plpp3  (Proteintech)

Journal: Cells

Article Title: Phospholipid Phosphatase 3 ( PLPP3 ) Induces Oxidative Stress to Accelerate Ovarian Aging in Pigs

doi: 10.3390/cells13171421

Figure Lengend Snippet: PLPP3 may be involved in regulating ovarian aging. ( A ) The mRNA levels of oxidative stress-associated genes SOD1 , CYP1B1 , and SOD2 , autophagy-associated genes p62 , ATG7 , BECN1 , and LC3B , and aging-associated genes SIRT1 , SIRT6 , and p16. The protein levels of SOD1, p62, ATG7, SIRT1, and SIRT6 measured by Western blotting ( B ) and quantified by grayscale analysis ( C ). ( D ) The volcano plot showing the differentially expressed genes between the aged and young ovaries of sows (log2FC > 1, p ≤ 0.05). ( E ) Functional annotation of differentially expressed genes by GO analysis. ( F ) The mRNA level of PLPP3 detected by qRT−PCR in sows’ aged and young ovaries. The protein level of PLPP3 measured by Western blotting ( G ) and quantified by grayscale analysis ( H ). ( I ) The mRNA level of PLPP3 in GCs with PLPP3 overexpression (OE− PLPP3 , 1000 ng/mL). ( J ) The volcano plot showing the differentially expressed genes with PLPP3 overexpression in GCs (log2FC > 1, p ≤ 0.05). ( K ) The functional annotation of differentially expressed genes by GO analysis. The graphs are shown as mean ± SD. The number of independent biological samples (represented as dots) used, and the p −values are indicated in the graphs.

Article Snippet: After GCs reaching 70–80% confluency, the overexpression plasmid (OE- PLPP3 , 1000 ng/mL) and siRNA (si- PLPP3 , 100 nM) of PLPP3 were transfected into GCs using LipofectamineTM 3000 (Thermo Scientific) for 24 h. Then, the RNA and protein were extracted from GCs for qRT-PCR and Western blotting analysis.

Techniques: Western Blot, Functional Assay, Quantitative RT-PCR, Over Expression

Journal: Cells

Article Title: Phospholipid Phosphatase 3 ( PLPP3 ) Induces Oxidative Stress to Accelerate Ovarian Aging in Pigs

doi: 10.3390/cells13171421

Figure Lengend Snippet: PLPP3 aggravates the oxidative stress and ferroptosis in GCs. ( A ) Representative images of ROS level in GCs with PLPP3 overexpression and PLPP3 knockdown (Scale bar: 200 μm). ( B ) The ROS level was quantified with OD (optical density) at 495 nm. ( C ) The mRNA levels of SOD1 , CAT , SOD2 , and CYP1B1 detected by qRT-PCR. The protein levels of SOD1 and CAT detected by Western blotting ( D ) and quantified by the gray value of protein bands ( E ). ( F ) The sub-cellular location and protein expressions of SOD1 (green) and PLPP3 (red) assessed by immunofluorescence analysis in ovaries of sows (Scale bar: Young, 100 μm; Aged, 50 μm). The measurement of MDA ( G ) and Fe ( H ) levels in GCs with PLPP3 overexpression and PLPP3 knockdown. ( I ) The mRNA levels of GPX4 , SLC7A11 , FTH1 , NOX1 , and FSP1 detected by qRT-PCR. The protein levels of FSP1, FTH1, and SLC7A11 detected by Western blotting ( J ) and quantified by the gray value of protein bands ( K ). The graphs are shown as mean ± SD. The number of independent biological samples (represented as dots) used, and the p -values are indicated in the graphs.

Article Snippet: After GCs reaching 70–80% confluency, the overexpression plasmid (OE- PLPP3 , 1000 ng/mL) and siRNA (si- PLPP3 , 100 nM) of PLPP3 were transfected into GCs using LipofectamineTM 3000 (Thermo Scientific) for 24 h. Then, the RNA and protein were extracted from GCs for qRT-PCR and Western blotting analysis.

Techniques: Over Expression, Knockdown, Quantitative RT-PCR, Western Blot, Immunofluorescence

Journal: Cells

Article Title: Phospholipid Phosphatase 3 ( PLPP3 ) Induces Oxidative Stress to Accelerate Ovarian Aging in Pigs

doi: 10.3390/cells13171421

Figure Lengend Snippet: PLPP3 increases the autophagy level in GCs. ( A ) Representative images of GFP-LC3 puncta in GCs with PLPP3 overexpression and knockdown (Scale bar: 50 μm). ( B ) The autophagy level was quantified by the number of autophagosomes (yellow dots) and autolysosomes (free red dots). ( C ) The mRNA levels of p62 , LC3B , ATG7 , BECN1 , and ATG5 detected by qRT-PCR. The protein levels of p62, LC3B, and ATG7 detected by Western blotting (ns = not significant) ( D ) and quantified by the gray value of protein bands ( E ). ( F ) The sub-cellular location and protein expressions of p62 (green) and PLPP3 (red) assessed by immunofluorescence analysis in ovaries of sows (Scale bar: 100 μm). The graphs are shown as mean ± SD. The number of independent biological samples (represented as dots) used, and the p -values are indicated in the graphs.

Article Snippet: After GCs reaching 70–80% confluency, the overexpression plasmid (OE- PLPP3 , 1000 ng/mL) and siRNA (si- PLPP3 , 100 nM) of PLPP3 were transfected into GCs using LipofectamineTM 3000 (Thermo Scientific) for 24 h. Then, the RNA and protein were extracted from GCs for qRT-PCR and Western blotting analysis.

Techniques: Over Expression, Knockdown, Quantitative RT-PCR, Western Blot, Immunofluorescence

Journal: Journal of Lipid Research

Article Title: Identification of two lipid phosphatases that regulate sphingosine-1-phosphate cellular uptake and recycling

doi: 10.1016/j.jlr.2022.100225

Figure Lengend Snippet: Genome-wide CRISPR/Cas9 KO screen identifies regulators of S1P cellular uptake and recycling into the sphingolipid synthesis pathway. A: Schematic of the genome-wide CRISPR/Cas9 KO screening strategy undertaken to identify genes required for S1P uptake and recycling. WT Cas9-expressing HeLa cells (gray); SPTLC1 KO Cas9-expressing HeLa cells (light blue). Cells were transduced (MOI of 0.25) with a lentivirus-based genome-wide CRISPR/Cas9 KO library and selected in puromycin to yield cells that each contained approximately one viral genome. Cells were grown in media containing S1P (orange) to induce Gb3 cell-surface expression and treated either with or without Shiga toxin. The genomic DNA of cells from each group was subjected to deep sequencing to identify the sgRNAs. B: Scatterplot showing the ranking of genes from MAGeCK analysis. X-axis indicates positive ranking of individual genes, and y-axis indicates -Log10 values of corresponding robust ranking aggregation (RRA) score. The 10 top-ranking genes are highlighted and labeled. Inset panel , scatterplot showing log-fold change for all genes. The genes for enzymes directly involved in Gb3 synthesis are marked with asterisks. The genes shown in red are involved in the S1P uptake and recycling pathway. C: Schematic showing sphingolipid synthesis pathway leading to the production of Gb3 in SPTLC1 KO HeLa cells. Top-ranking genes from the screen that are directly involved in Gb3 synthesis are marked with asterisks. PLPP3 (PPAP2B) and SGPP1 , which are not involved in de novo synthesis of Gb3 ( , , , ), are involved in the S1P uptake and recycling pathway. CRISPR, clustered regularly interspersed short palindromic repeats; Gb3, globotriaosylceramide; MAGeCK, Model-based Analysis of Genome-wide CRISPR-Cas9 KO; MOI, multiplicity of infection; PM, plasma membrane; S1P, sphingosine-1-phosphate; sgRNA, single guide RNA.

Article Snippet: HeLa cells were transfected either with pReceiver-M29 plasmids containing eGFP-SGPP1 , eGFP-SPHK1 , or eGFP-SPHK2 , or with pReceiver-M03 plasmids containing PLPP3 (PPAP2B)-eGFP or SGPL1-eGFP (GeneCopoeia, Rockville, MD) using the Lipofectamine 3000 reagent.

Techniques: Genome Wide, CRISPR, Expressing, Sequencing, Labeling, Infection

Journal: Journal of Lipid Research

Article Title: Identification of two lipid phosphatases that regulate sphingosine-1-phosphate cellular uptake and recycling

doi: 10.1016/j.jlr.2022.100225

Figure Lengend Snippet: PLPP3 or SGPP1 disruption reduces S1P-stimulated Gb3 cell-surface expression. A: Schematic of the potential roles of PLPP3 (PPAP2B) and SGPP1 in the S1P uptake and recycling pathway. B: Timeline of experiment used to measure Gb3 recovery after 2.5 μM myriocin and 3 μM S1P treatment in KD HeLa cells and WT HeLa cells. C: Validation of myriocin inhibition of de novo Gb3 synthesis and subsequent cell-surface expression in WT HeLa cells. WT HeLa cells were cultured with 2.5 μM myriocin for 7 days and cell-surface Gb3 was measured by flow cytometry. Data represent the mean ± SD (n = 3). D: Gb3 cell-surface expression recovery rate after supplemental S1P treatment in myriocin-treated PLPP3 KD HeLa cells and WT HeLa cells, based on flow cytometry. Experiments were performed twice using one PLPP3 KD clone and WT HeLa cells (n = 6). E: Gb3 cell-surface expression recovery rate after supplemental S1P treatment in myriocin-treated SGPP1 KD HeLa cells and WT HeLa cells, based on flow cytometry. Experiments were performed three times using two SGPP1 KD clones and WT HeLa cells (n = 9 for WT, n = 12 for SGPP1 ). D and E: Gb3 cell-surface expression in the cells treated only with myriocin (0 h of S1P supplementation) was used as a baseline and subtracted from each data value. Experiments were conducted in triplicate wells for each condition. Data are presented as mean ± SD. P values were determined by unpaired t-tests; ∗ P < 0.05, ∗∗ P < 0.01, ∗∗∗∗ P < 0.0001. ns, not significant. Gb3, globotriaosylceramide; KD, knockdown; PM, plasma membrane; Sph, sphingosine, S1P, sphingosine-1-phosphate.

Article Snippet: HeLa cells were transfected either with pReceiver-M29 plasmids containing eGFP-SGPP1 , eGFP-SPHK1 , or eGFP-SPHK2 , or with pReceiver-M03 plasmids containing PLPP3 (PPAP2B)-eGFP or SGPL1-eGFP (GeneCopoeia, Rockville, MD) using the Lipofectamine 3000 reagent.

Techniques: Expressing, Inhibition, Cell Culture, Flow Cytometry, Clone Assay

Journal: Journal of Lipid Research

Article Title: Identification of two lipid phosphatases that regulate sphingosine-1-phosphate cellular uptake and recycling

doi: 10.1016/j.jlr.2022.100225

Figure Lengend Snippet: SGPP1 localizes to the ER and PLPP3 (PPAP2B) localizes to the plasma membrane. A: eGFP-SGPP1 and (F) PLPP3 (PPAP2B)-eGFP were transfected into HeLa cells along with RFP-tagged organelle markers ( supplemental Table S1 ) for (B) ER and (G) plasma membrane (PM). C and H: Nuclei (blue) were stained by NucBlue Live Cell ReadyProbes Reagent (Hoechst 33,342). Cells were examined by confocal fluorescence microscopy. D and I: Merged images. E and J: Colocalization of fluorescent markers was determined by Manders overlap coefficient using ZEN Blue software (ZEN 2012 SP5). n=20–30 cells. RFP, red fluorescent protein.

Article Snippet: HeLa cells were transfected either with pReceiver-M29 plasmids containing eGFP-SGPP1 , eGFP-SPHK1 , or eGFP-SPHK2 , or with pReceiver-M03 plasmids containing PLPP3 (PPAP2B)-eGFP or SGPL1-eGFP (GeneCopoeia, Rockville, MD) using the Lipofectamine 3000 reagent.

Techniques: Transfection, Staining, Fluorescence, Microscopy, Software

Journal: Journal of Lipid Research

Article Title: Identification of two lipid phosphatases that regulate sphingosine-1-phosphate cellular uptake and recycling

doi: 10.1016/j.jlr.2022.100225

Figure Lengend Snippet: Proposed pathway for S1P cellular uptake and incorporation into the sphingolipid synthesis pathway. Plasma membrane–resident PLPP3 dephosphorylates exogenous S1P to sphingosine, which enters cells by a flip-flop mechanism and is phosphorylated to S1P by SPHKs 1 and 2. Intracellular S1P serves as a branch-point substrate for ER-resident SGPP1 and SGPL1. SGPP1 dephosphorylates S1P to sphingosine, which is utilized for ceramide synthesis (CERS2) and sequential modifications for glycosphingolipid production. SGPL1 irreversibly degrades S1P to hexadecenal and phosphoethanolamine, allowing exit of the substrate from the sphingolipid synthesis pathway. These enzymes work in concert to drive an S1P dephosphorylation-phosphorylation-dephosphorylation/degradation cycle. In the absence of sphingosine kinases, sphingosine may bypass the cycle and directly serve as a CERS2 substrate (dashed line), thereby being excluded from the SGPL1-mediated degradation pathway. UGCG, B4GALT5, and A4GALT are Golgi core glycosphingolipid synthesis enzymes for synthesis of Gb3. LAPTM4A, TM9SF2, SLC35A2, TMEM165, and GOLPH3 are positive regulators of Gb3 synthesis in the Golgi. AHR is a transcriptional activator of genes involved in glycosphingolipid synthesis. Deg, degradation; Dephos, dephosphorylation; Gb3, globotriaosylceramide; Phos, phosphorylation; PM, plasma membrane; Sph, sphingosine; S1P, sphingosine-1-phosphate

Article Snippet: HeLa cells were transfected either with pReceiver-M29 plasmids containing eGFP-SGPP1 , eGFP-SPHK1 , or eGFP-SPHK2 , or with pReceiver-M03 plasmids containing PLPP3 (PPAP2B)-eGFP or SGPL1-eGFP (GeneCopoeia, Rockville, MD) using the Lipofectamine 3000 reagent.

Techniques: De-Phosphorylation Assay

Journal: Cardiovascular Research

Article Title: DNA methylation of a PLPP3 MIR transposon-based enhancer promotes an osteogenic programme in calcific aortic valve disease

doi: 10.1093/cvr/cvy111

Figure Lengend Snippet: Expression of PLPP3 is reduced in CAVD. (A) Transcriptome of PLPPs in CTL (n = 12) and CAVD specimens (n = 12), the number in parentheses following the gene name represents the relative change in expression (*indicates P < 0.05). (B) mRNA levels for PLPP3 in CTL (n = 29) and CAVD (n = 39) (P < 0.0001). (C) Western blotting for PLPP3 showing a representative experiment and the quantification in CTL (n = 8) and CAVD (n = 9) (P = 0.01). (D) PLPP enzyme activity in CTL (n = 8) and CAVD (n = 8) (P = 0.03). (E) LysoPA relative levels according to PLPP3 mRNA levels (divided at median) (n = 10) (P = 0.007). (F) PLPP3 mRNA levels in pathologic aortic valves (CAVD) according to the state of mineralization (n = 7) (P = 0.007). G) PLPP3 mRNA levels in VICs isolated from CTL (n = 5) and CAVD (n = 5) (P = 0.004). Values are mean ± SEM, (B) Student t-test, (C–G) Wilcoxon-Mann-Whitney statistical analyses; CTL, control; CAVD, calcific aortic valve disease.

Article Snippet: Membranes were blocked with TBS-tween containing 5% non-fat dry milk and incubated with either PLPP3 (Novus Biologicals, ON, Canada) or β-ACTIN (Sigma-Aldrich, ON, Canada) primary antibodies overnight at 4°C.

Techniques: Expressing, Western Blot, Activity Assay, Isolation, MANN-WHITNEY, Control

Journal: Cardiovascular Research

Article Title: DNA methylation of a PLPP3 MIR transposon-based enhancer promotes an osteogenic programme in calcific aortic valve disease

doi: 10.1093/cvr/cvy111

Figure Lengend Snippet: 5meC level at cg02468627 inversely correlates with PLPP3 expression. (A) Genomic coordinates (hg19) of CpG sites (open circles) and their relationships with PLPP3 expression represented as –log10 (P-value). (B) Correlation analysis between the expression of PLPP3 and CpG methylation level (cg02468627) (n = 21) in CAVD (r = −0.51, P = 0.01). Bisulphite pyrosequencing of cg02468627 in CTL (n = 24) and CAVD (n = 43) (P < 0.0001) (C), and correlation analysis between methylation of cg02468627 with PLPP3 expression (n = 28) (r = −0.48, P = 0.009) (D). (E) Bisulphite pyrosequencing levels in pathologic aortic valves according to the state of mineralization (n = 8) (P = 0.03). Values are mean ± S.E.M.; (A) linear regression model, (B and D) Pearson’s coefficient, (C) student t-tests, (E) Wilcoxon-Mann-Whitney analysis; CTL, control; CAVD, calcific aortic valve disease.

Article Snippet: Membranes were blocked with TBS-tween containing 5% non-fat dry milk and incubated with either PLPP3 (Novus Biologicals, ON, Canada) or β-ACTIN (Sigma-Aldrich, ON, Canada) primary antibodies overnight at 4°C.

Techniques: Expressing, CpG Methylation Assay, Methylation, MANN-WHITNEY, Control

Journal: Cardiovascular Research

Article Title: DNA methylation of a PLPP3 MIR transposon-based enhancer promotes an osteogenic programme in calcific aortic valve disease

doi: 10.1093/cvr/cvy111

Figure Lengend Snippet: Intron 1 of PLPP3 contains an enhancer. (A) UCSC browser image (hg19) showing cg02468627 in highly conserved region containing a MIR3 sequence. (B–D) H3K4me1 (CTL n = 5, CAVD n = 5) (P = 0.42) (B), H3K4me3 (CTL n = 5, CAVD n = 5) (P = 0.15) (C), and H3K27me3 (CTL n = 5, CAVD n = 6) (P = 0.017) (D) levels at MIR3 locus (determined by quantitative ChIP). Values are mean ± S.E.M., Wilcoxon-Mann-Whitney analyses; CTL, control; CAVD, calcific aortic valve disease.

Article Snippet: Membranes were blocked with TBS-tween containing 5% non-fat dry milk and incubated with either PLPP3 (Novus Biologicals, ON, Canada) or β-ACTIN (Sigma-Aldrich, ON, Canada) primary antibodies overnight at 4°C.

Techniques: Sequencing, MANN-WHITNEY, Control

Journal: Cardiovascular Research

Article Title: DNA methylation of a PLPP3 MIR transposon-based enhancer promotes an osteogenic programme in calcific aortic valve disease

doi: 10.1093/cvr/cvy111

Figure Lengend Snippet: Intronic enhancer regulates PLPP3 expression. (A) Reporter assay constructions and activities (HEK293T) (n = 5) (P = 0.007). (B) Scheme showing dCas9-DNMT and dCas9-ΔDNMT for epigenome editing. C) Bisulphite pyrosequencing of intronic enhancer performed in HEK293T after epigenome editing (n = 5) (P = 0.03). (D) Reporter assay in response to epigenome editing (HEK293T) (n = 6) (P = 0.03). (E) Expression of PLPP3 in VICs in response to epigenome editing (n = 5) (P = 0.03). Values are mean ± S.E.M., Wilcoxon-Mann-Whitney analyses.

Article Snippet: Membranes were blocked with TBS-tween containing 5% non-fat dry milk and incubated with either PLPP3 (Novus Biologicals, ON, Canada) or β-ACTIN (Sigma-Aldrich, ON, Canada) primary antibodies overnight at 4°C.

Techniques: Expressing, Reporter Assay, MANN-WHITNEY

Journal: Cardiovascular Research

Article Title: DNA methylation of a PLPP3 MIR transposon-based enhancer promotes an osteogenic programme in calcific aortic valve disease

doi: 10.1093/cvr/cvy111

Figure Lengend Snippet: Lower expression of PLPP3 enhances the osteogenic transition of VICs. (A) siRNA-mediated knockdown on mRNA (n = 5) (P = 0.007) and protein levels. (B–D) PLPP3 siRNA on lysoPA-mediated gene expression of BMP2 (n = 11) (pAnova < 0.0001) (B), RUNX2 (n = 11) (pAnova < 0.0001) (C), and BGLAP (n = 9) (P < 0.0001) (D). (E and F) PLPP3 knockdown on lysoPA-induced mineralization of VIC cultures (n = 5) (day 7) (P = 0.0001) (E), and ALP activity (n = 7) (day 7) (P < 0.0001) (F). (G) Proposed working model showing that EZH2 (enzyme component of the PRC2 complex that increases H3K27me3) may associate with DNMT3 (de novo methylation of CpG) and promote silencing of PLPP3 intronic enhancer during the mineralization of the AV. Values are mean ± S.E.M.; (A) Wilcoxon-Mann-Whitney analysis, (B and C) Anova, post-hoc Tukey, (D–F) Kruskal-Wallis, post-hoc Steel; *P < 0.05; LysoPA: 10 µM; PO4 is mineralizing medium (PO4 2 mM); percentage of calcium in (E) is indicated and represents a surrogate of mineralization (hydroxyapatite of calcium) that is deposited by VICs.

Article Snippet: Membranes were blocked with TBS-tween containing 5% non-fat dry milk and incubated with either PLPP3 (Novus Biologicals, ON, Canada) or β-ACTIN (Sigma-Aldrich, ON, Canada) primary antibodies overnight at 4°C.

Techniques: Expressing, Knockdown, Gene Expression, Activity Assay, Methylation, MANN-WHITNEY

Journal: Cardiovascular Research

Article Title: DNA methylation of a PLPP3 MIR transposon-based enhancer promotes an osteogenic programme in calcific aortic valve disease

doi: 10.1093/cvr/cvy111

Figure Lengend Snippet: Expression of PLPP3 is reduced in CAVD. (A) Transcriptome of PLPPs in CTL (n = 12) and CAVD specimens (n = 12), the number in parentheses following the gene name represents the relative change in expression (*indicates P < 0.05). (B) mRNA levels for PLPP3 in CTL (n = 29) and CAVD (n = 39) (P < 0.0001). (C) Western blotting for PLPP3 showing a representative experiment and the quantification in CTL (n = 8) and CAVD (n = 9) (P = 0.01). (D) PLPP enzyme activity in CTL (n = 8) and CAVD (n = 8) (P = 0.03). (E) LysoPA relative levels according to PLPP3 mRNA levels (divided at median) (n = 10) (P = 0.007). (F) PLPP3 mRNA levels in pathologic aortic valves (CAVD) according to the state of mineralization (n = 7) (P = 0.007). G) PLPP3 mRNA levels in VICs isolated from CTL (n = 5) and CAVD (n = 5) (P = 0.004). Values are mean ± SEM, (B) Student t-test, (C–G) Wilcoxon-Mann-Whitney statistical analyses; CTL, control; CAVD, calcific aortic valve disease.

Article Snippet: Primers for PLPP3 (cat no. QT00052836), RUNX2 (cat no. QT00020517) , BGLAP (cat no. QT00232771), and BMP2 (cat no. QT00012544) were obtained from QIAGEN (ON, Canada).

Techniques: Expressing, Western Blot, Activity Assay, Isolation, MANN-WHITNEY, Control

Journal: Cardiovascular Research

Article Title: DNA methylation of a PLPP3 MIR transposon-based enhancer promotes an osteogenic programme in calcific aortic valve disease

doi: 10.1093/cvr/cvy111

Figure Lengend Snippet: 5meC level at cg02468627 inversely correlates with PLPP3 expression. (A) Genomic coordinates (hg19) of CpG sites (open circles) and their relationships with PLPP3 expression represented as –log10 (P-value). (B) Correlation analysis between the expression of PLPP3 and CpG methylation level (cg02468627) (n = 21) in CAVD (r = −0.51, P = 0.01). Bisulphite pyrosequencing of cg02468627 in CTL (n = 24) and CAVD (n = 43) (P < 0.0001) (C), and correlation analysis between methylation of cg02468627 with PLPP3 expression (n = 28) (r = −0.48, P = 0.009) (D). (E) Bisulphite pyrosequencing levels in pathologic aortic valves according to the state of mineralization (n = 8) (P = 0.03). Values are mean ± S.E.M.; (A) linear regression model, (B and D) Pearson’s coefficient, (C) student t-tests, (E) Wilcoxon-Mann-Whitney analysis; CTL, control; CAVD, calcific aortic valve disease.

Article Snippet: Primers for PLPP3 (cat no. QT00052836), RUNX2 (cat no. QT00020517) , BGLAP (cat no. QT00232771), and BMP2 (cat no. QT00012544) were obtained from QIAGEN (ON, Canada).

Techniques: Expressing, CpG Methylation Assay, Methylation, MANN-WHITNEY, Control

Journal: Cardiovascular Research

Article Title: DNA methylation of a PLPP3 MIR transposon-based enhancer promotes an osteogenic programme in calcific aortic valve disease

doi: 10.1093/cvr/cvy111

Figure Lengend Snippet: Intron 1 of PLPP3 contains an enhancer. (A) UCSC browser image (hg19) showing cg02468627 in highly conserved region containing a MIR3 sequence. (B–D) H3K4me1 (CTL n = 5, CAVD n = 5) (P = 0.42) (B), H3K4me3 (CTL n = 5, CAVD n = 5) (P = 0.15) (C), and H3K27me3 (CTL n = 5, CAVD n = 6) (P = 0.017) (D) levels at MIR3 locus (determined by quantitative ChIP). Values are mean ± S.E.M., Wilcoxon-Mann-Whitney analyses; CTL, control; CAVD, calcific aortic valve disease.

Article Snippet: Primers for PLPP3 (cat no. QT00052836), RUNX2 (cat no. QT00020517) , BGLAP (cat no. QT00232771), and BMP2 (cat no. QT00012544) were obtained from QIAGEN (ON, Canada).

Techniques: Sequencing, MANN-WHITNEY, Control

Journal: Cardiovascular Research

Article Title: DNA methylation of a PLPP3 MIR transposon-based enhancer promotes an osteogenic programme in calcific aortic valve disease

doi: 10.1093/cvr/cvy111

Figure Lengend Snippet: Intronic enhancer regulates PLPP3 expression. (A) Reporter assay constructions and activities (HEK293T) (n = 5) (P = 0.007). (B) Scheme showing dCas9-DNMT and dCas9-ΔDNMT for epigenome editing. C) Bisulphite pyrosequencing of intronic enhancer performed in HEK293T after epigenome editing (n = 5) (P = 0.03). (D) Reporter assay in response to epigenome editing (HEK293T) (n = 6) (P = 0.03). (E) Expression of PLPP3 in VICs in response to epigenome editing (n = 5) (P = 0.03). Values are mean ± S.E.M., Wilcoxon-Mann-Whitney analyses.

Article Snippet: Primers for PLPP3 (cat no. QT00052836), RUNX2 (cat no. QT00020517) , BGLAP (cat no. QT00232771), and BMP2 (cat no. QT00012544) were obtained from QIAGEN (ON, Canada).

Techniques: Expressing, Reporter Assay, MANN-WHITNEY

Journal: Cardiovascular Research

Article Title: DNA methylation of a PLPP3 MIR transposon-based enhancer promotes an osteogenic programme in calcific aortic valve disease

doi: 10.1093/cvr/cvy111

Figure Lengend Snippet: Lower expression of PLPP3 enhances the osteogenic transition of VICs. (A) siRNA-mediated knockdown on mRNA (n = 5) (P = 0.007) and protein levels. (B–D) PLPP3 siRNA on lysoPA-mediated gene expression of BMP2 (n = 11) (pAnova < 0.0001) (B), RUNX2 (n = 11) (pAnova < 0.0001) (C), and BGLAP (n = 9) (P < 0.0001) (D). (E and F) PLPP3 knockdown on lysoPA-induced mineralization of VIC cultures (n = 5) (day 7) (P = 0.0001) (E), and ALP activity (n = 7) (day 7) (P < 0.0001) (F). (G) Proposed working model showing that EZH2 (enzyme component of the PRC2 complex that increases H3K27me3) may associate with DNMT3 (de novo methylation of CpG) and promote silencing of PLPP3 intronic enhancer during the mineralization of the AV. Values are mean ± S.E.M.; (A) Wilcoxon-Mann-Whitney analysis, (B and C) Anova, post-hoc Tukey, (D–F) Kruskal-Wallis, post-hoc Steel; *P < 0.05; LysoPA: 10 µM; PO4 is mineralizing medium (PO4 2 mM); percentage of calcium in (E) is indicated and represents a surrogate of mineralization (hydroxyapatite of calcium) that is deposited by VICs.

Article Snippet: Primers for PLPP3 (cat no. QT00052836), RUNX2 (cat no. QT00020517) , BGLAP (cat no. QT00232771), and BMP2 (cat no. QT00012544) were obtained from QIAGEN (ON, Canada).

Techniques: Expressing, Knockdown, Gene Expression, Activity Assay, Methylation, MANN-WHITNEY