Journal: Molecular Medicine Reports

Article Title: Rs41291957 polymorphism in the promoter region of microRNA-143 serves as a prognostic biomarker for patients with intracranial hemorrhage

doi: 10.3892/mmr.2021.11928

Figure Lengend Snippet: TLR2 is a direct target gene of miR-143. (A) Computational analysis of the regulatory relationship between miR-143 and TLR2 mRNA. (B) Luciferase assay of THP-1 cells co-transfected with wild-type or mutant TLR2 mRNA, and miR-143 or miRNA controls. (C) Luciferase assay of HPASMC cells co-transfected with wild-type or mutant TLR2 mRNA, and miR-143 or miRNA controls. (D) Relative expression of miR-143 in THP-1 cells transfected with miR-143 mimics. (E) Relative expression of miR-143 in HPASMC cells transfected with miR-143 mimics. n=3. *P<0.05 vs. negative control group. TLR2, Toll-like receptor 2; miR-143, microRNA-143; miR-cont, microRNA-control; 3′-UTR, 3′-untranslated region; WT, wild-type; MUT, mutant; NC, negative control.

Article Snippet: THP-1 (human monocytic cells) and human pulmonary artery smooth muscle cells (HPASMC) obtained from American Type Culture Collection were cultured in DMEM (Gibco; Thermo Fisher Scientific, Inc.) supplemented with 10% fetal bovine serum (Invitrogen; Thermo Fisher Scientific, Inc.).

Techniques: Luciferase, Transfection, Mutagenesis, Expressing, Negative Control, Control

Journal: Molecular Medicine Reports

Article Title: Rs41291957 polymorphism in the promoter region of microRNA-143 serves as a prognostic biomarker for patients with intracranial hemorrhage

doi: 10.3892/mmr.2021.11928

Figure Lengend Snippet: IL-16 mRNA is not a target of miR-143. (A) Computational analysis of the regulatory relationship between miR-143 and IL-16 mRNA. (B) Luciferase assay of THP-1 cells co-transfected with wild-type or mutant IL-16 mRNA, and miR-143 or miRNA controls. (C) Luciferase assay of HPASMC cells co-transfected with wild-type or mutant IL-16 mRNA, and miR-143 or miRNA controls. IL-16, interleukin-16; miR-143, microRNA-143; 3′-UTR, 3′-untranslated region; WT, wild-type; MUT, mutant.

Article Snippet: THP-1 (human monocytic cells) and human pulmonary artery smooth muscle cells (HPASMC) obtained from American Type Culture Collection were cultured in DMEM (Gibco; Thermo Fisher Scientific, Inc.) supplemented with 10% fetal bovine serum (Invitrogen; Thermo Fisher Scientific, Inc.).

Techniques: Luciferase, Transfection, Mutagenesis

Journal: Circulation research

Article Title: Altered Smooth Muscle Cell Histone Acetylome by the SPHK2/S1P Axis Promotes Pulmonary Hypertension

doi: 10.1161/CIRCRESAHA.123.322740

Figure Lengend Snippet: (A) Representative immunocytochemistry images of pSPHK2 (pink), actin (green, cytoplasmic marker) and DAPI (blue, nuclear) coimmunostaining in EMAP II treated (2 hr) or vehicle treated fixed hPASMCs, scale bar is 20 μm, n=3. (B) Representative immunoblot probed for pSPHK2, tubulin and lamin B in cytoplasmic and nuclear fractions of hPASMCs following EMAP II treatment for 0, 2 and 4 hours, n=3. (C) Representative immunoblot probed for pSPHK2 and lamin B in nuclear fractions of hPASMCs following EMAP II treatment (150 minutes) with or without SPHK2 inhibitor (D) quantification of nuclear pSPHK2/lamin B, n=3. (E) ELISA-nuclear C18-S1P levels normalized against 1 μg of nuclear proteins in the nuclear fractions of hPASMCs following EMAP II for 15 or 150 minutes with or without SPHK2 inhibitor, n=3 or 4/group. P values are calculated using Kruskal-Wallis against control or Kolmogorov-Smirnov non-parametric test and results are shown as median and inter-quartile range.

Article Snippet: Primary human PASMCs (hPASMCs), pulmonary microvascular endothelial cells (hPMVECs) purchased from Lonza (Walkersville, MD) and iPAH: PASMCs were cultured in complete growth medium or conditioned media in a humidified atmosphere or 1% O 2 with 5% CO 2 at 37° C. For all studies, passages 5–10 were used for hPASMCs and hPMVECs.

Techniques: Immunocytochemistry, Marker, Western Blot, Enzyme-linked Immunosorbent Assay, Control

Journal: Circulation research

Article Title: Altered Smooth Muscle Cell Histone Acetylome by the SPHK2/S1P Axis Promotes Pulmonary Hypertension

doi: 10.1161/CIRCRESAHA.123.322740

Figure Lengend Snippet: (A) Schematic diagram representing the collection of vascular endothelial cells (ECs) conditioned media (ECM) from ECs grown in 1%O2 or room air to treat vascular smooth muscle cells (SMCs) and, (B) representative dot blot probed for secreted EMAP II expression in ECM. (C) Representative immunoblot probed for KLF4, SPHK2, tubulin, Ac-H3K9 and total histone H3 in whole cell lysates of normoxia or hypoxia ECM with or without EMAP II neutralizing antibody treated hPASMCs pre-transfected with siRNA mediated SPHK2 or scramble silencing and, (D) quantification of KLF4/Tubulin, n=3 and (E) quantification of Ac-H3K9/total histone H3, n=3. (F) KLF4 expression levels normalized against 18S rRNA in normoxia or hypoxia ECM with or without EMAP II neutralizing antibody treated hPASMCs pre-transfected with siRNA mediated SPHK2 or scramble silencing, n=3–4. (G) EMAP II secreted by vascular ECs promote SPHK2/Ac-H3K9/KLF4 signaling in vascular SMCs that may promote PASMCs proliferation. P values are calculated using Kruskal-Wallis against Hy ECM+Scr or Kolmogorov-Smirnov non-parametric test if not mentioned otherwise, and results are shown as median and inter-quartile range.

Article Snippet: Primary human PASMCs (hPASMCs), pulmonary microvascular endothelial cells (hPMVECs) purchased from Lonza (Walkersville, MD) and iPAH: PASMCs were cultured in complete growth medium or conditioned media in a humidified atmosphere or 1% O 2 with 5% CO 2 at 37° C. For all studies, passages 5–10 were used for hPASMCs and hPMVECs.

Techniques: Dot Blot, Expressing, Western Blot, Transfection

Journal: Circulation research

Article Title: Altered Smooth Muscle Cell Histone Acetylome by the SPHK2/S1P Axis Promotes Pulmonary Hypertension

doi: 10.1161/CIRCRESAHA.123.322740

Figure Lengend Snippet: (A) Representative immunoblot probed for AIMP1 (precursor form of EMAP II) and Tubulin in protein lysates of human iPAH or FDL, n=19–20/group and, (B) quantitation of AIMP1 (AIMP1/Tubulin) in protein lysates of human iPAH or FDL, n=19–20/group. (C) Representative immunoblot probed for Ac-H3K9, total H3 or tubulin in hPASMCs following EMAP II treatment for 0, 1, 2, 4 and 6 hours and (D) quantitation of Ac-H3K9 expression levels normalized against total H3 in hPASMCs, n=4. (E) Representative immunoblot probed for Ac-H3K9, total H3 or tubulin in hPMVECs following EMAP II treatment for 0, 1, 2, 4 and 6 hours and (F) quantitation of Ac-H3K9 expression levels normalized against total H3 in hPMVECs, n=3. P values are calculated using unpaired t-test or Kolmogorov-Smirnov non-parametric testing and results are shown as means ± SEM or median and inter-quartile range.

Article Snippet: Primary human PASMCs (hPASMCs), pulmonary microvascular endothelial cells (hPMVECs) purchased from Lonza (Walkersville, MD) and iPAH: PASMCs were cultured in complete growth medium or conditioned media in a humidified atmosphere or 1% O 2 with 5% CO 2 at 37° C. For all studies, passages 5–10 were used for hPASMCs and hPMVECs.

Techniques: Western Blot, Quantitation Assay, Expressing

Journal: Circulation research

Article Title: Altered Smooth Muscle Cell Histone Acetylome by the SPHK2/S1P Axis Promotes Pulmonary Hypertension

doi: 10.1161/CIRCRESAHA.123.322740

Figure Lengend Snippet: (A) Representative immunoblot probed for Ac-H3K9, total H3, SPHK2 and tubulin in whole cell lysates of hPASMCs following siRNA mediated SPHK2 silencing and post-transfection EMAP II treatment for 4 hours and (B) quantitation of Ac-H3K9/total H3 and (C) quantification of SPHK2/tubulin, n=4. (D) Volcano plot showed the log2-fold changes and statistical significance of hyperacetylated H3K9 regions calculated after differential binding analysis of EMAP II treated vs control hPASMCs. Pink points indicate significantly hyperacetylated H3K9 regions in EMAP II (right to 0) or in control (left to 0). FDR=0.05, n=2 (E) Genome wide distribution of differentially enriched hyperacetylated H3K9 peaks (log2-fold change > 1, p value < 0.05) n=2. (F) Number of peaks of Ac-H3K9 normalized to IgG in with or without SPHK2 inhibitor and EMAP II treated (2–3 hours) hPASMCs, n=2. (G) Gene Ontology results using differentially enriched Ac-H3K9 peaks in EMAP II treated hPASMCs, n=2. (H) Cell proliferation rate in hPASMCs treated with vehicle or EMAP II following SPHK2 inhibitor treatment for 24 hours, n=3. P values are calculated using Kruskal-Wallis against control or Kolmogorov-Smirnov non-parametric test and results are shown as means ± SEM or median and inter-quartile range.

Article Snippet: Primary human PASMCs (hPASMCs), pulmonary microvascular endothelial cells (hPMVECs) purchased from Lonza (Walkersville, MD) and iPAH: PASMCs were cultured in complete growth medium or conditioned media in a humidified atmosphere or 1% O 2 with 5% CO 2 at 37° C. For all studies, passages 5–10 were used for hPASMCs and hPMVECs.

Techniques: Western Blot, Transfection, Quantitation Assay, Binding Assay, Control, Genome Wide

Journal: Circulation research

Article Title: Altered Smooth Muscle Cell Histone Acetylome by the SPHK2/S1P Axis Promotes Pulmonary Hypertension

doi: 10.1161/CIRCRESAHA.123.322740

Figure Lengend Snippet: (A) The Venn’s diagram of differential acetylated sites in control vs EMAP II (total) (purple), EMAP II vs iSPHK2+EMAP II (yellow) and control vs EMAPII only in 5’UTR and upstream with fold enrichment greater than 2 (green). The red circle indicates the potential gene set with potential upstream candidate regulatory elements that would be differentially acetylated by EMAP II through SPHK2 in hPASMCs. Venn diagram is created using Venny 2.1 (an online interactive tool), n=2/group (B) Snapshot of IGV view of KLF4 gene in Ac-H3K9 CUT&RUN data of with or without SPHK2 inhibitor and EMAP II treated (2–3 hours) hPASMCs. (cCRE= candidate Cis-Regulatory Elements) n=2/group (C) Representative immunoblot probed for KLF4, SPHK2 and tubulin in whole cell lysates of hPASMCs following siRNA mediated SPHK2 silencing and post-transfection EMAP II treatment for 6–8 hours, and (D) quantitation of KLF4/tubulin and (E) KLF4 expression levels normalized against 18S rRNA in hPASMC cells following siRNA mediated SPHK2 silencing and EMAP II treatment for 6 hours, n=4. P values are calculated using Kolmogorov-Smirnov non-parametric testing and results are shown as median and inter-quartile range.

Article Snippet: Primary human PASMCs (hPASMCs), pulmonary microvascular endothelial cells (hPMVECs) purchased from Lonza (Walkersville, MD) and iPAH: PASMCs were cultured in complete growth medium or conditioned media in a humidified atmosphere or 1% O 2 with 5% CO 2 at 37° C. For all studies, passages 5–10 were used for hPASMCs and hPMVECs.

Techniques: Control, Western Blot, Transfection, Quantitation Assay, Expressing

Journal: Circulation research

Article Title: Altered Smooth Muscle Cell Histone Acetylome by the SPHK2/S1P Axis Promotes Pulmonary Hypertension

doi: 10.1161/CIRCRESAHA.123.322740

Figure Lengend Snippet: (A) RNA-seq data of SPHK2, KLF4 and AIMP1 in iPAH: PASMCs and non-iPAH:PASMCs in log2-fold of count per million (cpm). Following two-way ANOVA, Sidak’s multiple comparisons test for logarithmic values, n=4. (B) Representative immunoblot probed for KLF4, SPHK2, tubulin, Ac-H3K9 and total histone H3 in whole cell lysates of non: iPAH or iPAH PASMCs with scramble or SPHK2 siRNA transfection and, quantification of (C) Ac-H3K9/total histone H3 and, (D) KLF4/Tubulin, n=3 (E) KLF4 expression levels normalized against 18S rRNA in non: iPAH or iPAH PASMCs with scramble or SPHK2 siRNA transfection, n=4. (F) Cell proliferation rate of non: iPAH or iPAH PASMC with or without iSPHK2 pretreatment for 24 hours, n=4. (G) The proposed model: Endothelial monocyte activating polypeptide II (EMAP II) plays a key role in reawakening pluripotency factor, KLF4 in human pulmonary artery smooth muscle cells (PASMCs) through stimulation of the nuclear SPHK2/S1P epigenetic modulating axis, suggesting that cooperation between SPHK2 and EMAP II could be a major driving force for epigenetic-mediated vascular PASMCs reprogramming and remodeling in PH. Ablation of SPHK2 expression confers protection against PH by rescuing the global and local transcription machinery from histone acetylation and activation of the pluripotency factor, KLF4. P values are calculated using Kruskal-Wallis against iPAH or Kolmogorov-Smirnov non-parametric test if not mentioned otherwise, and results are shown as median and inter-quartile range.

Article Snippet: Primary human PASMCs (hPASMCs), pulmonary microvascular endothelial cells (hPMVECs) purchased from Lonza (Walkersville, MD) and iPAH: PASMCs were cultured in complete growth medium or conditioned media in a humidified atmosphere or 1% O 2 with 5% CO 2 at 37° C. For all studies, passages 5–10 were used for hPASMCs and hPMVECs.

Techniques: RNA Sequencing, Western Blot, Transfection, Expressing, Activation Assay

Journal: Molecular Therapy. Nucleic Acids

Article Title: MicroRNA-212-5p, an anti-proliferative miRNA, attenuates hypoxia and sugen/hypoxia-induced pulmonary hypertension in rodents

doi: 10.1016/j.omtn.2022.06.008

Figure Lengend Snippet: Expression of miR-212-5p is induced in PASMCs and the lung of PH subjects Expression of miR-212-5p was measured by qRT-PCR analysis in the following samples. (A) PASMCs of human PH patients (n = 4) or normal donors (control, n = 9). (B) PASMCs isolated from control mice (N, exposed to room air, n = 8) or mice with hypoxia-induced PH (H, 10% O 2 , 3 weeks, n = 7). (C) Lungs of control mice (N, exposed to room air, n = 5 for each time point) or mice exposed to hypoxia for 1, 2, or 3 weeks (H, 10% O 2 , n = 5 for each time point). (D) Lungs of control rats (CTRL, given DMSO and stayed in room air, n = 6) or rats with Sugen/hypoxia-induced severe PH (SuHx, n = 5). Data are presented as mean ± SEM. ∗ or ∗∗, versus CTRL or N. ∗p < 0.05, ∗∗p < 0.01.

Article Snippet: MiR-212-5p is an “anti-proliferative” miRNA in PASMC (A) Normal human PASMCs (hPASMCs, Lonza) were transfected with miR-212-5p or negative control miR inhibitor (Anti-miR-212 versus Anti-Neg).

Techniques: Expressing, Quantitative RT-PCR, Control, Isolation

Journal: Molecular Therapy. Nucleic Acids

Article Title: MicroRNA-212-5p, an anti-proliferative miRNA, attenuates hypoxia and sugen/hypoxia-induced pulmonary hypertension in rodents

doi: 10.1016/j.omtn.2022.06.008

Figure Lengend Snippet: MiR-212-5p is an “anti-proliferative” miRNA in PASMC (A) Normal human PASMCs (hPASMCs, Lonza) were transfected with miR-212-5p or negative control miR inhibitor (Anti-miR-212 versus Anti-Neg). BrdU incorporation assay was performed. Proliferation level of PASMCs transfected with negative control miR inhibitor (Anti-Neg) was set as 1 and served as controls. Proliferation level of PASMCs transfected with miR-212-5p inhibitor (Anti-miR-212) was compared with that of control cells. Inhibition of miR-212-5p induced PASMC proliferation in vitro in room air. (B) Normal hPASMC (Lonza) were transfected with miR-212-5p or negative control miR mimic and then exposed to room air (N) or hypoxia (H, 1% O 2 ) for 24 h. BrdU incorporation assay was performed to determine the relative proliferation level of these cells. Proliferation level of PASMCs transfected with negative control miR mimic (Neg mimic) and exposed to room air was set as 1 and served as controls. Proliferation levels of PASMCs in other groups were compared with that of control cells. miR-212-5p mimic significantly suppressed PASMC proliferation in both room air and hypoxia. (C) Normal hPASMCs (Lonza) were infected with adenoviral particles that express miR-212-5p (Ad-miR212, 100 PFUs/cell) or control adenoviral particles (Ad-Ctrl, 100 PFUs/cell) on day 1. Then cells were collected for cell counting on day 3 and 5, respectively. Overexpression of miR-212-5p significantly suppressed cell growth in room air. (D) Human PASMCs of PH patient (PH hPASMCs) were transfected with miR-212-5p or negative control miR mimic. BrdU incorporation assay was performed and relative cell proliferation level was compared, as described above. miR-212-5p mimic also significantly suppressed proliferation of PH hPASMCs. Data are presented as mean ± SEM. ∗ or ∗∗ versus room air Anti-Neg, Neg mimic control, or Ad-Ctrl, respectively; ## versus hypoxic Neg mimic control. ∗p < 0.05, ∗∗ or ##p < 0.01.

Article Snippet: MiR-212-5p is an “anti-proliferative” miRNA in PASMC (A) Normal human PASMCs (hPASMCs, Lonza) were transfected with miR-212-5p or negative control miR inhibitor (Anti-miR-212 versus Anti-Neg).

Techniques: Transfection, Negative Control, BrdU Incorporation Assay, Control, Inhibition, In Vitro, Infection, Cell Counting, Over Expression

Journal: Molecular Therapy. Nucleic Acids

Article Title: MicroRNA-212-5p, an anti-proliferative miRNA, attenuates hypoxia and sugen/hypoxia-induced pulmonary hypertension in rodents

doi: 10.1016/j.omtn.2022.06.008

Figure Lengend Snippet: SMC-specific knockout of miR-212 significantly exacerbates hypoxia-induced PH in mice (A) The strategy to generate smooth muscle cell (SMC)-specific miR-212 knockout mice and mouse genotyping (B). (C) The expression levels of miR-212-5p in freshly isolated mouse PASMCs (mPASMCs) from sm-212 −/ − mice (n = 4) and their 212-fl/fl littermates (n = 4). sm-212 −/− and their 212-fl/fl littermates were exposed to room air or hypoxia (10% O 2 ) for 3 weeks and then PH indices were measured. SMC-specific knockout of miR-212 significantly exacerbated hypoxia-induced RVSP elevation (D), RV hypertrophy (E), and pulmonary vessel wall thickening (F–G). V: vessels. Data are presented as mean ± SEM. ∗, ∗∗, or ∗∗∗ versus fl/fl control in room air; ## or ### versus fl/fl control in hypoxia; ∗p < 0.05, ∗∗ or ##p < 0.01, ###p < 0.001.

Article Snippet: MiR-212-5p is an “anti-proliferative” miRNA in PASMC (A) Normal human PASMCs (hPASMCs, Lonza) were transfected with miR-212-5p or negative control miR inhibitor (Anti-miR-212 versus Anti-Neg).

Techniques: Knock-Out, Expressing, Isolation, Control

Journal: American journal of physiology. Heart and circulatory physiology

Article Title: Electroporation-mediated delivery of catalytic oligodeoxynucleotides for manipulation of vascular gene expression

doi: 10.1152/ajpheart.00350.2003

Figure Lengend Snippet: Effects of DNAzymes on PKC-ε protein levels in cultured cells. A: Western blots of hPASMCs. Representative Western blots for PKC-ε (top) or β-tubulin (bottom) from hPASMCs treated with liposome alone, wtDNAzyme, mDNAzyme, or sDNAzyme are shown. B: quantification of protein levels in hPASMCs. Western blots from 5 separate transfection experiments using hPASMCs were digitized, analyzed by densitometry using NIH Image, and normalized to liposome only treatment. Values are expressed as normalized averages ± SE. A Mann-Whitney U-test gave a P value of 0.016 for the wtDNAzyme-induced PKC-ε decrease compared with the other conditions. C: Western blots of A7r5 cells. Representative Western blots for PKC-ε (top) or β-tubulin (bottom) from rat A7r5 cells treated with liposome alone, wtDNAzyme, mDNAzyme, or sDNAzyme are shown. D: quantification of protein levels in A7r5 cells. Western blots from 3 separate transfection experiments using A7r5 cells were digitized, analyzed by densitometry using NIH Image, and normalized to liposome only treatment, as described in B. *P = 0.003 for PKC-ε levels in wtDNAzyme-treated cells vs. all other conditions by Mann-Whitney U-test.

Article Snippet: Cell culture and transfection Human pulmonary artery smooth muscle cells (hPASMC; a gift of Dr. Paul Babal, Department of Pharmacology, University of South Alabama) and A7r5 cells (ATCC No. CRL-1444) were maintained in 100-mm plates containing DMEM with 10% FBS, 1× Kanamycin, and 1× antimycotic solution (Life Technologies; Gaithersberg, MD) at 37°C and 5% CO 2 .

Techniques: Cell Culture, Western Blot, Transfection, MANN-WHITNEY

Journal: Circulation Research

Article Title: Cyclin D-CDK4 Disulfide Bond Attenuates Pulmonary Vascular Cell Proliferation

doi: 10.1161/CIRCRESAHA.122.321836

Figure Lengend Snippet: Cyclin D-CDK4 (cyclin-dependent kinase 4) forms an inducible intermolecular disulfide dimer. A , An Affymetrix GeneChip microarray with gene enrichment pathway analysis of approximately the top 1000 genes of defined biological function with an altered expression between mice exposed to chronic hypoxia (10% oxygen) or normoxia (21% oxygen) for 3 days, n=6 mice per group. The top 20 gene-enriched pathways are shown based on pathway analysis performed in R studio using the gseGO function of the clusterProfiler package. B , CDK4, cyclin D1, and cyclin D3 form oxidant-induced intermolecular disulfide bonds. Monomeric and disulfide dimeric (indicated by black arrows) CDK4, cyclin D1, or cyclin D3 were detected by nonreducing immunoblotting in human pulmonary arterial smooth muscle cells (HPASMCs) after treatment with H 2 O 2 . Vinculin was used as a loading control. C , The percentage of CDK4, cyclin D1, and cyclin D3 observed as a disulfide dimer in response to H 2 O 2 treatment. As the data sample size with an n<6 cannot be reliably tested for normality, P values are calculated using a nonparametric Kruskal-Wallis test followed by Dunn multiple comparisons to compare H 2 O 2 -induced disulfide formation with 0 µmol/L control (n=5 independent experiments); the results are shown as means±SEM. D , Cyclin D expression is higher in G1 phase cells than quiescent cells, causing an increase in disulfide cyclin D-CDK4. Monomeric and disulfide dimeric CDK4 were detected by nonreducing immunoblotting of HPASMCs synchronized into the G0/1 phase by serum starvation (0.1% FBS) for 72 hours, followed by stimulation with 10% FBS for 6 or 12 hours. HPASMCs were then treated with H 2 O 2 for 15 minutes. Cyclin D1, cyclin D3, and vinculin (loading control) were detected by immunoblotting under reducing conditions. E , Quantification of the percentage of CDK4 observed as a disulfide dimer. As the data sample size with an n<6 cannot be reliably tested for normality, P values are calculated using a nonparametric Kruskal-Wallis test followed by Dunn multiple comparisons to compare H 2 O 2 -induced disulfide formation with 0 µmol/L control within cell cycle phase (n=3 independent experiments); the results are shown as means±SEM. F , Auranofin potentiates accumulation of the oxidant-induced cyclin D1-CDK4 disulfide bond. Monomeric and disulfide dimeric (indicated by black arrows) CDK4 and cyclin D1 were detected by nonreducing immunoblotting in HPASMCs after pretreatment with the thioredoxin reductase inhibitor, auranofin, for 25 minutes followed by the addition of H 2 O 2 for 15 minutes. GAPDH was used as a loading control. G , The proportion of CDK4 and cyclin D1 observed as a disulfide dimer after treatment with auranofin and H 2 O 2 . P values are calculated using a nonparametric Kruskal-Wallis test followed by Dunn multiple comparisons to compare disulfide formation in response to auranofin treatment within 0, 50, 100 or 200 µM H 2 O 2 -treatment group (n=5 independent experiments); the results are shown as means±SEM.

Article Snippet: PAH and control HPASMCs were cultured in smooth muscle cell growth medium-2 (PromoCell, No. C-39262).

Techniques: Microarray, Expressing, Western Blot, Control

Journal: Circulation Research

Article Title: Cyclin D-CDK4 Disulfide Bond Attenuates Pulmonary Vascular Cell Proliferation

doi: 10.1161/CIRCRESAHA.122.321836

Figure Lengend Snippet: The intermolecular disulfide bond forms between CDK4 (cyclin-dependent kinase 4) C135 and cyclin D1 C7/8. A , The cyclin D1-CDK4 crystal structure (PDB 2W96) showing the locations of CDK4 C135 and cyclin D1 C7/8 in black. The distance (Å) between cysteine residues was measured using PyMOL. The cyclin D1 protein structure and CDK4 protein structure are shown in gray and green, respectively. B , C7/8A cyclin D1 and C135A CDK4 are redox-dead. Immunoblots show disulfide dimeric cyclin D1 and CDK4 (indicated by black arrows) in human pulmonary arterial smooth muscle cells (HPASMCs) overexpressed with wild-type (WT) or C135A CDK4-FLAG and WT or C7/8A cyclin D1-HA and treated with H 2 O 2 . Cyclin D1 (HA-tag) and CDK4 (FLAG-tag) were detected by nonreducing immunoblotting. Red arrows indicate additional artificial bands. C , The percentage of cyclin D1 and CDK4 observed as a disulfide dimer in overexpressed HPASMCs. As the data sample size with an n<6 cannot be reliably tested for normality, P values are calculated using unpaired 2-tailed nonparametric Mann-Whitney U test to compare between vehicle and H 2 O 2 treatment for each mutant; nonparametric Kruskal-Wallis test followed by Dunn multiple comparisons was used to compare disulfide formation in response to 200 µM H 2 O 2 treatment between each mutant and WT (n=5 independent experiments); the results are shown as means±SEM. D , BLAST alignments show CDK4 C135 is not conserved between cell cycle CDK proteins. E , BLAST alignments show cyclin D1 C7/8 is conserved between cyclin D subtypes. All sequences were obtained from the UniProt database with identification codes presented in brackets. F , C5/6A cyclin D3 and C135A CDK4 are redox-dead. Disulfide dimeric cyclin D3 and CDK4 (indicated by black arrows) in HPASMCs overexpressed with WT or C135A CDK4-FLAG and WT or C5/6A cyclin D3-HA. Cells were treated with H 2 O 2 for 15 minutes. Cyclin D3 and CDK4 (FLAG-tag) were detected by nonreducing immunoblotting. Red arrows indicate additional artificial bands. G , The percentage of cyclin D3 and CDK4 observed as a disulfide dimer in overexpressed HPASMCs. P values are calculated using unpaired 2-tailed nonparametric Mann-Whitney U test to compare between H 2 O 2 treatment and vehicle for each mutant; nonparametric Kruskal-Wallis test followed by Dunn multiple comparisons was used to compare disulfide formation in response to 200 µM H 2 O 2 treatment between each mutant and WT (n=5 independent experiments); the results are shown as means±SEM. H and I , Analysis of an extracted ion chromatogram from an LC-MS/MS precursor ion survey scan identified an ion of ( H ) m/z 681.55 4+ and ( I ) m/z 656.82 4+ , which correspond with the peptide 127 GLDFLHANCIVHR 139 in CDK4 bound through a disulfide bond to the peptide 1 MELLCCEGTR 10 in cyclin D3, as shown in the red boxes. The cyclin D3 peptide was identified with dioxidation of the free cysteine residue, and acetylation of the N-terminal, either with the N-terminal methionine ( H ) present, or ( I ) absent.

Article Snippet: PAH and control HPASMCs were cultured in smooth muscle cell growth medium-2 (PromoCell, No. C-39262).

Techniques: Western Blot, FLAG-tag, MANN-WHITNEY, Mutagenesis, Liquid Chromatography with Mass Spectroscopy, Residue

Journal: Circulation Research

Article Title: Cyclin D-CDK4 Disulfide Bond Attenuates Pulmonary Vascular Cell Proliferation

doi: 10.1161/CIRCRESAHA.122.321836

Figure Lengend Snippet: Oxidation decreases cyclin D-CDK4 (cyclin-dependent kinase) substrate phosphorylation and induces G1 phase cell cycle arrest. A , Oxidation of human pulmonary arterial smooth muscle cells (HPASMCs), induced by acute H 2 O 2 treatment, decreased. Rb (retinoblastoma) phosphorylation at S780, S795, or S807-811, and total Rb were detected by immunoblotting under reducing conditions. B , Quantification of Rb phosphorylation, normalized to total Rb. As the data sample size with an n<6 cannot be reliably tested for normality, P values are calculated using a nonparametric Kruskal-Wallis test followed by Dunn post hoc multiple comparisons to compare Rb phosphorylation in response to H 2 O 2 with the 0 µmol/L control (n=5 independent experiments); the results are shown as means±SEM. C , Histograms show cell cycle analysis using flow cytometry of propidium iodide-stained HPASMCs. Continuously cycling cells were maintained in DMEM growth medium (10% FBS; upper left), while G0/1 phase synchronized cells were maintained in starvation media (0.1% FBS) for 40 hours (upper right). Synchronized cells were stimulated to enter the cell cycle with 10% FBS alone for 24 hours (middle left), FBS plus 50 µmol/L H 2 O 2 for 24 hours (middle right), FBS plus DMSO vehicle (bottom left), or FBS plus 10 µmol/L palbociclib for 24 hours (bottom right). The proportion of cells in each phase of the cell cycle at this time point was analyzed by the Watson Pragmatic cell cycle model and an average from 4 to 5 independent experiments is provided. D , H 2 O 2 causes G1 phase cell cycle arrest. Graph shows the proportion of synchronized cells in each cell cycle phase with time after stimulation with 10% FBS alone (solid lines) or 10% FBS plus 50 µmol/L H 2 O 2 (dashed lines). P values are calculated using unpaired 2-tailed nonparametric Mann-Whitney U test to compare between vehicle and H 2 O 2 treatment in cell cycle phases at each time point (0 and 8 hours, n=5 experimental sample per group; 16, 24, or 28 hours, n=4 experimental samples per group); the results are shown as means±SEM. E , Proliferation of HPASMCs treated with 50 µmol/L H 2 O 2 or vehicle. Cells were seeded in xCELLigence real-time cell analysis (RTCA) E-plates at 3×10 4 cells/well in starvation media (0.1% FBS). After 40 hours, cells were stimulated with 10% FBS with or without H 2 O 2 . Proliferation rate of HPASMCs after treatment was quantified using area under the curve. As the data sample size with an n<6 cannot be reliably tested for normality, the P value is calculated using an unpaired 2-tailed nonparametric Mann-Whitney U test to compare between area under the curve for 0 and 50 µmol/L (n=4 independent experiments/ biological replicates per group, including n=2 performed in technical replicates for 0 µmol/L, altogether comprising n=6 replicates for 0 µmol/L and n=4 replicates for 50 µmol/L); the results are shown as means±SEM.

Article Snippet: PAH and control HPASMCs were cultured in smooth muscle cell growth medium-2 (PromoCell, No. C-39262).

Techniques: Phospho-proteomics, Western Blot, Control, Cell Cycle Assay, Flow Cytometry, Staining, MANN-WHITNEY, Cell Analysis

Journal: Circulation Research

Article Title: Cyclin D-CDK4 Disulfide Bond Attenuates Pulmonary Vascular Cell Proliferation

doi: 10.1161/CIRCRESAHA.122.321836

Figure Lengend Snippet: Idiopathic pulmonary arterial hypertension (PAH; IPAH) pulmonary arteries and human pulmonary arterial smooth muscle cells (HPASMCs) have less CDK4 (cyclin-dependent kinase 4) disulfide than donor controls. A , Formation of the CDK4 disulfide bond is decreased in pulmonary arteries of IPAH patients compared with healthy volunteer controls. Nonreducing immunoblots show monomeric and disulfide dimeric CDK4, while reducing immunoblots show CDK4 expression with vinculin used as a loading control. Quantification is provided for the percentage of CDK4 observed as a disulfide dimer. The data passed the normality test performed by the Shapiro-Wilk test. P values were calculated using the parametric unpaired 2-tailed t test to compare between 2 groups (control, n=8; IPAH, n=9), and the results are shown as means±SEM. B , IPAH HPASMCs have an attenuated response to oxidant treatment compared with donor control cells. HPASMCs were treated with H 2 O 2 for 15 minutes in DMEM growth medium. The formation of disulfide cyclin D-CDK4 was detected by nonreducing immunoblotting probed for CDK4, cyclin D1, and cyclin D3. The graph shows quantification of the percentage of CDK4 that is detected as a disulfide dimer. The data passed the normality test performed by the Shapiro-Wilk test. P values were calculated using the parametric 2-way ANOVA followed by Sidak post hoc multiple comparisons test (n=6 per group); the results are shown as means±SEM. C , IPAH HPASMCs accumulate less disulfide CDK4 than donor control cells in response to auranofin treatment. HPASMCs were treated with auranofin for 1 hour in serum-free DMEM. Monomeric and disulfide dimeric CDK4, cyclin D1, and cyclin D3 were detected by nonreducing immunoblotting. The graph shows quantification of the proportion of CDK4 that was observed to form a disulfide (n=6 per group). The data did not pass the normality test performed by the Shapiro-Wilk test. P values are calculated using an unpaired 2-tailed nonparametric Mann-Whitney U test to compare 0.5 or 1 μM Aur-treatment between Control and IPAH groups (n=6 per group). The results are shown as means±SEM. D , HPASMCs isolated from patients with IPAH have an increased proliferation rate compared with donor control HPASMCs. Proliferation was measured by electrical impedance in real time using xCELLigence (representative of n=6 independent biological replicates per group, in technical duplicate). Proliferation rate was then analyzed using area under the curve (AUC). The data passed the normality test performed by the Shapiro-Wilk test. P value is calculated using the parametric unpaired 2-tailed t test to compare between 2 groups (n=6 independent biological replicates per group, in technical duplicate), and the results are shown as means±SEM.

Article Snippet: PAH and control HPASMCs were cultured in smooth muscle cell growth medium-2 (PromoCell, No. C-39262).

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