Journal: Frontiers in Pharmacology

Article Title: Profilin 1 Induces Tumor Metastasis by Promoting Microvesicle Secretion Through the ROCK 1/p-MLC Pathway in Non-Small Cell Lung Cancer

doi: 10.3389/fphar.2022.890891

Figure Lengend Snippet: PFN1 is correlated with NSCLC metastasis and could promote NSCLC cell migration in vitro . (A) Representative IHC images of PFN1 expression on the NSCLC tissues. (B) The staining index of PFN1 on the tissue chip. ** p < 0.01. (C) Representative IHC images of PFN1 expression on the tissue chip. (D) The expression of PFN1 in TCGA LUAD data. ** p < 0.01. (E) The Kaplan–Meier survival analysis of PFN1 in NSCLC patients. (Data source: TCGA LUAD dataset) (F,G) Wound healing assays conducted to evaluate the migration ability of PFN1 -overexpressing (F) and PFN1 knockdown (KD) (G) H1299 cells. ** p < 0.01; scale bar, 500 μm. (H,I) Transwell migration assays conducted to evaluate the migration of PFN1 -overexpressing (H) and PFN1 KD (I) H1299 cells. ** p < 0.01; scale bar, 500 μm. EV, empty vector; OE, PFN1 overexpression; NC, negative control; si-1/ 2, PFN1 siRNA1 1/2.

Article Snippet: PFN1 siRNA and control scramble siRNA were synthesized by Guangzhou RiboBio Co. (Guangzhou, China), and the sequences are listed in .

Techniques: Migration, In Vitro, Expressing, Staining, Knockdown, Plasmid Preparation, Over Expression, Negative Control

Journal: Frontiers in Pharmacology

Article Title: Profilin 1 Induces Tumor Metastasis by Promoting Microvesicle Secretion Through the ROCK 1/p-MLC Pathway in Non-Small Cell Lung Cancer

doi: 10.3389/fphar.2022.890891

Figure Lengend Snippet: PFN1 could promote MVs secretion in NSCLC. (A) Heatmap of differentially expressed proteins between EV and PFN1 OE cells. (B) GO enrichment analysis of differentially expressed proteins. (C) COG/KOG analysis of differentially expressed proteins. (D) MVs extracted from EV-expressing and PFN1 -overexpressing cells, using continuous differential centrifugation, identified using transmission electron microscopy. Scale bar, 100 nm. (E,F) Flow cytometry (E) and western blotting (F) were used to quantify MVs in PFN1 -overexpressing and EV-expressing cells. ARF6 and actin were used as MV markers. (G) Expression of PFN1 and annexin A1 in lung tumor tissues detected using immunofluorescence. (H) The staining index of p-MLC on the tissue chip. ** p < 0.01. (I) Representative IHC images of p-MLC expression. (J) Spearman rank correlation analysis was used to assess the relationship between PFN1 and p-MLC expression on the tissue chip; p and r values are shown in the plot.

Article Snippet: PFN1 siRNA and control scramble siRNA were synthesized by Guangzhou RiboBio Co. (Guangzhou, China), and the sequences are listed in .

Techniques: Expressing, Centrifugation, Transmission Assay, Electron Microscopy, Flow Cytometry, Western Blot, Immunofluorescence, Staining

Journal: Frontiers in Pharmacology

Article Title: Profilin 1 Induces Tumor Metastasis by Promoting Microvesicle Secretion Through the ROCK 1/p-MLC Pathway in Non-Small Cell Lung Cancer

doi: 10.3389/fphar.2022.890891

Figure Lengend Snippet: MVs derived from PFN1 OE cells promote migration in NSCLC cells. (A) MVs collected from sera of patients with NSCLC quantified using flow cytometry. ** p < 0.01. (B) Protein expression of ARF6 and β-actin in MVs collected from sera of patients with NSCLC detected using western blotting. (C) Effect of PFN1 -overexpressing cell supernatants on cell migration evaluated through wound healing assays. ** p < 0.01; scale bar, 500 μm. (D) PKH67-labeled MVs taken up by H1299 cells. DAPI was used to stain the nuclei of H1299 cells. Scale bar, 500 μm. (E,F) Wound healing (E) and Transwell migration (F) assays conducted to evaluate the migration of H1299 cells after treatment with MVs derived from EV-expressing and PFN1 -overexpressing cells; ** p < 0.01; scale bar, 500 μm.

Article Snippet: PFN1 siRNA and control scramble siRNA were synthesized by Guangzhou RiboBio Co. (Guangzhou, China), and the sequences are listed in .

Techniques: Derivative Assay, Migration, Flow Cytometry, Expressing, Western Blot, Labeling, Staining

Journal: Frontiers in Pharmacology

Article Title: Profilin 1 Induces Tumor Metastasis by Promoting Microvesicle Secretion Through the ROCK 1/p-MLC Pathway in Non-Small Cell Lung Cancer

doi: 10.3389/fphar.2022.890891

Figure Lengend Snippet: PFN1 promotes in vivo NSCLC metastasis by elevating MV secretion. (A) Schematic illustration of the mouse model of metastatic tumor established to determine the role of PFN1 in tumor metastasis. (B) Body weight changes in mice after intracardiac injection of PFN1 -overexpressing and EV-expressing cell lines. (C,D) Representative images of lung (C) and liver (D) metastases of the mouse model. The number of metastases is displayed in the right-hand side graph. * p < 0.05, ** p < 0.01. (E) Representative images of HE-stained lung tissues of the mouse model. (F) Representative IHC images of PFN1 and p-MLC expression in lung tissues. The staining index is shown in the right-hand side graph. ** p < 0.01. (G) Representative images of HE-stained liver tissues of the mouse model. (H) Representative IHC images of PFN1 and p-MLC expression in liver tissues. The staining index is shown in the right-hand side graph. ** p < 0.01. (I) Body weight changes in mice after intracardiac injection of H1299 cells and MVs. (J) Representative images of lung metastases of the mouse model. The number of metastases is shown in the bottom graph. * p < 0.05. (K) Representative images of HE-stained lung tissues of the mouse model. (L) Representative IHC images of PFN1 and p-MLC expression in lung tissues. The staining index is shown in the right-hand side graph; * p < 0.05.

Article Snippet: PFN1 siRNA and control scramble siRNA were synthesized by Guangzhou RiboBio Co. (Guangzhou, China), and the sequences are listed in .

Techniques: In Vivo, Injection, Expressing, Staining

Journal: Frontiers in Pharmacology

Article Title: Profilin 1 Induces Tumor Metastasis by Promoting Microvesicle Secretion Through the ROCK 1/p-MLC Pathway in Non-Small Cell Lung Cancer

doi: 10.3389/fphar.2022.890891

Figure Lengend Snippet: Mechanisms underlying the promotion of MLC phosphorylation by PFN1. (A,B) Protein expression after PFN1 overexpression (A) and knockdown (B) measured using western blotting. (C) Protein expression in PFN1 mutants measured using western blotting. (D) PFN1 interactions with ROCK1/2 confirmed using co-IP. (E) Protein expression after treatment with Y27632 (10 µM) measured using western blotting. (F) Effect of PFN1 on ROCK1 activity. ** p < 0.01. (G) Effect of PFN1 on ROCK2 activity. (H) Flow cytometry measuring changes in the amount of MVs after treatment with Y27632; * p < 0.05.

Article Snippet: PFN1 siRNA and control scramble siRNA were synthesized by Guangzhou RiboBio Co. (Guangzhou, China), and the sequences are listed in .

Techniques: Phospho-proteomics, Expressing, Over Expression, Knockdown, Western Blot, Co-Immunoprecipitation Assay, Activity Assay, Flow Cytometry

Journal: Frontiers in Pharmacology

Article Title: Profilin 1 Induces Tumor Metastasis by Promoting Microvesicle Secretion Through the ROCK 1/p-MLC Pathway in Non-Small Cell Lung Cancer

doi: 10.3389/fphar.2022.890891

Figure Lengend Snippet: ROCK1 inhibitor Y27632 partially reversed the promotion of lung cancer metastasis by PFN1 in vitro and in vivo . (A,B) Wound healing assays conducted to evaluate the effect of Y27632 (A) and Y27632 combined with MVs (B) on cell migration. ** p < 0.01; scale bar, 500 μm. (C) Transwell migration assays conducted to evaluate the effect of Y27632 and Y27632 combined with MVs on cell migration. ** p < 0.01; scale bar, 500 μm. (D) Schematic diagram of the mouse model of metastatic tumor established to determine the effect of Y27632 on PFN1-induced lung cancer metastasis. (E) Body weight changes in mice after intracardiac injection of PFN1 -overexpressing H1299 cells and intraperitoneal injection of Y27632 (10 mg/kg). (F) Representative images of lung and liver metastatic tissue in mice. The number of metastatic nodules is shown in the right-hand side graph. * p < 0.05. (G,H) Representative images of HE-stained lung (G) and liver (H) metastases. (I) Representative IHC images of PFN1 and p-MLC expression in lung tissues. The staining index is shown in the right-hand side graph. ** p < 0.01. (J) Representative IHC images of PFN1 and p-MLC expression in liver tissues. The staining index is shown in the right-hand side graph; ** p < 0.01.

Article Snippet: PFN1 siRNA and control scramble siRNA were synthesized by Guangzhou RiboBio Co. (Guangzhou, China), and the sequences are listed in .

Techniques: In Vitro, In Vivo, Migration, Injection, Staining, Expressing

Journal: Frontiers in Pharmacology

Article Title: Profilin 1 Induces Tumor Metastasis by Promoting Microvesicle Secretion Through the ROCK 1/p-MLC Pathway in Non-Small Cell Lung Cancer

doi: 10.3389/fphar.2022.890891

Figure Lengend Snippet: Schematic diagram of the role of PFN1 in NSCLC metastasis. In the initiation stage of NSCLC, cells with upregulated PFN1 secret more MVs through PFN1 interactions with the ROCK/p-MLC pathway. These MVs contain numerous oncogenenic moleculars, which could enhance migration abilities of PFN1 normal expressed NSCLC cells, and untimately promote progression and metastasis of NSCLC.

Article Snippet: PFN1 siRNA and control scramble siRNA were synthesized by Guangzhou RiboBio Co. (Guangzhou, China), and the sequences are listed in .

Techniques: Migration

Journal: Cell Death and Differentiation

Article Title: A novel LncRNA HITT forms a regulatory loop with HIF-1α to modulate angiogenesis and tumor growth

doi: 10.1038/s41418-019-0449-8

Figure Lengend Snippet: HITT prevents tumor growth in vivo by inhibiting angiogenesis in a HIF-1α dependent manner. The expression levels of HITT in human colorectal tumors (T) of different stages (B) relative to their paired adjacent normal controls (N) (n = 46, a, b) were determined by qRT-PCR. Tumor volumes at the indicated dates (c), as well as images (left) and tumor weights (right) at 4 weeks (d) of HCT116/vector (Vect.), HIC116/HITT, HCT116/(Vect. + si-HIF-1α), and HCT116/(HITT + si-HIF-1α) xenografts. The average values are present as bar graphs (means ± SD) (n = 6 for each group). Representative immunohistochemistry staining for Ki-67 (top) and CD31 (bottom) in the indicated xenografts (e), and the quantification of bar graphs (f). g HIF-1α and VEGF protein (upper panel) and VEGF mRNA (lower panel) levels were measured by WB and qRT-PCR, respectively, in HCT116 xenografts. Representative images are presented. h, i VEGF protein levels were measured by ELISA in clone stably transfected with HITT and vector controls after HIF-1α knockdown (KD) or ectopic HIF-1α expression under hypoxia. The KD efficiency of HIF-1α was confirmed by WB. j Representative images of HUVECs are presented (left) and quantification of HUVEC tube formation assays is presented in the bar graph (right). Data derived from three independent experiments are presented as mean ± SEM in the bar graph. Values in controls were normalized to 1 (a, f, g). *P < 0.05; **P < 0.01; ##P < 0.01, compared with hypoxia-treated controls (h–j). Vect. vector control, Ctl. control. Scale bar 50 μm. See also Fig. S1–S3

Article Snippet: siRNA specifically targeting HITT, YB-1, HIF-1α, HIF-2α, or Ago2 , and nonspecific si-scramble control were synthesized by GenePharma (Shanghai, China).

Techniques: In Vivo, Expressing, Quantitative RT-PCR, Plasmid Preparation, Immunohistochemistry, Staining, Enzyme-linked Immunosorbent Assay, Stable Transfection, Transfection, Knockdown, Derivative Assay, Control

Journal: Cell Death and Differentiation

Article Title: A novel LncRNA HITT forms a regulatory loop with HIF-1α to modulate angiogenesis and tumor growth

doi: 10.1038/s41418-019-0449-8

Figure Lengend Snippet: HITT inhibits HIF-1α expression and activity. HIF-1α protein was detected by WB of lysates from control and HITT stably transfected lines (a). Quantification of WB bands was conducted by ImageJ (b). HIF-1α mRNA relative to 18 s was detected by qRT-PCR (lower panel, b). The KD efficiency of HITT was confirmed by qRT-PCR (c). HIF-1α protein levels were measured by WB after HITT KD under normoxia in the presence or absence of proteasome inhibitor MG132 (d). Quantification of WB bands was conducted by ImageJ (e). HIF-1α mRNA relative to 18 s was detected by qRT-PCR (lower panel, e). Hypoxic response element (HRE) luciferase activity (f), and expression levels of VEGF and Angiogenin-1 (g), were measured by luciferase reporter assay or qRT-PCR, respectively, under the indicated conditions. Data derived from three independent experiments are presented as mean ± SEM in the bar graph. Values in controls were normalized to 1 (b, c, e–g). *P < 0.05; **P < 0.01; ##P < 0.01, compared with hypoxia-treated controls (b, f–g). Vect. vector control. See also Fig. S4

Article Snippet: siRNA specifically targeting HITT, YB-1, HIF-1α, HIF-2α, or Ago2 , and nonspecific si-scramble control were synthesized by GenePharma (Shanghai, China).

Techniques: Expressing, Activity Assay, Control, Stable Transfection, Transfection, Quantitative RT-PCR, Luciferase, Reporter Assay, Derivative Assay, Plasmid Preparation

Journal: Cell Death and Differentiation

Article Title: A novel LncRNA HITT forms a regulatory loop with HIF-1α to modulate angiogenesis and tumor growth

doi: 10.1038/s41418-019-0449-8

Figure Lengend Snippet: HITT inhibits HIF-1α translation. a Representative images of HIF-1α protein levels detected by WB (left) and quantification of HIF-1α band intensities by ImageJ (right) for vector control and HITT ectopic expression HCT116 cells, in the presence of CHX, for the indicate time periods under hypoxia. HIF-1α levels in the CHX untreated group were normalized to 1. b HIF-1α protein levels were determined by WB after ectopic HITT expression in the presence or absence of MG132 in HCT116 and HeLa cells under hypoxia. c, d Polysomes in cytoplasmic extracts were fractionated through sucrose gradients, and mRNA levels of HIF-1α and GAPDH in HCT116 cells were measured by qRT-PCR in gradient fractions after HITT overexpression or KD. Representative data, as a percentage of total RNA of interest in the gradient from three independent experiments, are presented. e, f Affinity purification of biotinylated l-azidohomoalanine (AHA)-labeled acutely synthesized proteins from cell lysates. Purified HIF-1α and α-tubulin proteins were detected by WB

Article Snippet: siRNA specifically targeting HITT, YB-1, HIF-1α, HIF-2α, or Ago2 , and nonspecific si-scramble control were synthesized by GenePharma (Shanghai, China).

Techniques: Plasmid Preparation, Control, Expressing, Quantitative RT-PCR, Over Expression, Affinity Purification, Labeling, Synthesized, Purification

Journal: Cell Death and Differentiation

Article Title: A novel LncRNA HITT forms a regulatory loop with HIF-1α to modulate angiogenesis and tumor growth

doi: 10.1038/s41418-019-0449-8

Figure Lengend Snippet: HITT inhibits HIF-1α translation by competing with YB-1 for binding with HIF-1α 5′-UTR. a The expression levels of HIF-1α were measured by WB in HITT stable lines, with or without YB-1 KD, under hypoxia in HCT116 and HeLa cells. The KD efficiency of YB-1 was confirmed by WB. b Schematic of the positions of high-stringency YB-1 binding sites in HITT predicted by a bioinformatics online tool (http://rbpmap.technion.ac.il/). c Biotin-HITT pull-down using HeLa cell lysates showed that YB-1 bands were present in Biotin-HITT precipitates. YB-1-associated HITT (d) and HIF-1α 5′-UTR (e, f), as measured by qRT-PCR following CLIP YB-1 after HITT overexpression or KD, under normoxia or hypoxia, in HCT116 cells, as indicated in the figures. GAPDH mRNA and CLIP IgG were used as negative controls. g Schematic of the positions of high-stringency YB-1 binding sites in HITT (left). CLIP assay was used to detect the binding of YB-1 with full-length or fragmented HITT in 4T1 cells, a cell line with no endogenous HITT, after ectopic expression of the indicated HITT. CLIP IgG and 18 s were used as negative controls (lower panel). Similar expression levels of HITT were confirmed by qRT-PCR (upper panel). h Schematic of full-length and fragmented HITT, and the predicted YB-1 binding sites (upper panel). HIF-1α protein levels were measured by WB after transfection with full-length (1–2050) HITT and MT (1–1029) HITT (lower right panel). Similar expression levels of HITT were confirmed by qRT-PCR (lower left panel). i Schematic of positions of all potential YB-1 binding sites in HIF-1α 5′-UTR predicted by a bioinformatics online tool (http://rbpmap.technion.ac.il/)(Left). Luciferase reporter containing HIF-1α 5′-UTR (1–256) or two truncated mutants (1–138: M1 and 139–256: M2) was used to assess translational efficiency after HITT overexpression with or without YB-1 KD (right). Data derived from three independent experiments are presented as mean ± SEM in the bar graph (d–i). Values in controls were normalized to 1. **P < 0.01 (d–i); #P < 0.05, ##P < 0.01, compared with hypoxia-treated controls (d, e, and i). N.S. not significant (d–g). See also Fig. S5

Article Snippet: siRNA specifically targeting HITT, YB-1, HIF-1α, HIF-2α, or Ago2 , and nonspecific si-scramble control were synthesized by GenePharma (Shanghai, China).

Techniques: Binding Assay, Expressing, Quantitative RT-PCR, Over Expression, Transfection, Luciferase, Derivative Assay

Journal: Cell Death and Differentiation

Article Title: A novel LncRNA HITT forms a regulatory loop with HIF-1α to modulate angiogenesis and tumor growth

doi: 10.1038/s41418-019-0449-8

Figure Lengend Snippet: HIF-1α lowers HITT’s stability via negative feedback. a Hypoxia-responsive lncRNAs reported previously and HITT were measured by qRT-PCR under hypoxia relative to normoxia. HIF-1α expression was determined by WB, and HITT was measured by qRT-PCR in samples derived from cells subjected to dose- (b) and time-dependent (c) hypoxia treatment. The expression levels of HITHITT and HITT promoter luciferase reporter activity were detected by qRT-PCR and luciferase reporter assay, respectively, in HCT116 cells after HIF-1α KD (d) or overexpression (e) under hypoxia. The efficiency of HIF-1α overexpression and KD was confirmed by WB. f The half-lives of HITT and GAPDH mRNA were measured by qRT-PCR in the presences of ActD in HCT116 cells. Data derived from three independent experiments are represented as mean ± SEM in the bar graph. *P < 0.05, **P < 0.01, N.S. not significant. See also Fig. S6

Article Snippet: siRNA specifically targeting HITT, YB-1, HIF-1α, HIF-2α, or Ago2 , and nonspecific si-scramble control were synthesized by GenePharma (Shanghai, China).

Techniques: Quantitative RT-PCR, Expressing, Derivative Assay, Luciferase, Activity Assay, Reporter Assay, Over Expression

Journal: Cell Death and Differentiation

Article Title: A novel LncRNA HITT forms a regulatory loop with HIF-1α to modulate angiogenesis and tumor growth

doi: 10.1038/s41418-019-0449-8

Figure Lengend Snippet: HIF-1α promotes HITT decay via MiR-205-mediated RNA degradation. a MiR-205 was predicted to be a potent microRNA that can bind HITT by two independent bioinformatics tools: http://www.mircode.org/index.php and http://annolnc.cbi.pku.edu.cn/index.jsp. b Expression levels of MiR-205 normalized to U6 were measured by qRT-PCR after HIF-1α KD under hypoxia. Relative expression levels of HITT were determined by qRT-PCR after transfection of MiR-205 inhibitors (Inh., c) or MiR-205 mimics (d) into HCT116 cells. MiR-205 levels normalized to U6 were measured by qRT-PCR. e The expression levels of HITT were determined by qRT-PCR in HCT116 cells after HIF-1α KD with or without MiR-205 inhibitor treatment. f–h Schematic of the hypothetical duplexes formed by interactions between the binding site in HITT (top), MiR-205 (middle), and mutated HITT (bottom) (f). The activities of wild type (WT) or MiR-205-binding defective mutant (MT) HITT luciferase reporters were detected in HCT116 cells after transfection of MiR-205 inhibitor (Inh., g) or mimics (h) under normoxia or hypoxia, as indicated in the figures. i Biotinylated precursor MiR-205 was transfected into HCT116 cells. MiR-205-associated endogenous HITT relative to total HITT was determined by qRT-PCR following CLIP biotin. CLIP GAPDH and nonbiotinylated MiR-205 were used as negative controls. j The expression levels of HITT were determined by qRT-PCR in HCT116 cells after Ago2 KD (right). KD efficiency was confirmed by WB (left). k Ago2-associated HITT levels relative to total HITT were determined by qRT-PCR following formaldehyde cross-linked IP of Ago2 in HCT116 cells under normoxia and hypoxia, with or without treatment with MiR-205 inhibitors (Inh.). GAPDH and IgG were used as negative controls. l Relative expression levels of HITT determined by qRT-PCR after treatment with MiR-205 mimics in control and Ago2 KD HCT116 cells. m HITT’s half-life was determined by qRT-PCR after treatment with MiR-205 inhibitor (Inh.) alone, Ago2 KD alone, or a combination of the two in the presences of RNA synthesis inhibitor ActD under hypoxia in HCT116 cells. Data derived from three independent experiments are presented as mean ± SEM in the bar graph. Values in controls were normalized to 1. *P < 0.05; **P < 0.01; #P < 0.05, compared with hypoxia treatment controls (c, e, g, k). N.S. not significant (e, g–i, k, l). See also Fig. S7

Article Snippet: siRNA specifically targeting HITT, YB-1, HIF-1α, HIF-2α, or Ago2 , and nonspecific si-scramble control were synthesized by GenePharma (Shanghai, China).

Techniques: Expressing, Quantitative RT-PCR, Transfection, Binding Assay, Mutagenesis, Luciferase, Control, Derivative Assay

Journal: Cell Death and Differentiation

Article Title: A novel LncRNA HITT forms a regulatory loop with HIF-1α to modulate angiogenesis and tumor growth

doi: 10.1038/s41418-019-0449-8

Figure Lengend Snippet: The association of HITT with HIF-1α and VEGF levels in human colon cancer tissues. a Representative image of HIF-1α protein expression in human colon cancer tissue samples (T) and paired adjacent normal controls (N). The average fold-change of HIF-1α protein (b) and VEGF mRNA (c) levels normalized to 18S rRNA were detected by qRT-PCR and WB in human colorectal cancer tissue samples, (T) and paired adjacent normal controls (n = 46). Correlation analysis of HITT with HIF-1α protein (d) and VEGF mRNA (e). f Schematic diagram of the proposed HITT–HIF-1α feedback loop that controls hypoxia-induced angiogenesis and tumor growth. Under normoxia, HITT does not undergo HIF-1α/MiR-205-mediated destabilization. Thus, HITT acts as a molecular decoy for YB-1, such that it is associated with YB-1 via favorable binding sequences, titrating YB-1 away from HIF-1α 5′-UTR, resulting in the attenuation of HIF-1α protein translation. Under hypoxia, HITT is targeted by Ago2/MiR-205 for degradation in a HIF-1α-dependent manner, consequently diminishing the inhibitory effect of HITT on HIF-1α transcription, resulting in angiogenesis and tumor growth

Article Snippet: siRNA specifically targeting HITT, YB-1, HIF-1α, HIF-2α, or Ago2 , and nonspecific si-scramble control were synthesized by GenePharma (Shanghai, China).

Techniques: Expressing, Quantitative RT-PCR, Binding Assay

Journal: Virologica Sinica

Article Title: HSV-2-encoded miRNA-H4 Regulates Cell Cycle Progression and Act-D-induced Apoptosis in HeLa Cells by Targeting CDKL2 and CDKN2A

doi: 10.1007/s12250-019-00101-8

Figure Lengend Snippet: Production of HSV-2 LAT-encoded miRNA-H4. A Schematic of the HSV-2 genome expanded with details of the LAT locus. miRNA-H4 maps with antisense to the noncoding region of ICP34.5. L long, S short, IR internal repeat, TR terminal repeat, UL unique long region, US unique short region. B Secondary structure adopted by pri-miRNA precursor of miRNA-H4 which includes miRNA-H4-5p and miRNA-H4-3p according to the miRbase.

Article Snippet: miRNA-H4-5p mimics (MC-5p), miRNA-H4-5p inhibitor (MI-5p), miRNA-H4-3p inhibitor (MI-3p), miRNA inhibitor negative control (MI-NC), miRNA-H4 negative control (miRNA-NC), siRNAs against human CDKN2A (siRNA-p16), and CDKL2 (siRNA-p65) were all synthesized and purified by RiboBio (Guangzhou, China). siRNAs targeting nonspecific sequences were used as negative control (siRNA-NC). miRNA and siRNAs were all transfected using Lipofectamine 2000 (Invitrogen).

Techniques:

Journal: Virologica Sinica

Article Title: HSV-2-encoded miRNA-H4 Regulates Cell Cycle Progression and Act-D-induced Apoptosis in HeLa Cells by Targeting CDKL2 and CDKN2A

doi: 10.1007/s12250-019-00101-8

Figure Lengend Snippet: miRNA expression and Act-D-induced apoptosis in HeLa-H4 cells. A Different cell lines were subjected to qRT-PCR to detect the relative expression of miRNA-H4-5p. B Different cell lines were subjected to qRT-PCR to detect the relative expression of miRNA-H4-3p. C The activity of caspase-3 in HeLa cells transfected with or without transfection, accompanied by Act-D treatment. D Apoptosis was assessed using FAM in HeLa cells with or without transfection, accompanied by Act-D treatment. E HeLa cells with or without transfection treated with Hoechst 33342 were imaged under a fluorescent microscope. Cells with bright blue nucleus were recognized as apoptotic.

Article Snippet: miRNA-H4-5p mimics (MC-5p), miRNA-H4-5p inhibitor (MI-5p), miRNA-H4-3p inhibitor (MI-3p), miRNA inhibitor negative control (MI-NC), miRNA-H4 negative control (miRNA-NC), siRNAs against human CDKN2A (siRNA-p16), and CDKL2 (siRNA-p65) were all synthesized and purified by RiboBio (Guangzhou, China). siRNAs targeting nonspecific sequences were used as negative control (siRNA-NC). miRNA and siRNAs were all transfected using Lipofectamine 2000 (Invitrogen).

Techniques: Expressing, Quantitative RT-PCR, Activity Assay, Transfection, Microscopy

Journal: Virologica Sinica

Article Title: HSV-2-encoded miRNA-H4 Regulates Cell Cycle Progression and Act-D-induced Apoptosis in HeLa Cells by Targeting CDKL2 and CDKN2A

doi: 10.1007/s12250-019-00101-8

Figure Lengend Snippet: Validation of miRNA-H4-5p targets. A Sequence alignment between miRNA-H4-5p and 3′-UTR of CDKN2A or CDKL2. Binding sites are indicated in dotted lines and the corresponding mutants are labeled in solid lines. All complementary pairing bases are indicated in the vertical curve. B, C Changes in luciferase enzyme viability. HeLa cells co-transfected with luciferase reporter containing either wild type (WT) or mutant type (mut) 3′-UTR of (B) CDKN2A or (C) CDKL2 along with MC-5p or miRNA-H4 negative control. Relative viability of luciferase enzyme = Renilla reniformis/firefly luciferase. D Effects of MC-5p or MI-5p on the expression of CDKN2A and CDKL2 in HeLa cells were examined using Western blot analysis with GAPDH as the internal control. Differences were judged to be statistically significant when *P < 0.05, **P < 0.01. E, F Expression of CDKN2A and CDKL2 mRNA was analyzed using qRT-PCR. G The effects of siRNAs on cell proliferation were determined by MTT assay 24 h and 36 h after transfection. H The relative number of EdU positive cells was estimated from siRNA-p16 and siRNA-p65 transfections, cells with positive EdU were found to have accumulated in the S phase.

Article Snippet: miRNA-H4-5p mimics (MC-5p), miRNA-H4-5p inhibitor (MI-5p), miRNA-H4-3p inhibitor (MI-3p), miRNA inhibitor negative control (MI-NC), miRNA-H4 negative control (miRNA-NC), siRNAs against human CDKN2A (siRNA-p16), and CDKL2 (siRNA-p65) were all synthesized and purified by RiboBio (Guangzhou, China). siRNAs targeting nonspecific sequences were used as negative control (siRNA-NC). miRNA and siRNAs were all transfected using Lipofectamine 2000 (Invitrogen).

Techniques: Biomarker Discovery, Sequencing, Binding Assay, Labeling, Luciferase, Transfection, Mutagenesis, Negative Control, Expressing, Western Blot, Control, Quantitative RT-PCR, MTT Assay

Journal: Virologica Sinica

Article Title: HSV-2-encoded miRNA-H4 Regulates Cell Cycle Progression and Act-D-induced Apoptosis in HeLa Cells by Targeting CDKL2 and CDKN2A

doi: 10.1007/s12250-019-00101-8

Figure Lengend Snippet: miRNA expression and its regulatory function on target genes in HSV-2-infected HeLa cells. A Expression of miRNA-H4-5p in different cell lines after HSV-2 infection were detected using qRT-PCR. B Expression of CDKN2A and CDKL2 in different cell lines after HSV-2 infection were examined using Western blot analysis with GAPDH as the internal control.

Article Snippet: miRNA-H4-5p mimics (MC-5p), miRNA-H4-5p inhibitor (MI-5p), miRNA-H4-3p inhibitor (MI-3p), miRNA inhibitor negative control (MI-NC), miRNA-H4 negative control (miRNA-NC), siRNAs against human CDKN2A (siRNA-p16), and CDKL2 (siRNA-p65) were all synthesized and purified by RiboBio (Guangzhou, China). siRNAs targeting nonspecific sequences were used as negative control (siRNA-NC). miRNA and siRNAs were all transfected using Lipofectamine 2000 (Invitrogen).

Techniques: Expressing, Infection, Quantitative RT-PCR, Western Blot, Control

Journal: Biochemistry and Biophysics Reports

Article Title: Distal extracellular teneurin region (teneurin C-terminal associated peptide; TCAP) possesses independent intracellular calcium regulating actions, in vitro : A potential antagonist of corticotropin-releasing factor (CRF)

doi: 10.1016/j.bbrep.2022.101397

Figure Lengend Snippet: siRNA oligonucleotides used to knockdown teneurin and TCAP expression . A. The 270 oligonucleotide is designed to knockdown mouse TCAPs 1–4 although has reduced homology with TCAP-3. B. The 721 oligonucleotide was designed to be more specific to mouse TCAP-3. C. Negative control oligonucleotides.

Article Snippet: SiRNA sequences for TCAP (terminal exon) and the negative controls were synthesized by Genosys Inc., annealed, phosphorylated and cloned into pLKO.1puro (Sigma) ( ).

Techniques: Knockdown, Expressing, Negative Control

Journal: Biochemistry and Biophysics Reports

Article Title: Distal extracellular teneurin region (teneurin C-terminal associated peptide; TCAP) possesses independent intracellular calcium regulating actions, in vitro : A potential antagonist of corticotropin-releasing factor (CRF)

doi: 10.1016/j.bbrep.2022.101397

Figure Lengend Snippet: RT-PCR primers to amplify TCAPs and Teneurins.

Article Snippet: SiRNA sequences for TCAP (terminal exon) and the negative controls were synthesized by Genosys Inc., annealed, phosphorylated and cloned into pLKO.1puro (Sigma) ( ).

Techniques:

Journal: Biochemistry and Biophysics Reports

Article Title: Distal extracellular teneurin region (teneurin C-terminal associated peptide; TCAP) possesses independent intracellular calcium regulating actions, in vitro : A potential antagonist of corticotropin-releasing factor (CRF)

doi: 10.1016/j.bbrep.2022.101397

Figure Lengend Snippet: Summary of Pharmacological Agents and their Actions on TCAP associated Ca2+ activity.

Article Snippet: SiRNA sequences for TCAP (terminal exon) and the negative controls were synthesized by Genosys Inc., annealed, phosphorylated and cloned into pLKO.1puro (Sigma) ( ).

Techniques: Activity Assay, Biomarker Discovery, Inhibition, Blocking Assay, Concentration Assay

Journal: Biochemistry and Biophysics Reports

Article Title: Distal extracellular teneurin region (teneurin C-terminal associated peptide; TCAP) possesses independent intracellular calcium regulating actions, in vitro : A potential antagonist of corticotropin-releasing factor (CRF)

doi: 10.1016/j.bbrep.2022.101397

Figure Lengend Snippet: Effect of TCAP and Teneurin expression on N38 cells transfected with TCAP-based siRNAs. A. N38 cells after transfection with either the pLuc negative control, or p270 and p721 siRNA containing plasmids. Scale bar: 50 μM. B. Expression product of histone H3, TCAP and teneurin after transfection. C. Quantification of the data provided in ‘B’. TCAP-1 and teneurin-1 is not expressed by N38 cells. The level of significance was determined using a one-way analysis of variance (ANOVA) using Tukey's post hoc test. n = 4 for all analyses. (*p < 0.05; **p < 0.01; ***p < 0.001).

Article Snippet: SiRNA sequences for TCAP (terminal exon) and the negative controls were synthesized by Genosys Inc., annealed, phosphorylated and cloned into pLKO.1puro (Sigma) ( ).

Techniques: Expressing, Transfection, Negative Control

Journal: Biochemistry and Biophysics Reports

Article Title: Distal extracellular teneurin region (teneurin C-terminal associated peptide; TCAP) possesses independent intracellular calcium regulating actions, in vitro : A potential antagonist of corticotropin-releasing factor (CRF)

doi: 10.1016/j.bbrep.2022.101397

Figure Lengend Snippet: Intracellular calcium flux by TCAP-1 and CRF in immortalized neurons . A. Dose-response curves showing the acute effect of increasing concentrations of CRF or TCAP-1 on intracellular Ca2+ as established by Fluo-4 in mHypoE-38 cells where CRF shows dose-dependent increase in cytosolic Ca2+ concentrations and TCAP has an opposite action. Data represent the mean ± SEM (n = 4–5 replicates per treatment dose) B. Cells showed normal behaviour after treatment. Representative differential interference contrast (DIC) (left) and Fluo-4 fluorescence images of N38 neurons (right). The scale bar indicates 20 μm. C-E: TCAP-1 and CRF induce opposing calcium responses in embryonic mouse hypothalamic neurons. C. Superimposed Fluo-4 calcium responses following treatment with I) ACSF, II) CRF (100 nM); III) sc-TCAP (100 nM), and IV) TCAP (100 nM). The red trace represents the mean of the individual grey traces (n = 5–6 each), and each grey trace represents each of the average calcium response of 5 cells from the same experiment. D. Overlay of summary data shown in C. E. Summary graph showing percent change from baseline from data obtained in B and C, data assessed at the 15 min time point. Data represent the mean ± SEM (n = 5–6 replicates per experiment). **P < 0.01, ****P < 0.0001. One-way ANOVA with Tukey's post-hoc test was used. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)

Article Snippet: SiRNA sequences for TCAP (terminal exon) and the negative controls were synthesized by Genosys Inc., annealed, phosphorylated and cloned into pLKO.1puro (Sigma) ( ).

Techniques: Fluorescence

Journal: Biochemistry and Biophysics Reports

Article Title: Distal extracellular teneurin region (teneurin C-terminal associated peptide; TCAP) possesses independent intracellular calcium regulating actions, in vitro : A potential antagonist of corticotropin-releasing factor (CRF)

doi: 10.1016/j.bbrep.2022.101397

Figure Lengend Snippet: TCAP-1 antagonizes CRF pre-treatment-mediated increases in cytosolic Ca2+ concentrations. A. Fluo-4 measurements in response to a 5 m CRF (1 nM) pre-treatment followed by a 5 m treatment with I) CRF (1 nM); II) TCAP-1 (100 nM): III) sc-TCAP (100 nM). The red trace represents the mean of the individual grey traces (n = 5 each), and each grey trace represents the mean Ca2+ response of 5 cells from the same experiment. B. Overlay of summary data shown in A. C. Quantification showing percent change from baseline from data obtained in B and C, summary data assessed at the 15 min time point. Data represent the mean ± SEM (n = 5–6 replicates per experiment). **P < 0.01, ***P < 0.001. One-way ANOVA with Tukey's post-hoc test. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)

Article Snippet: SiRNA sequences for TCAP (terminal exon) and the negative controls were synthesized by Genosys Inc., annealed, phosphorylated and cloned into pLKO.1puro (Sigma) ( ).

Techniques:

Journal: Biochemistry and Biophysics Reports

Article Title: Distal extracellular teneurin region (teneurin C-terminal associated peptide; TCAP) possesses independent intracellular calcium regulating actions, in vitro : A potential antagonist of corticotropin-releasing factor (CRF)

doi: 10.1016/j.bbrep.2022.101397

Figure Lengend Snippet: Teneurin TCAP-1 pre-treatment prevents CRF-mediated increases in cytosolic Ca2+ concentrations. A. Superimposed Fluo-4 Ca2+ measurements in response to a 5 m TCAP-1 (100 nM) treatment followed by a 5 min treatment with I) TCAP (100 nM), II) Sc-TCAP (100 nM); III) CRF (1 nM). The red trace represents the average of the individual grey traces (n = 5 each), and each grey trace represents the average calcium response of 5 cells from the same experiment. B. Summary graph of data shown in B for direct comparison of mean traces. C. Quantification indicating percent change from baseline from data obtained in B and C, where the mean data was assessed at the 15 m time point. Data represent the mean ± SEM (n = 5–6 replicates per experiment). ns = not significant. One-way ANOVA with Tukey's post-hoc test. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)

Article Snippet: SiRNA sequences for TCAP (terminal exon) and the negative controls were synthesized by Genosys Inc., annealed, phosphorylated and cloned into pLKO.1puro (Sigma) ( ).

Techniques: Comparison

Journal: Biochemistry and Biophysics Reports

Article Title: Distal extracellular teneurin region (teneurin C-terminal associated peptide; TCAP) possesses independent intracellular calcium regulating actions, in vitro : A potential antagonist of corticotropin-releasing factor (CRF)

doi: 10.1016/j.bbrep.2022.101397

Figure Lengend Snippet: TCAP-1-mediated Ca2+ channel and transporter actions determined by antagonist interaction. TCAP-1 treatment (10 m; 10 nM) are indicated by Fluo-4 Ca2+ activity in N38 cells with following antagonist treatment: I) Cells pretreated for 20 m with ruthedium red (RuR; 50 μM) followed by 10 m treatment with RuR and TCAP-1 (100 nM), II. Cells pre-treated for 20 m with benzamil (BZ; 100 μM) followed by 1 m treatment with BZ and TCAP-1 (100 nM), III) Cells pretreated for 20 m with thapsigargin (TGX; 100 nM) followed by 10 m treatment with TGX and TCAP-1 (100 nM). IV) Cells pretreated for 20 m VDT (vanadate; 100 nM) followed by a 10 m treatment with VDT and TCAP-1 (100 nM). Note: The red trace represents the average of the individual grey traces (n = 5 each), each grey trace represents the average calcium response of 5 cells from the same experiment. B. Summary graph of data shown in A, for comparison this graph includes the TCAP only treatment data shown in A. C. Summary graph showing percent change from baseline from data obtained in B and C, summary data assessed at the 15 m time point. Data represent the mean ± SEM (n = 4–6 replicates per experiment). *P < 0.05, ***P < 0.001. One-way ANOVA with a Dunnett's post-hoc test. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)

Article Snippet: SiRNA sequences for TCAP (terminal exon) and the negative controls were synthesized by Genosys Inc., annealed, phosphorylated and cloned into pLKO.1puro (Sigma) ( ).

Techniques: Activity Assay, Comparison

Journal: Biochemistry and Biophysics Reports

Article Title: Distal extracellular teneurin region (teneurin C-terminal associated peptide; TCAP) possesses independent intracellular calcium regulating actions, in vitro : A potential antagonist of corticotropin-releasing factor (CRF)

doi: 10.1016/j.bbrep.2022.101397

Figure Lengend Snippet: TCAP induces MT Ca2+ uptake and prevents CRF-induced MT Ca2+ release. A. Representative DIC and Rhod-2 fluorescence images of N38 neurons. Scale bar: 20 μm. B. Rhod-2 MT Ca2+ responses following treatment with I) ACSF, II) TCAP-1 (100 nM), CRF (1 nM) and IV) TCAP + CRF (100 nM, 1 nM respectively); cells were pre-treated with TCAP-1 (100 nM) for 20 m prior start of experiment. The red trace represents the average of the individual grey traces (n = 5–6 each), and each grey trace represents the average Ca2+ response of 5 cells from the same experiment. C. Summary graph of data shown in B. D) Quantification of results obtained in B and C, summary data assessed at the 15 min time point. Data represent the mean ± SEM (n = 4–5 replicates per experiment). *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001. One-way ANOVA with Tukey's post-hoc test was used. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)

Article Snippet: SiRNA sequences for TCAP (terminal exon) and the negative controls were synthesized by Genosys Inc., annealed, phosphorylated and cloned into pLKO.1puro (Sigma) ( ).

Techniques: Fluorescence

Journal: Biochemistry and Biophysics Reports

Article Title: Distal extracellular teneurin region (teneurin C-terminal associated peptide; TCAP) possesses independent intracellular calcium regulating actions, in vitro : A potential antagonist of corticotropin-releasing factor (CRF)

doi: 10.1016/j.bbrep.2022.101397

Figure Lengend Snippet: TCAP prevents CRF-induced depolarization of the MT membrane potential (v). A) Representative DIC and Rhodamine 123 (Rhod-123) fluorescence images of N38 neurons. Scale bar: 20 μm. B) Rhod-123 measurements of changes in ψm in N38 cells in response to: B I) ACSF, II) TCAP (100 nM), III) CRF (1 nM), IV) Sc-TCAP (100 nM), and V) TCAP + CRF; cells were pre-treated with TCAP-1 (100 nM) for 20 m prior start of experiment. Note: The red trace represents the average of the individual grey traces (n = 5 each), each grey trace represents the average calcium response of 5 cells from the same experiment. Cells were loaded/recorded in quench mode and an increase in fluorescence indicates ψm depolarization and a decrease in fluorescence indicates ψm hyperpolarization. C) Summary graph of data shown in A. D) Quantification of results obtained in A and B, summary data assessed at the 10 min time point. Data represent the mean ± SEM (n = 4–6 replicates per experiment). **P < 0.01, ***P < 0.001, ****P < 0.0001. One-way ANOVA with Tukey's post-hoc test was used. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)

Article Snippet: SiRNA sequences for TCAP (terminal exon) and the negative controls were synthesized by Genosys Inc., annealed, phosphorylated and cloned into pLKO.1puro (Sigma) ( ).

Techniques: Membrane, Fluorescence

Journal: Biochemistry and Biophysics Reports

Article Title: Distal extracellular teneurin region (teneurin C-terminal associated peptide; TCAP) possesses independent intracellular calcium regulating actions, in vitro : A potential antagonist of corticotropin-releasing factor (CRF)

doi: 10.1016/j.bbrep.2022.101397

Figure Lengend Snippet: TCAP-1 increases intracellular NADH and ATP levels in N38 cells. A. Summary trace showing change in resorufin fluorescence over time following TCAP-1 (100 nM) or ACSF treatment. B. Summary graph showing change in the rate of resorufin formation C. ATP turnover Data represent the mean ± SEM (n = 4–5 replicates per exp eriment). Data were analyzed by a two-way ANOVA followed by a Sidak post-hoc test (A), and a one-way ANOVA followed by a Tukey's post-hoc test (B). Data were considered statistically significant with an a priori hypothesis of P < 0.05 (*P < 0.05, **P < 0.01, ****P < 0.0001).

Article Snippet: SiRNA sequences for TCAP (terminal exon) and the negative controls were synthesized by Genosys Inc., annealed, phosphorylated and cloned into pLKO.1puro (Sigma) ( ).

Techniques: Fluorescence

Journal: Biochemistry and Biophysics Reports

Article Title: Distal extracellular teneurin region (teneurin C-terminal associated peptide; TCAP) possesses independent intracellular calcium regulating actions, in vitro : A potential antagonist of corticotropin-releasing factor (CRF)

doi: 10.1016/j.bbrep.2022.101397

Figure Lengend Snippet: Actions of pharmacological antagonists on TCAP-1 actions on calcium flux . A. Schematic of the actions of Ca2+ channel and transporter antagonists and their intracellular targets. Antagonist abbreviations are shown in red text with red squares on associated channels and transporters. B. Summary of TCAP-1 actions on intracellular Ca2+ flux based on the data shown in ‘A’. TCAP-1 reduces cytosolic Ca2+ concentrations by plasma membrane efflux and mitochondrial influx. Abbreviations: BZ; benzamil, CaU; Ca2+ uniporter, NCX; Na+/Ca2+ exchanger, RuR; ruthenium red, TGX; thapsigargin, VDT; vanadate. Figure prepared using BioRender software. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)

Article Snippet: SiRNA sequences for TCAP (terminal exon) and the negative controls were synthesized by Genosys Inc., annealed, phosphorylated and cloned into pLKO.1puro (Sigma) ( ).

Techniques: Clinical Proteomics, Membrane, Software