enhances IR-induced p53/p21 signaling and (2) PR55α inhibits p16 transcription independently of p53 function.

Techniques Used: Alternative Splicing, Activation Assay, Ubiquitin Proteomics, Inhibition, Expressing

PR55α suppresses p16 protein expression in normal and cancer cells originating from the human exocrine pancreas. ( A ) Human pancreatic normal ductal (HPNE) cells were stably transduced with a retroviral vector expressing a Dox-inducible PR55α cDNA (PR55α). As a control, HPNE cells stably transduced with a relevant empty retroviral vector were included in the analysis (Control). Following the induction of ectopic PR55α expression by Dox (1 μg/ml) for 2 days, cells were exposed to 10 Gy of ionizing radiation (IR), incubated for the indicated hours, and analyzed by immunoblotting for the levels of p16, p14, p53, and p21. GAPDH in the lysates was measured as an internal control. ( B ) Human pancreatic ductal adenocarcinoma cells (CD18/HPAF) were stably transduced with a lentiviral vector expressing a Dox-inducible shRNA against either PR55α (PR55α-shRNA) or an irrelevant negative control (Control-shRNA). Following induction of the shRNA with Dox (2 μg/ml) for 5 days, the cells were exposed to 10 Gy IR, incubated for the indicated hours, and analyzed by immunoblotting for the levels of the indicated proteins. GAPDH in the lysates was again used as an internal control. The difference in the p16 levels between HPNE-Control and HPNE-PR55α cells, as well as between the CD18/HPAF-Control-shRNA and CD18/HPAF-PR55α-shRNA cells were determined to be statistically significant (HPNE-Control vs. HPNE-PR55α, p < 0.001; CD18/HPAF-Control-shRNA vs. CD18/HPAF-PR55α-shRNA, p = 0.004).

Figure Legend Snippet: PR55α suppresses p16 protein expression in normal and cancer cells originating from the human exocrine pancreas. ( A ) Human pancreatic normal ductal (HPNE) cells were stably transduced with a retroviral vector expressing a Dox-inducible PR55α cDNA (PR55α). As a control, HPNE cells stably transduced with a relevant empty retroviral vector were included in the analysis (Control). Following the induction of ectopic PR55α expression by Dox (1 μg/ml) for 2 days, cells were exposed to 10 Gy of ionizing radiation (IR), incubated for the indicated hours, and analyzed by immunoblotting for the levels of p16, p14, p53, and p21. GAPDH in the lysates was measured as an internal control. ( B ) Human pancreatic ductal adenocarcinoma cells (CD18/HPAF) were stably transduced with a lentiviral vector expressing a Dox-inducible shRNA against either PR55α (PR55α-shRNA) or an irrelevant negative control (Control-shRNA). Following induction of the shRNA with Dox (2 μg/ml) for 5 days, the cells were exposed to 10 Gy IR, incubated for the indicated hours, and analyzed by immunoblotting for the levels of the indicated proteins. GAPDH in the lysates was again used as an internal control. The difference in the p16 levels between HPNE-Control and HPNE-PR55α cells, as well as between the CD18/HPAF-Control-shRNA and CD18/HPAF-PR55α-shRNA cells were determined to be statistically significant (HPNE-Control vs. HPNE-PR55α, p < 0.001; CD18/HPAF-Control-shRNA vs. CD18/HPAF-PR55α-shRNA, p = 0.004).

Techniques Used: Expressing, Stable Transfection, Transduction, Retroviral, Plasmid Preparation, Control, Incubation, Western Blot, shRNA, Negative Control

PR55α inhibits p16 expression by suppressing its mRNA transcription. ( A ) PR55α does not affect p16 protein stability. CD18/HPAF cells stably transduced with Dox-inducible PR55α-shRNA or Control-shRNA were cultivated in media containing 2 μg/ml Dox for 5 days to induce the shRNA, after which cells were exposed to cycloheximide (CHX, 15 μg/ml) to halt protein synthesis. Cell lysates collected at the indicated time points after CHX addition were analyzed for changes in p16 protein levels. The α-tubulin protein has a long half-life and was used as an internal control. Relative p16 protein levels were determined after normalization with the α-tubulin levels and these normalized values were used to calculate the protein half-life of p16. Half-lives were estimated by linear regression analysis of p16 normalized levels against time using SigmaPlot. ( B ) PR55α inhibits p16 mRNA expression. Left panel: HPNE expressing the Dox-inducible PR55α (HPNE/PR55α), or empty vector (HPNE/Control) was treated with Dox (1 μg/ml) for 3 days. Right panel: CD18/HPAF cells expressing the Dox-inducible PR55α shRNA (B4-2, B4-11) or Control shRNA (Control) were treated with Dox (2 μg/ml) for 5 days. At the end of each treatment, RNA was isolated and analyzed by quantitative RT-PCR for differences in p16 mRNA. The relative abundance of the p16 mRNA was calculated by normalizing the p16 mRNA levels with those of the GAPDH mRNA, with the data represented as mean ± S.D. (bar graphs). Statistical significance was calculated by a Student’s t -test (HPNE cells) or one-way ANOVA (CD18/HPAF). The difference with the Control group ( n = 6/group) was determined to be statistically significant at * p < 0.001 or ** p < 0.005. ( C ) PR55α suppresses p16 promoter activity. HPNE and CD18/HPAF cells in the presence/absence of ectopic PR55α and PR55α-shRNA expression, respectively, were co-transfected with a Firefly luciferase reporter under the control of the p16 promoter and a control Renilla luciferase reporter driven by the SV40 promoter. Two days after transfection, Firefly, and Renilla luciferase activities were measured separately in each lysate, as described in the Materials and Methods. p16 promoter activity was calculated by normalizing the activity of Firefly luciferase over that of Renilla luciferase. The graphs show relative p16 promoter activities in the indicated cell samples and are expressed as the mean ± S.D. of two independent experiments done in duplicates. * Statistically significant in a Student’s t -test with p < 0.001. ( D ) HPNE/Control and HPNE/PR55α cells were incubated in the presence of 1 μg/ml Dox for 48 h to induce PR55α expression, after which cells were transfected with either a non-targeting siRNA ( Control ) or AUF1 siRNA. Two days later, cells were analyzed by immunoblotting for differences in levels of AUF1 and p16. GAPDH was used as an internal standard. The levels of p16 and GAPDH were quantified using Fiji-ImageJ software and relative p16 levels in the samples were determined after normalizing it with GAPDH levels.

Figure Legend Snippet: PR55α inhibits p16 expression by suppressing its mRNA transcription. ( A ) PR55α does not affect p16 protein stability. CD18/HPAF cells stably transduced with Dox-inducible PR55α-shRNA or Control-shRNA were cultivated in media containing 2 μg/ml Dox for 5 days to induce the shRNA, after which cells were exposed to cycloheximide (CHX, 15 μg/ml) to halt protein synthesis. Cell lysates collected at the indicated time points after CHX addition were analyzed for changes in p16 protein levels. The α-tubulin protein has a long half-life and was used as an internal control. Relative p16 protein levels were determined after normalization with the α-tubulin levels and these normalized values were used to calculate the protein half-life of p16. Half-lives were estimated by linear regression analysis of p16 normalized levels against time using SigmaPlot. ( B ) PR55α inhibits p16 mRNA expression. Left panel: HPNE expressing the Dox-inducible PR55α (HPNE/PR55α), or empty vector (HPNE/Control) was treated with Dox (1 μg/ml) for 3 days. Right panel: CD18/HPAF cells expressing the Dox-inducible PR55α shRNA (B4-2, B4-11) or Control shRNA (Control) were treated with Dox (2 μg/ml) for 5 days. At the end of each treatment, RNA was isolated and analyzed by quantitative RT-PCR for differences in p16 mRNA. The relative abundance of the p16 mRNA was calculated by normalizing the p16 mRNA levels with those of the GAPDH mRNA, with the data represented as mean ± S.D. (bar graphs). Statistical significance was calculated by a Student’s t -test (HPNE cells) or one-way ANOVA (CD18/HPAF). The difference with the Control group ( n = 6/group) was determined to be statistically significant at * p < 0.001 or ** p < 0.005. ( C ) PR55α suppresses p16 promoter activity. HPNE and CD18/HPAF cells in the presence/absence of ectopic PR55α and PR55α-shRNA expression, respectively, were co-transfected with a Firefly luciferase reporter under the control of the p16 promoter and a control Renilla luciferase reporter driven by the SV40 promoter. Two days after transfection, Firefly, and Renilla luciferase activities were measured separately in each lysate, as described in the Materials and Methods. p16 promoter activity was calculated by normalizing the activity of Firefly luciferase over that of Renilla luciferase. The graphs show relative p16 promoter activities in the indicated cell samples and are expressed as the mean ± S.D. of two independent experiments done in duplicates. * Statistically significant in a Student’s t -test with p < 0.001. ( D ) HPNE/Control and HPNE/PR55α cells were incubated in the presence of 1 μg/ml Dox for 48 h to induce PR55α expression, after which cells were transfected with either a non-targeting siRNA ( Control ) or AUF1 siRNA. Two days later, cells were analyzed by immunoblotting for differences in levels of AUF1 and p16. GAPDH was used as an internal standard. The levels of p16 and GAPDH were quantified using Fiji-ImageJ software and relative p16 levels in the samples were determined after normalizing it with GAPDH levels.

Techniques Used: Expressing, Stable Transfection, Transduction, shRNA, Control, Plasmid Preparation, Isolation, Quantitative RT-PCR, Activity Assay, Transfection, Luciferase, Incubation, Western Blot, Software

PR55α expression increases RB phosphorylation in normal and malignant pancreatic cells. ( A ) A schematic depiction of the regulation of the p16/RB cascade by PR55α-controlled PP2A enzymes. PR55α suppresses the expression of p16, which acts as the primary inhibitor of the CDK4/6 kinases that phosphorylate and inactivate RB, thereby resulting in its dissociation from the E2F transcriptional factor. The net effect of PR55α is promoting the G1/S transition by allowing the phosphorylation of RB and the release of E2F. ( B ) HPNE cells transduced with Dox-inducible PR55α, or control vector were incubated with 1 μg/ml Dox for 3 days to induce ectopic PR55α expression. The cells were exposed to 10 Gy IR and then incubated for the times indicated. Harvested cells were analyzed for RB-Ser708 phosphorylation, total RB level, and GAPDH (as an internal control). ( C ) CD18/HPAF-transduced with the Control or PR55α shRNA were incubated with 2 μg/ml Dox for 5 days to knockdown PR55α expression, after which cells were analyzed by immunoblotting for differences in RB-Ser708 phosphorylation, total RB, and GAPDH.

Figure Legend Snippet: PR55α expression increases RB phosphorylation in normal and malignant pancreatic cells. ( A ) A schematic depiction of the regulation of the p16/RB cascade by PR55α-controlled PP2A enzymes. PR55α suppresses the expression of p16, which acts as the primary inhibitor of the CDK4/6 kinases that phosphorylate and inactivate RB, thereby resulting in its dissociation from the E2F transcriptional factor. The net effect of PR55α is promoting the G1/S transition by allowing the phosphorylation of RB and the release of E2F. ( B ) HPNE cells transduced with Dox-inducible PR55α, or control vector were incubated with 1 μg/ml Dox for 3 days to induce ectopic PR55α expression. The cells were exposed to 10 Gy IR and then incubated for the times indicated. Harvested cells were analyzed for RB-Ser708 phosphorylation, total RB level, and GAPDH (as an internal control). ( C ) CD18/HPAF-transduced with the Control or PR55α shRNA were incubated with 2 μg/ml Dox for 5 days to knockdown PR55α expression, after which cells were analyzed by immunoblotting for differences in RB-Ser708 phosphorylation, total RB, and GAPDH.

Techniques Used: Expressing, Phospho-proteomics, Transduction, Control, Plasmid Preparation, Incubation, shRNA, Knockdown, Western Blot

PR55α inhibits IR-induced cellular senescence. ( A ) Ectopic PR55α overexpression prevents senescence induction by IR in normal HPNE cells. HPNE/PR55α and HPNE/control cells were incubated in the presence of 1 μg/ml Dox for 2 days (to induce ectopic PR55α in the HPNE/PR55α cells) and then exposed to 7 Gy IR or left unirradiated as control ( 0 Gy ). When the second radiation dose was applied, the interval was 24 hours between the two doses. Seven days post-IR, cells were assessed for SA-β-gal activity and photographed. Scale bar = 1 μm. The bar graph expresses the percent of senescent cells in the indicated samples and represents the mean ± S.D. of two separate experiments with each done in duplicate samples. ( B ) PR55α-knockdown sensitizes CD18/HPAF pancreatic cancer cells to senescence induction by IR. CD18/HPAF cells expressing Dox-inducible PR55α-shRNA or Control-shRNA were cultivated in the presence of 2 μg/ml Dox for 5 days, to allow time to silence PR55α expression, and then exposed to 7 Gy IR, or left unirradiated as a control ( 0 Gy ). After 7 days, the cells were assessed for SA-β-gal activity and photographed. Scale bar = 1 μm. The graphs express the percent of senescent cells in the indicated samples and represent the mean ± S.D. of two separate experiments with each one in duplicate samples. ( C , D ) Normal HPNE and CD18/HPAF pancreatic cancer cells, with/without PR55α manipulation, were exposed to 7 Gy IR, or left unirradiated as a control ( 0 day ). When the second radiation dose was applied, the interval was 24 hours between the two doses. The irradiated cells were incubated for the times indicated and analyzed by immunoblotting for the differences in levels of p16, p14, p53, and p21. GAPDH was used as an internal control.

Figure Legend Snippet: PR55α inhibits IR-induced cellular senescence. ( A ) Ectopic PR55α overexpression prevents senescence induction by IR in normal HPNE cells. HPNE/PR55α and HPNE/control cells were incubated in the presence of 1 μg/ml Dox for 2 days (to induce ectopic PR55α in the HPNE/PR55α cells) and then exposed to 7 Gy IR or left unirradiated as control ( 0 Gy ). When the second radiation dose was applied, the interval was 24 hours between the two doses. Seven days post-IR, cells were assessed for SA-β-gal activity and photographed. Scale bar = 1 μm. The bar graph expresses the percent of senescent cells in the indicated samples and represents the mean ± S.D. of two separate experiments with each done in duplicate samples. ( B ) PR55α-knockdown sensitizes CD18/HPAF pancreatic cancer cells to senescence induction by IR. CD18/HPAF cells expressing Dox-inducible PR55α-shRNA or Control-shRNA were cultivated in the presence of 2 μg/ml Dox for 5 days, to allow time to silence PR55α expression, and then exposed to 7 Gy IR, or left unirradiated as a control ( 0 Gy ). After 7 days, the cells were assessed for SA-β-gal activity and photographed. Scale bar = 1 μm. The graphs express the percent of senescent cells in the indicated samples and represent the mean ± S.D. of two separate experiments with each one in duplicate samples. ( C , D ) Normal HPNE and CD18/HPAF pancreatic cancer cells, with/without PR55α manipulation, were exposed to 7 Gy IR, or left unirradiated as a control ( 0 day ). When the second radiation dose was applied, the interval was 24 hours between the two doses. The irradiated cells were incubated for the times indicated and analyzed by immunoblotting for the differences in levels of p16, p14, p53, and p21. GAPDH was used as an internal control.

Techniques Used: Over Expression, Control, Incubation, Activity Assay, Knockdown, Expressing, shRNA, Irradiation, Western Blot

Knockdown of PR55α does not result in senescence induction by IR in pancreatic cancer cells that lack p16 expression. ( A ) The CDKN2A locus, which encodes the p16 and p14 genes, is deleted from AsPC-1 pancreatic cancer cells . Log-phase growing AsPC-1 cells were analyzed for the presence of p16 and p14 proteins with CD18/HPAF pancreatic cancer cells as a positive control. ( B ) AsPC-1 cells stably transduced with the Dox-inducible PR55α-shRNA or Control-shRNA were induced with 2 μg/ml Dox for 3 days to knock down PR55α. The cells were then exposed to 7 Gy IR, or left unirradiated (0 time point), and incubated for an additional 1, 4 and 7 days. The cells were analyzed by immunoblotting for the levels of PR55α, p53, and p21. GAPDH level was measured as an internal loading control. ( C ) The irradiated cells incubated for 7 days were analyzed for senescence by SA-β-gal activity assay and photographed. Scale bar = 1 μm.

Figure Legend Snippet: Knockdown of PR55α does not result in senescence induction by IR in pancreatic cancer cells that lack p16 expression. ( A ) The CDKN2A locus, which encodes the p16 and p14 genes, is deleted from AsPC-1 pancreatic cancer cells . Log-phase growing AsPC-1 cells were analyzed for the presence of p16 and p14 proteins with CD18/HPAF pancreatic cancer cells as a positive control. ( B ) AsPC-1 cells stably transduced with the Dox-inducible PR55α-shRNA or Control-shRNA were induced with 2 μg/ml Dox for 3 days to knock down PR55α. The cells were then exposed to 7 Gy IR, or left unirradiated (0 time point), and incubated for an additional 1, 4 and 7 days. The cells were analyzed by immunoblotting for the levels of PR55α, p53, and p21. GAPDH level was measured as an internal loading control. ( C ) The irradiated cells incubated for 7 days were analyzed for senescence by SA-β-gal activity assay and photographed. Scale bar = 1 μm.

Techniques Used: Knockdown, Expressing, Positive Control, Stable Transfection, Transduction, shRNA, Control, Incubation, Western Blot, Irradiation, Activity Assay

PR55α level is much lower in human normal tissue specimens of older individuals compared to younger individuals and inversely correlates with p16 levels. Human normal tissue specimens derived from various organs/sites were analyzed for differences in PR55α and p16 expression by IHC. ( A ) Representative images of adjacent tissue sections stained with anti-PR55α and anti-p16 antibodies. OSTSE–tonsil; GMST-seminiferous tubules; ET-thyroid; GIE-esophagus. Young, ≤43 y/o; Old, ≥68 y/o; ( B ) Box plot shows the H-Score quantification of PR55α and p16 expression from adjacent sections.

Figure Legend Snippet: PR55α level is much lower in human normal tissue specimens of older individuals compared to younger individuals and inversely correlates with p16 levels. Human normal tissue specimens derived from various organs/sites were analyzed for differences in PR55α and p16 expression by IHC. ( A ) Representative images of adjacent tissue sections stained with anti-PR55α and anti-p16 antibodies. OSTSE–tonsil; GMST-seminiferous tubules; ET-thyroid; GIE-esophagus. Young, ≤43 y/o; Old, ≥68 y/o; ( B ) Box plot shows the H-Score quantification of PR55α and p16 expression from adjacent sections.

Techniques Used: Derivative Assay, Expressing, Staining

other:

Article Title: Proximity-dependent mapping of the HCMV US28 interactome identifies RhoGEF signaling as a requirement for efficient viral reactivation
Article Snippet: Cells were co-transfected with 50ng pcDNA3.1(-) control, or pcDNA3.1-US28-HA or US28-TurboID along with 10ng of pRL-SV40 ( R luc) and 50ng pGL4 firefly luciferase reporter vectors (SRE and SRF) using Fugene4K (Promega).

Article Title: Proximity-dependent mapping of the HCMV US28 interactome identifies RhoGEF signaling as a requirement for efficient viral reactivation
Article Snippet: Reporter plasmids pRL-SV40 Renilla luciferase ( R luc), pGL4.33[luc2P/SRE/Hygro] and pGL4.34[luc2P/SRF-RE/Hygro] containing SRE and SRF responsive elements driving luciferase expression were purchased from Promega.

Article Title: Combinatorial mutagenesis of rapidly evolving residues yields super-restrictor antiviral proteins
Article Snippet: A total of 4.0 ng each of PB2, PB1, and PA and 1.0 ng of NP, all in pCAGGS expression vector, as well as 20 ng pHH21-vNP-FF-Luc (firefly luciferase), 50 ng of pRL-SV40-R luc (Promega), and varying input (50 ng for five-site screen and 15 ng for four-site screen) of MxA plasmids were cotransfected into HEK 293T cells using the reagent TransIT-LT1 (Mirus Bio) in a 96-well plate format.

Transfection:

Article Title: Influenza A Virus Strains Differ in Sensitivity to the Antiviral Action of Mx-GTPase
Article Snippet: .. To provide a measure of transfection efficiency, 0.1 μg of the Renilla luciferase-encoding plasmid pRL-SV40-R luc (Promega, Madison, WI) was cotransfected. ..

Luciferase:

Plasmid Preparation:

Image Search Results

Journal: Aging (Albany NY)

Article Title: PR55α-controlled protein phosphatase 2A inhibits p16 expression and blocks cellular senescence induction by γ-irradiation

doi: 10.18632/aging.205619

Figure Lengend Snippet: Working model for the regulation of the p16/RB pathway and senescence induction by PR55α. Black lines depict our current understanding of the respective roles of the p16/RB and p53/p21 pathways in the promotion of cellular senescence in response to genotoxic stressors [ , ]. The CDKN2A locus produces both the p16 (INK4a) and p14 (ARF) proteins using both separate promoters and alternative splicing. The p16 protein blocks the CDK4/6 kinases leading to RB activation, which is required for G1 cell cycle arrest and senescence induction. The p14 protein stabilizes p53 by inhibiting the MDM2 E3 ubiquitin ligase, resulting in p21 (a p53 target gene) induction and subsequent inhibition of CDK2 and CDK4/6, which also leads to RB activation that promotes G1 cell cycle arrest and senescence. We have previously reported that p53 negatively regulates PR55α protein stability . However, the p53 mutational status had no detectable impact on the effects of PR55α on the expression of p16 and induction of senescence by IR. Red lines depict novel findings presented in this report: (1) PR55α-controlled PP2A enhances IR-induced p53/p21 signaling and (2) PR55α inhibits p16 transcription independently of p53 function.

Article Snippet: Next, this plasmid was digested with SacII and BglII and then self-ligated to eliminate the segment −164 to +17 containing the p16 initiation codon. pRL-SV40-Luc (Promega) is a control Renilla luciferase reporter that contains the SV40 promoter. pGL2-p16-Luc and pRL-SV40-Luc were co-transfected into the cells using Lipofectamine 2000 (Invitrogen, Waltham, MA, USA) according to the manufacturer’s suggestions.

Techniques: Alternative Splicing, Activation Assay, Ubiquitin Proteomics, Inhibition, Expressing

Journal: Aging (Albany NY)

Article Title: PR55α-controlled protein phosphatase 2A inhibits p16 expression and blocks cellular senescence induction by γ-irradiation

doi: 10.18632/aging.205619

Figure Lengend Snippet: PR55α suppresses p16 protein expression in normal and cancer cells originating from the human exocrine pancreas. ( A ) Human pancreatic normal ductal (HPNE) cells were stably transduced with a retroviral vector expressing a Dox-inducible PR55α cDNA (PR55α). As a control, HPNE cells stably transduced with a relevant empty retroviral vector were included in the analysis (Control). Following the induction of ectopic PR55α expression by Dox (1 μg/ml) for 2 days, cells were exposed to 10 Gy of ionizing radiation (IR), incubated for the indicated hours, and analyzed by immunoblotting for the levels of p16, p14, p53, and p21. GAPDH in the lysates was measured as an internal control. ( B ) Human pancreatic ductal adenocarcinoma cells (CD18/HPAF) were stably transduced with a lentiviral vector expressing a Dox-inducible shRNA against either PR55α (PR55α-shRNA) or an irrelevant negative control (Control-shRNA). Following induction of the shRNA with Dox (2 μg/ml) for 5 days, the cells were exposed to 10 Gy IR, incubated for the indicated hours, and analyzed by immunoblotting for the levels of the indicated proteins. GAPDH in the lysates was again used as an internal control. The difference in the p16 levels between HPNE-Control and HPNE-PR55α cells, as well as between the CD18/HPAF-Control-shRNA and CD18/HPAF-PR55α-shRNA cells were determined to be statistically significant (HPNE-Control vs. HPNE-PR55α, p < 0.001; CD18/HPAF-Control-shRNA vs. CD18/HPAF-PR55α-shRNA, p = 0.004).

Techniques: Expressing, Stable Transfection, Transduction, Retroviral, Plasmid Preparation, Control, Incubation, Western Blot, shRNA, Negative Control

Journal: Aging (Albany NY)

Article Title: PR55α-controlled protein phosphatase 2A inhibits p16 expression and blocks cellular senescence induction by γ-irradiation

doi: 10.18632/aging.205619

Figure Lengend Snippet: PR55α inhibits p16 expression by suppressing its mRNA transcription. ( A ) PR55α does not affect p16 protein stability. CD18/HPAF cells stably transduced with Dox-inducible PR55α-shRNA or Control-shRNA were cultivated in media containing 2 μg/ml Dox for 5 days to induce the shRNA, after which cells were exposed to cycloheximide (CHX, 15 μg/ml) to halt protein synthesis. Cell lysates collected at the indicated time points after CHX addition were analyzed for changes in p16 protein levels. The α-tubulin protein has a long half-life and was used as an internal control. Relative p16 protein levels were determined after normalization with the α-tubulin levels and these normalized values were used to calculate the protein half-life of p16. Half-lives were estimated by linear regression analysis of p16 normalized levels against time using SigmaPlot. ( B ) PR55α inhibits p16 mRNA expression. Left panel: HPNE expressing the Dox-inducible PR55α (HPNE/PR55α), or empty vector (HPNE/Control) was treated with Dox (1 μg/ml) for 3 days. Right panel: CD18/HPAF cells expressing the Dox-inducible PR55α shRNA (B4-2, B4-11) or Control shRNA (Control) were treated with Dox (2 μg/ml) for 5 days. At the end of each treatment, RNA was isolated and analyzed by quantitative RT-PCR for differences in p16 mRNA. The relative abundance of the p16 mRNA was calculated by normalizing the p16 mRNA levels with those of the GAPDH mRNA, with the data represented as mean ± S.D. (bar graphs). Statistical significance was calculated by a Student’s t -test (HPNE cells) or one-way ANOVA (CD18/HPAF). The difference with the Control group ( n = 6/group) was determined to be statistically significant at * p < 0.001 or ** p < 0.005. ( C ) PR55α suppresses p16 promoter activity. HPNE and CD18/HPAF cells in the presence/absence of ectopic PR55α and PR55α-shRNA expression, respectively, were co-transfected with a Firefly luciferase reporter under the control of the p16 promoter and a control Renilla luciferase reporter driven by the SV40 promoter. Two days after transfection, Firefly, and Renilla luciferase activities were measured separately in each lysate, as described in the Materials and Methods. p16 promoter activity was calculated by normalizing the activity of Firefly luciferase over that of Renilla luciferase. The graphs show relative p16 promoter activities in the indicated cell samples and are expressed as the mean ± S.D. of two independent experiments done in duplicates. * Statistically significant in a Student’s t -test with p < 0.001. ( D ) HPNE/Control and HPNE/PR55α cells were incubated in the presence of 1 μg/ml Dox for 48 h to induce PR55α expression, after which cells were transfected with either a non-targeting siRNA ( Control ) or AUF1 siRNA. Two days later, cells were analyzed by immunoblotting for differences in levels of AUF1 and p16. GAPDH was used as an internal standard. The levels of p16 and GAPDH were quantified using Fiji-ImageJ software and relative p16 levels in the samples were determined after normalizing it with GAPDH levels.

Techniques: Expressing, Stable Transfection, Transduction, shRNA, Control, Plasmid Preparation, Isolation, Quantitative RT-PCR, Activity Assay, Transfection, Luciferase, Incubation, Western Blot, Software

Journal: Aging (Albany NY)

Article Title: PR55α-controlled protein phosphatase 2A inhibits p16 expression and blocks cellular senescence induction by γ-irradiation

doi: 10.18632/aging.205619

Figure Lengend Snippet: PR55α expression increases RB phosphorylation in normal and malignant pancreatic cells. ( A ) A schematic depiction of the regulation of the p16/RB cascade by PR55α-controlled PP2A enzymes. PR55α suppresses the expression of p16, which acts as the primary inhibitor of the CDK4/6 kinases that phosphorylate and inactivate RB, thereby resulting in its dissociation from the E2F transcriptional factor. The net effect of PR55α is promoting the G1/S transition by allowing the phosphorylation of RB and the release of E2F. ( B ) HPNE cells transduced with Dox-inducible PR55α, or control vector were incubated with 1 μg/ml Dox for 3 days to induce ectopic PR55α expression. The cells were exposed to 10 Gy IR and then incubated for the times indicated. Harvested cells were analyzed for RB-Ser708 phosphorylation, total RB level, and GAPDH (as an internal control). ( C ) CD18/HPAF-transduced with the Control or PR55α shRNA were incubated with 2 μg/ml Dox for 5 days to knockdown PR55α expression, after which cells were analyzed by immunoblotting for differences in RB-Ser708 phosphorylation, total RB, and GAPDH.

Techniques: Expressing, Phospho-proteomics, Transduction, Control, Plasmid Preparation, Incubation, shRNA, Knockdown, Western Blot

Journal: Aging (Albany NY)

Article Title: PR55α-controlled protein phosphatase 2A inhibits p16 expression and blocks cellular senescence induction by γ-irradiation

doi: 10.18632/aging.205619

Figure Lengend Snippet: PR55α inhibits IR-induced cellular senescence. ( A ) Ectopic PR55α overexpression prevents senescence induction by IR in normal HPNE cells. HPNE/PR55α and HPNE/control cells were incubated in the presence of 1 μg/ml Dox for 2 days (to induce ectopic PR55α in the HPNE/PR55α cells) and then exposed to 7 Gy IR or left unirradiated as control ( 0 Gy ). When the second radiation dose was applied, the interval was 24 hours between the two doses. Seven days post-IR, cells were assessed for SA-β-gal activity and photographed. Scale bar = 1 μm. The bar graph expresses the percent of senescent cells in the indicated samples and represents the mean ± S.D. of two separate experiments with each done in duplicate samples. ( B ) PR55α-knockdown sensitizes CD18/HPAF pancreatic cancer cells to senescence induction by IR. CD18/HPAF cells expressing Dox-inducible PR55α-shRNA or Control-shRNA were cultivated in the presence of 2 μg/ml Dox for 5 days, to allow time to silence PR55α expression, and then exposed to 7 Gy IR, or left unirradiated as a control ( 0 Gy ). After 7 days, the cells were assessed for SA-β-gal activity and photographed. Scale bar = 1 μm. The graphs express the percent of senescent cells in the indicated samples and represent the mean ± S.D. of two separate experiments with each one in duplicate samples. ( C , D ) Normal HPNE and CD18/HPAF pancreatic cancer cells, with/without PR55α manipulation, were exposed to 7 Gy IR, or left unirradiated as a control ( 0 day ). When the second radiation dose was applied, the interval was 24 hours between the two doses. The irradiated cells were incubated for the times indicated and analyzed by immunoblotting for the differences in levels of p16, p14, p53, and p21. GAPDH was used as an internal control.

Techniques: Over Expression, Control, Incubation, Activity Assay, Knockdown, Expressing, shRNA, Irradiation, Western Blot

Journal: Aging (Albany NY)

Article Title: PR55α-controlled protein phosphatase 2A inhibits p16 expression and blocks cellular senescence induction by γ-irradiation

doi: 10.18632/aging.205619

Figure Lengend Snippet: Knockdown of PR55α does not result in senescence induction by IR in pancreatic cancer cells that lack p16 expression. ( A ) The CDKN2A locus, which encodes the p16 and p14 genes, is deleted from AsPC-1 pancreatic cancer cells . Log-phase growing AsPC-1 cells were analyzed for the presence of p16 and p14 proteins with CD18/HPAF pancreatic cancer cells as a positive control. ( B ) AsPC-1 cells stably transduced with the Dox-inducible PR55α-shRNA or Control-shRNA were induced with 2 μg/ml Dox for 3 days to knock down PR55α. The cells were then exposed to 7 Gy IR, or left unirradiated (0 time point), and incubated for an additional 1, 4 and 7 days. The cells were analyzed by immunoblotting for the levels of PR55α, p53, and p21. GAPDH level was measured as an internal loading control. ( C ) The irradiated cells incubated for 7 days were analyzed for senescence by SA-β-gal activity assay and photographed. Scale bar = 1 μm.

Techniques: Knockdown, Expressing, Positive Control, Stable Transfection, Transduction, shRNA, Control, Incubation, Western Blot, Irradiation, Activity Assay

Journal: Aging (Albany NY)

Article Title: PR55α-controlled protein phosphatase 2A inhibits p16 expression and blocks cellular senescence induction by γ-irradiation

doi: 10.18632/aging.205619

Figure Lengend Snippet: PR55α level is much lower in human normal tissue specimens of older individuals compared to younger individuals and inversely correlates with p16 levels. Human normal tissue specimens derived from various organs/sites were analyzed for differences in PR55α and p16 expression by IHC. ( A ) Representative images of adjacent tissue sections stained with anti-PR55α and anti-p16 antibodies. OSTSE–tonsil; GMST-seminiferous tubules; ET-thyroid; GIE-esophagus. Young, ≤43 y/o; Old, ≥68 y/o; ( B ) Box plot shows the H-Score quantification of PR55α and p16 expression from adjacent sections.

Techniques: Derivative Assay, Expressing, Staining

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