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: EBioMedicine

Article Title: IL-33 Initiates Vascular Remodelling in Hypoxic Pulmonary Hypertension by up-Regulating HIF-1α and VEGF Expression in Vascular Endothelial Cells

doi: 10.1016/j.ebiom.2018.06.003

Figure Lengend Snippet: Effects of co-culture with HPAECs on proliferation and migration of HPASMCs in the presence/absence of IL-33. (A) IL-33 alone did not affect proliferation of HPASMCs, while positive control PDGF promoted the proliferation of HPASMCs ( n = 6 each group). *** p < 0.001, ### p < 0.001. (B) The effect of IL-33 on proliferation of HPASMCs (labelled SMC) when co-cultured with HPAECs (labelled EC) ( n = 6 each group). *** p < 0.001, ### p < 0.001. (C) The effect of IL-33 on migration of HPASMCs (labelled SMC) when co-cultured with HPAECs (labelled EC) ( n = 6 each group). *** p < 0.001.

Article Snippet: Proliferation of human pulmonary artery smooth muscle cells (HPASMCs) was measured using a commercial kit (Cell Counting Kit-8, Dojindo, Japan).

Techniques: Co-Culture Assay, Migration, Positive Control, Cell Culture

Journal: The FASEB Journal

Article Title: CircNAP1L4 regulates pulmonary artery smooth muscle cell proliferation via the NAP1L4‐mediated super‐enhancer‐driven glycolysis gene hexokinase II (HK II) in pulmonary hypertension

doi: 10.1096/fj.202400585rrr

Figure Lengend Snippet: FIGURE 1 The expression and characteristics of circNAP1L4 in HPASMCs and PH patients' plasma. (A) Genomic location and back- splice junction site of circNAP1L4. (B) Quantitative RT–PCR (qRT–PCR) analysis of circNAP1L4 expression in hypoxic HPASMCs (n = 7). (C) Analysis of the RNA levels of circNAP1L4 and linear NAP1L4 after treatment with RNase R (n = 3). (D) Analysis of the RNA abundance of circNAP1L4 and linear NAP1L4 treated with actinomycin D (2 μg/mL) at the indicated time points (n = 6). (E) FISH was performed to determine the circNAP1L4 distribution and expression in HPASMCs. Scale bar, 50 μm. (F) qRT–PCR analyses of circNAP1L4 in plasma from control (n = 15) and PH patients (n = 25). All tests were performed at least three times, and the values are presented as the mean ± SEM. Statistical analysis was performed with one-way ANOVA or Student's t-test. *p < .05, **p < .01, ***p < .001. HYP, hypoxic; NOR, normoxia; PH, pulmonary hypertension.

Article Snippet: See the T erm s and C onditions (https://onlinelibrary.w iley.com /term s-and-conditions) on W iley O nline L ibrary for rules of use; O A articles are governed by the applicable C reative C om m ons L icense 6 of 21 | WANG et al. HPASMCs were incubated with anti- NAP1L4 antibody (1:100, 16 018- 1- AP; Proteintech, IL, USA) and anti- Ki67 antibody (1:100, M00254- 3; Boster, Wuhan, China) at 4°C overnight.

Techniques: Expressing, Clinical Proteomics, Quantitative RT-PCR, Control

Journal: The FASEB Journal

Article Title: CircNAP1L4 regulates pulmonary artery smooth muscle cell proliferation via the NAP1L4‐mediated super‐enhancer‐driven glycolysis gene hexokinase II (HK II) in pulmonary hypertension

doi: 10.1096/fj.202400585rrr

Figure Lengend Snippet: FIGURE 2 CircNAP1L4 inhibits hypoxia-induced HPASMC proliferation and glycolysis. (A) CCK8 assays were performed for HPASMCs overexpressing circNAP1L4 (n = 6). (B) EdU (red) incorporation and immunofluorescence analysis of Ki67 (green) in HPASMCs overexpression circNAP1L4 (n = 5–6). Scale bar, 50 μm. (C) Fluorescence-activated cell sorting analyses were used to detect the number of HPASMCs in each phase of the cell cycle. The bar graph shows the number of cells in each cell cycle phase. (D) Protein levels of cyclin A, cyclin D, and PCNA in HPASMCs overexpressing circNAP1L4 (n = 6). (E) Protein levels of HK II and PKM2 in HPASMCs transfected with circNAP1L4 overexpression (n = 6). (F) Extracellular acidification rate (ECAR) of HPASMCs overexpressing circNAP1L4 was measured via the Seahorse XFe24 platform (n = 3). (G) Lactate, pyruvate production, and ATP content in HPASMCs treated circNAP1L4 (n = 6). All tests were performed at least three times, and the values are presented as the mean ± SEM. Statistical analysis was performed with one-way ANOVA. *p < .05, **p < .01, ***p < .001. HYP, hypoxia; NC, negative control; NOR, normoxia.

Article Snippet: See the T erm s and C onditions (https://onlinelibrary.w iley.com /term s-and-conditions) on W iley O nline L ibrary for rules of use; O A articles are governed by the applicable C reative C om m ons L icense 6 of 21 | WANG et al. HPASMCs were incubated with anti- NAP1L4 antibody (1:100, 16 018- 1- AP; Proteintech, IL, USA) and anti- Ki67 antibody (1:100, M00254- 3; Boster, Wuhan, China) at 4°C overnight.

Techniques: Immunofluorescence, Over Expression, Fluorescence, FACS, Transfection, Negative Control

Journal: The FASEB Journal

Article Title: CircNAP1L4 regulates pulmonary artery smooth muscle cell proliferation via the NAP1L4‐mediated super‐enhancer‐driven glycolysis gene hexokinase II (HK II) in pulmonary hypertension

doi: 10.1096/fj.202400585rrr

Figure Lengend Snippet: FIGURE 3 CircNAP1L4 inhibits NAP1L4 nuclear translocation by interacting with NAP1L4. (A) qRT–PCR was used to detect NAP1L4 mRNA expression in HPASMCs transfected overexpressing circNAP1L4 (n = 6). (B) Protein levels of NAP1L4 in HPASMCs overexpression circNAP1L4 (n = 6). (C) Immunofluorescence analysis of NAP1L4 expression in HPASMCs overexpression circNAP1L4. Scale bar, 50 μm. (D) The translocation of NAP1L4 between the nucleus and cytoplasm in HPASMCs overexpression circNAP1L4 (n = 4). (E) Interactions between circNAP1L4 and the NAP1L4 protein were predicted by using catRAPID website (http://s.tartaglialab.com/page/catrapid_omics2_ group). (F) RNA pull-down assays were used to detect the interaction between circNAP1L4 (nucleotide position [nt] 989–1040) and the NAP1L4 protein. (G) RIP enrichment was determined as circNAP1L4 associated with the NAP1L4 IP relative to the input control (n = 5). (H) Immunofluorescence was used to observe the colocalization of circNAP1L4 and NAP1L4 proteins in the cytoplasm. Scale bar, 50 μm. All tests were performed at least three times, and the values are presented as the mean ± SEM. Statistical analysis was performed with Student's t-test. *p < .05, ***p < .001. HYP, hypoxia; NC, negative control; NOR, normoxia.

Article Snippet: See the T erm s and C onditions (https://onlinelibrary.w iley.com /term s-and-conditions) on W iley O nline L ibrary for rules of use; O A articles are governed by the applicable C reative C om m ons L icense 6 of 21 | WANG et al. HPASMCs were incubated with anti- NAP1L4 antibody (1:100, 16 018- 1- AP; Proteintech, IL, USA) and anti- Ki67 antibody (1:100, M00254- 3; Boster, Wuhan, China) at 4°C overnight.

Techniques: Translocation Assay, Quantitative RT-PCR, Expressing, Transfection, Over Expression, Immunofluorescence, Control, Negative Control

Journal: The FASEB Journal

Article Title: CircNAP1L4 regulates pulmonary artery smooth muscle cell proliferation via the NAP1L4‐mediated super‐enhancer‐driven glycolysis gene hexokinase II (HK II) in pulmonary hypertension

doi: 10.1096/fj.202400585rrr

Figure Lengend Snippet: FIGURE 4 NAP1L4 enhances the expression of the SE-modified glycolysis gene HK II in hypoxic HPASMCs. (A) Coimmunoprecipitation (Co-IP) assay verified the interaction of H3K27ac and NAP1L4. (B) qRT–PCR was used to detect HK II and PKM2 mRNA expression levels in HPASMCs treated with JQ1 (250 nM; S7110; Selleck, USA) (n = 6). (C) Gene tracks depicting the SE region of HK II in lung primary cells and lung tissue with measured H3K27ac, or H3K4me1 marks. (D) Protein levels of HK II in HPASMCs with NAP1L4 knockdown (n = 6). (E) CEBPB was identified as a candidate transcription factor, and Western blot analysis was used to verify the expression of CEBPB in hypoxia (n = 6). (F) HPASMCs were subjected to ChIP analysis using antibodies against H3K27ac, H3K4me1, and CEBPB. The association with the SE region (E1–E3) of HK II was quantified by qRT–PCR (n = 3–4). (G) HPASMCs were subjected to ChIP analysis using antibodies against H3K27ac, H3K4me1, and CEBPB. The association with the promoter region (△1–△5) of HK II was quantified by qRT–PCR (n = 3). All tests were performed at least three times, and the values are presented as the mean ± SEM. Statistical analysis was performed with one-way ANOVA or Student's t-test. *p < .05, **p < .01, ***p < .001. HYP, hypoxic; NC, negative control; NOR, normoxia.

Article Snippet: See the T erm s and C onditions (https://onlinelibrary.w iley.com /term s-and-conditions) on W iley O nline L ibrary for rules of use; O A articles are governed by the applicable C reative C om m ons L icense 6 of 21 | WANG et al. HPASMCs were incubated with anti- NAP1L4 antibody (1:100, 16 018- 1- AP; Proteintech, IL, USA) and anti- Ki67 antibody (1:100, M00254- 3; Boster, Wuhan, China) at 4°C overnight.

Techniques: Expressing, Modification, Co-Immunoprecipitation Assay, Quantitative RT-PCR, Knockdown, Western Blot, Negative Control