Journal: Journal of cellular physiology

Article Title: Mechanisms controlling neurite outgrowth in a pheochromocytoma cell line: the role of TRPC channels.

doi: 10.1002/jcp.22855

Figure Lengend Snippet: Fig. 1. Comparison of TRPC expression levels in control and NGF-differentiated PC12 cells. A: Representative bright field images of undifferentiated(control)andNGF-differentiated(NGF)PC12cellsareshown.B:RT-PCRanalysisofTRPCexpressioninPC12cells.PCRproducts amplified using TRPC specific primer sets were separated on ethidium bromide stained agarose gels. A representative gel image is shown. C: Comparison of TRPC expression in control (C) and NGF-differentiated PC12 cells (NGF). Each experiment was repeated 7–9 times.

Article Snippet: Conversely, neurite outgrowth was markedly facilitated in NGF-differentiated, TRPC5-defficient PC12 cells overexpressing an shRNA–TRPC5–GFP construct (OriGene, Rockville, MD; Supplemental Fig. 2A–2C) and reduced in NGFdifferentiated, TRPC1-defficient PC12 cells overexpressing shRNA–TRPC1–GFP (OriGene, Rockville, MD; Supplemental Fig. 2D).

Techniques: Comparison, Expressing, Control, Staining

Journal: Journal of cellular physiology

Article Title: Mechanisms controlling neurite outgrowth in a pheochromocytoma cell line: the role of TRPC channels.

doi: 10.1002/jcp.22855

Figure Lengend Snippet: Fig. 2. Western blot analysis of TRPC protein expression in PC12 cells. A: Monoclonal anti-TRPC1 antibody (a generous gift from Dr. Leonidas Tsiokas) was used to detect the TRPC1 protein in control (C) and NGF-differentiated PC12 cells (NGF). The specificity of monoclonal anti-TRPC1 was confirmed using mouse recombinant TRPC proteins. B: TRPC5 protein expression was detected only in undifferentiated PC12 cells. C: NGF pretreatment up-regulated TRPC6 in PC12 cells. In (A)–(C), the lanes labeled mTRPC1, mTRPC5, and hTRPC6 represent recombinant proteins used as controls. ‘‘C’’ denotes control, protein extracts of untreated PC12 cells. ‘‘NGF’’ labels protein extracts isolated from PC12 cells treated with NGF for 48 h. D: Comparison of relative TRPC protein expression levels in control and NGF-differentiated PC12 cells. Each experiment was repeated 10–16 times.

Article Snippet: Conversely, neurite outgrowth was markedly facilitated in NGF-differentiated, TRPC5-defficient PC12 cells overexpressing an shRNA–TRPC5–GFP construct (OriGene, Rockville, MD; Supplemental Fig. 2A–2C) and reduced in NGFdifferentiated, TRPC1-defficient PC12 cells overexpressing shRNA–TRPC1–GFP (OriGene, Rockville, MD; Supplemental Fig. 2D).

Techniques: Western Blot, Expressing, Control, Recombinant, Labeling, Isolation, Comparison

Journal: Journal of cellular physiology

Article Title: Mechanisms controlling neurite outgrowth in a pheochromocytoma cell line: the role of TRPC channels.

doi: 10.1002/jcp.22855

Figure Lengend Snippet: Fig. 3. DN-TRPC6 facilitates NGF-induced neurite outgrowth in PC12 cells. A: Comparison of total neurite length per cell in PC12 cells electroporated with a mixture of the YFP plasmid and the pcDNA3 plasmid with or without an insert as indicated. Each group consisted of 35–312 cells. B: Comparison of total neurite length per cell inPC12 cells electroporated with GFP-tagged TRPC constructasindicated. C: Representative images demonstrating the effect of GFP-TRPC6 and DN–GFP–TRPC6 overexpression on the total neurite length per cell. In these pseudocolor images, the red color corresponds to the highest YFP fluorescence, whereas the blue color indicates the lowest YFP fluorescence.

Article Snippet: Conversely, neurite outgrowth was markedly facilitated in NGF-differentiated, TRPC5-defficient PC12 cells overexpressing an shRNA–TRPC5–GFP construct (OriGene, Rockville, MD; Supplemental Fig. 2A–2C) and reduced in NGFdifferentiated, TRPC1-defficient PC12 cells overexpressing shRNA–TRPC1–GFP (OriGene, Rockville, MD; Supplemental Fig. 2D).

Techniques: Comparison, Plasmid Preparation, Over Expression

Journal: Journal of cellular physiology

Article Title: Mechanisms controlling neurite outgrowth in a pheochromocytoma cell line: the role of TRPC channels.

doi: 10.1002/jcp.22855

Figure Lengend Snippet: Fig. 5. The effect of hyperforin on neurite outgrowth and TRPC expression in NGF-differentiated PC12 cells. A: Comparison of the total neurite length per cell in hyperforin-treated PC12 cells (n U 405–635). B, C, and D: Hyperforin-induced changes in relative TRPC channel expression in PC12 cells (n U 4 in each group). E: Representative images of gels used during the RT-PCR analysis. HF, hyperforin; Control, undifferentiated PC12 cells; NGF, NGF-differentiated PC12 cells.

Article Snippet: Conversely, neurite outgrowth was markedly facilitated in NGF-differentiated, TRPC5-defficient PC12 cells overexpressing an shRNA–TRPC5–GFP construct (OriGene, Rockville, MD; Supplemental Fig. 2A–2C) and reduced in NGFdifferentiated, TRPC1-defficient PC12 cells overexpressing shRNA–TRPC1–GFP (OriGene, Rockville, MD; Supplemental Fig. 2D).

Techniques: Expressing, Comparison, Reverse Transcription Polymerase Chain Reaction, Control

Journal: Journal of cellular physiology

Article Title: Mechanisms controlling neurite outgrowth in a pheochromocytoma cell line: the role of TRPC channels.

doi: 10.1002/jcp.22855

Figure Lengend Snippet: Fig. 4. Electrophysiological characterization of GTPgS-activated currents inTRPC5-overexpressing cells. A: Currents with TRPC5-like current–voltage relationships were observed in the round TRPC5 expressing PC12 cells (n U 8). B: Heteromeric TRPC1–TRPC5 current–voltage relationships were seen in the TRPC5- overexpressing PC12 cells exhibiting neurites (n U 8). The insets in the quadrant II show representative images of TRPC5-expressing PC12 cells, whereas the insets in the quadrant IV show sample current–voltage relationships of the homomeric TRPC5 channel or the TRPC1–TRPC5 heteromeric channel heterologously expressed in HEK cells. The solid line corresponds to the averaged current amplitudes, whereas the vertical bars represent the SEM.

Article Snippet: Conversely, neurite outgrowth was markedly facilitated in NGF-differentiated, TRPC5-defficient PC12 cells overexpressing an shRNA–TRPC5–GFP construct (OriGene, Rockville, MD; Supplemental Fig. 2A–2C) and reduced in NGFdifferentiated, TRPC1-defficient PC12 cells overexpressing shRNA–TRPC1–GFP (OriGene, Rockville, MD; Supplemental Fig. 2D).

Techniques: Expressing

Journal: Journal of cellular physiology

Article Title: Mechanisms controlling neurite outgrowth in a pheochromocytoma cell line: the role of TRPC channels.

doi: 10.1002/jcp.22855

Figure Lengend Snippet: Fig. 6. Receptor tyrosine kinase TrkA is not involved in the NGF- induced up-regulation of TRPC1 and TRPC6. A: RT-PCR analysis of TRPC1 (upper panel) and TRPC6 (lower panel) expression in NGF-treated PC12 cells in the absence or presence of 500 nM GW441756, a potent TrkA inhibitor. B: Comparisons of the relative expression levels of TRPC1 and TRPC6 (left, n U 6–7), and the total neurite length per cell (right, n U 276–290) in NGF treated PC12 cells in the absence or presence of 500 nM GW441756.

Article Snippet: Conversely, neurite outgrowth was markedly facilitated in NGF-differentiated, TRPC5-defficient PC12 cells overexpressing an shRNA–TRPC5–GFP construct (OriGene, Rockville, MD; Supplemental Fig. 2A–2C) and reduced in NGFdifferentiated, TRPC1-defficient PC12 cells overexpressing shRNA–TRPC1–GFP (OriGene, Rockville, MD; Supplemental Fig. 2D).

Techniques: Reverse Transcription Polymerase Chain Reaction, Expressing

Journal: Journal of cellular physiology

Article Title: Mechanisms controlling neurite outgrowth in a pheochromocytoma cell line: the role of TRPC channels.

doi: 10.1002/jcp.22855

Figure Lengend Snippet: Fig. 7. IKK2 inhibitors abolished NGF-mediated increases of TRPC1 and TRPC6 channel expression in NGF-treated PC12 cells. A: Two different IKK2 inhibitors, BMS (BMS-345541, 10 mM) and IMD (IMD-0354, 1 mM), potently inhibited neurite outgrowth in NGF-differentiated PC12 cells (n U 94–383). B: RT-PCR analysis of TRPC expression in the IKK2 inhibitor treated NGF-differentiated PC12 cells (upper and middle panel). The lower panel shows the effect of the tested IKK2 inhibitors on basal TRPC1 and TRPC5 expression levels in control PC12 cells. TRPC6 expression level was very low in control PC12 cells and was not significantly altered in IKK2 inhibitor experiments (see summary in (C)). Representative gels are shown. C: The summary of RT-PCR analysis of relative TRPC expression in IKK2 inhibitor treated groups. ‘‘NGF R BMS’’ stands for NGF-differentiated PC12 cells treated with (BMS-345541, 10 mM). ‘‘NGF R IMD’’ stands for NGF-differentiated PC12 cells treated with (IMD-0354, 1 mM). ‘‘C’’ denotes undifferentiated PC12 cells. ‘‘NGF’’ denotes NGF differentiated PC12 cells. Each experiment was repeated 4–13 times.

Article Snippet: Conversely, neurite outgrowth was markedly facilitated in NGF-differentiated, TRPC5-defficient PC12 cells overexpressing an shRNA–TRPC5–GFP construct (OriGene, Rockville, MD; Supplemental Fig. 2A–2C) and reduced in NGFdifferentiated, TRPC1-defficient PC12 cells overexpressing shRNA–TRPC1–GFP (OriGene, Rockville, MD; Supplemental Fig. 2D).

Techniques: Expressing, Reverse Transcription Polymerase Chain Reaction, Control

Journal: Journal of cellular physiology

Article Title: Mechanisms controlling neurite outgrowth in a pheochromocytoma cell line: the role of TRPC channels.

doi: 10.1002/jcp.22855

Figure Lengend Snippet: Fig. 8. The effect of overexpression of a constitutively active mutant of IKK2 (S177E/S181E, IKK2ca) on TRPC channel expression in PC12 cells. A: IKK2ca-overexpressing undifferentiated PC12 cells exhibited longer neurites than non-transfected cells at 48 h post-electroporation time. Shownisanoverlayofthefluorescenceandcorrespondingbrightfieldimages.TheYFPfluorescenceisshowninpseudo-color.B:Comparisonofthe total neurite length per cell in pcDNA3 and IKK2ca-overexpressing undifferentiated PC12 cells (n U 231–267). C and D: The RT-PCR analysis of TRPC1 and TRPC6 expression in PC12 cells expressing either pcDNA3 or IKK2ca. E, F, and G: The relative changes in the expression of TRPC channels induced by the overexpression of IKK2ca. Each experiment was repeated 10–12 times.

Article Snippet: Conversely, neurite outgrowth was markedly facilitated in NGF-differentiated, TRPC5-defficient PC12 cells overexpressing an shRNA–TRPC5–GFP construct (OriGene, Rockville, MD; Supplemental Fig. 2A–2C) and reduced in NGFdifferentiated, TRPC1-defficient PC12 cells overexpressing shRNA–TRPC1–GFP (OriGene, Rockville, MD; Supplemental Fig. 2D).

Techniques: Over Expression, Mutagenesis, Expressing, Transfection, Electroporation, Reverse Transcription Polymerase Chain Reaction

Journal: Nature communications

Article Title: Transcriptional and epigenetic rewiring by the NUP98::KDM5A fusion oncoprotein directly activates CDK12.

doi: 10.1038/s41467-025-59930-9

Figure Lengend Snippet: Fig. 7 | CDK12-controlled DNA repair is a vulnerability in NUP98::KDM5A-driven leukemia. A Cell viability assay of primary human NUP98-rearranged AML cells, healthy donor BM MNC and CD34+ progenitors treated with indicated con- centrations of THZ531 for 3 days (n = 3 technical replicates). B GI50 values from cell viability assays of murine AML cells treated with THZ531 for 3 days. C RT-qPCR analysis of dTAG-NUP98::KDM5A cells treated with THZ531 (2 µM, 24 h) showing log2FC values (n = 3 technical replicates). D Gene set enrichment analysis of RNA- seq data from a doxycycline-controlled NUP98::KDM5A cell line (Tet-Off) after 5 days of doxycycline-induced NUP98::KDM5A downregulation compared to DMSO. E Time series plot of Tet-Off RNA-seq data showing gene expression levels of DNA double strand break repair genes at day 3 and day 5 after NUP98::KDM5A downregulation normalized to NUP98::KDM5A-expressing cells, highlighting selected genes (left), with a density plot of normalized expression of all genes

Article Snippet: For the generation of the CDK12 wild type overexpression construct pCR8/GW_TOPO_Cdk12_Nterm_FlagHa (Addgene plasmid #127177) expressing the mouse Cdk12 transgene (NM_001109626.1 isoform) was cloned into pLX303-Gw (Addgene plasmid #25897) using Gateway LR Clonase II EnzymeMix (Invitrogen, USA) according the manufacturer’s protocol.

Techniques: Viability Assay, Quantitative RT-PCR, RNA Sequencing, Gene Expression, Expressing

Journal: MedComm

Article Title: Hemidesmosome Mutations Contribute to the Onset and Severity of Acquired Autoimmune Bullous Diseases

doi: 10.1002/mco2.70627

Figure Lengend Snippet: Pathogenic variants in hemidesmosome assembly‐related genes in pemphigoid disease spectrum decrease skin‐related protein expression. (A) Pattern diagram showing the location and pattern of pathogenic antibody deposition for different PD subtypes. (B) Representative immunohistochemistry images of BP, LigA, DH, and EBA mutation‐carrying and control patient samples stained for ITGA6 , LAMC2 , EPPK1 , and ITGB4 , n = 10, 14 for mutations and controls. Scale bar: 150 µm. (C) Quantitative analysis of IHC results for BP samples. (D) Quantitative analysis of IHC results for LigA samples, n = 11, 11 for mutations and controls. p‐ values are calculated using a two‐tailed Student's t ‐test. * p ≤ 0.05, ** p ≤ 0.01. Each sample is represented as one dot.

Article Snippet: For RNA sequencing, 1 × 10 6 cells were seeded into 12‐well plates and cultured for 24 h. Stable Ker‐CT cell lines overexpressing wild‐type (WT) ITGA6 or ITGA6 variants were generated by lentivirus‐mediated transduction and selected with hygromycin (50 μg/ml; HY‐K1051, MCE).

Techniques: Expressing, Immunohistochemistry, Mutagenesis, Control, Staining, Two Tailed Test

Journal: MedComm

Article Title: Hemidesmosome Mutations Contribute to the Onset and Severity of Acquired Autoimmune Bullous Diseases

doi: 10.1002/mco2.70627

Figure Lengend Snippet: Hemidesmosome‐related gene ITGA6 homologue ina‐1 mutation affects protein expression in C. elegans models. (A) Diagram of ina‐1 mutation pattern in C. elegans models and positions of residues mutated by CRISPR‐Cas technology. (B) Diagram of the lam‐2 mutation pattern in C. elegans models and positions of residues mutated by CRISPR‐Cas technology. (C) Representative images of homozygous ina‐1 wild‐type and mutation type embryos for CRISPR‐Cas knock‐in ina‐1 ::wrmScarlet and vab‐10a::GFP with the developmental process from L1, L2, L3, and L4 larvae to adult indicated on the left and right, respectively. Scale bar: 50 µm. Panels 1–4 show wrmScarlet, GFP, and merged signals for the turn of wild‐type C. elegans from L1–L4 larvae to adult (Y1 and Y2 are both adult locations), respectively. (D) Representative images of homozygous embryos for CRISPR‐Cas knock‐in lam‐2 ::wrmScarlet and vab‐10a::GFP with the developmental process from L1–L4 larvae to adult. Scale bar: 50 µm. Each sample is represented as one dot. (E) Quantitative analysis of confocal microscopy results for ina‐1 ::wrmScarlet C. elegans samples shows stable expression of fluorescence signals from L1–L4 larvae to adult. (F) Quantitative analysis of confocal microscopy results for lam‐2 ::wrmScarlet mutants shows stable expression of fluorescence signals from L1–L4 larvae to adults.

Article Snippet: For RNA sequencing, 1 × 10 6 cells were seeded into 12‐well plates and cultured for 24 h. Stable Ker‐CT cell lines overexpressing wild‐type (WT) ITGA6 or ITGA6 variants were generated by lentivirus‐mediated transduction and selected with hygromycin (50 μg/ml; HY‐K1051, MCE).

Techniques: Mutagenesis, Expressing, CRISPR, Knock-In, Confocal Microscopy, Fluorescence

Journal: MedComm

Article Title: Hemidesmosome Mutations Contribute to the Onset and Severity of Acquired Autoimmune Bullous Diseases

doi: 10.1002/mco2.70627

Figure Lengend Snippet: ITGA6 homologue ina‐1 mutation mediated the PD‐like phenotype in C. elegans models. (A) Components and structures of mammalian hemidesmosome type in the skin of PD patients. (B) Components and structures of CeHDs in epidermis adjacent to body‐wall muscles. (C) Transmission electron microscopy (TEM) results revealed that hemidesmosome structures were destroyed in patients with different PD forms ( n = 6, including BP = 3, DH = 2, LigA = 1) and healthy controls ( n = 3). Desmosome structures were normal and served as controls. Black arrows indicate abnormal hemidesmosome structures and normal desmosome structures. Pat 2 represents high magnifications of the region highlighted by dashed rectangles in BP1, BP2, BP3, DH1, DH2, and LigA1. Scale bar: 500 nm. (D) Representative image of ina‐1 wild‐type (WT) shows clear epidermal and muscle junction structure border and normal cuticle basal layer in C. elegans . Scale bar: 1 µm. Scale bar: 500 nm. (E) Epidermal and muscle structure border and basal cuticle layer were basically normal with mild separation phenomena in the ina‐1 type (MUT1). Scale bar: 1 µm; 500 nm.

Article Snippet: For RNA sequencing, 1 × 10 6 cells were seeded into 12‐well plates and cultured for 24 h. Stable Ker‐CT cell lines overexpressing wild‐type (WT) ITGA6 or ITGA6 variants were generated by lentivirus‐mediated transduction and selected with hygromycin (50 μg/ml; HY‐K1051, MCE).

Techniques: Mutagenesis, Muscles, Transmission Assay, Electron Microscopy

Journal: Immunity

Article Title: Adoptive T Cell Therapy Targeting Different Gene Products Reveals Diverse and Context-Dependent Immune Evasion in Melanoma.

doi: 10.1016/j.immuni.2020.07.007

Figure Lengend Snippet: Figure 4. Diverse Mechanisms of Antigen Loss in esACT-Recurrent Melanomas (A–C) TYRP1-NFhgp100 (A), RAB38-NFhgp100 (B), and CDK4R24C-NFhgp100 (C) mRNA expression by RNA-seq (log2 fold change) in indicated non-treated (NT) and esACT-recurrent HC.Pmel/ melanomas. Additional depiction of complete regression status (minimal tumor area % 1 mm2), presence, and mutations status of CRISPitope cassette (NFhgp100 tag) in genomic DNA and mNeon expression assessed by flow cytometry. Statistics: ***FDR < 0.001; *FDR < 0.05, n.s.: not significant; unpaired two-sided t test. (D) Histograms exemplarily showing mNeon expression by flow cytometry in non-treated (NT) and recurrent (R) HC.Pmel/.TYRP1-NFhgp100 melanomas. (E) Western blot analysis of TYRP1, FLAG, MITF, and b-Actin from non-treated (NT) and recurrent (R) HC.Pmel/.TYRP1-NFhgp100 melanomas with antigen loss (R1-4, R8). (F and G) Histograms exemplarily showing mNeon expression by flow cytometry in non-treated (NT) and recurrent (R) HC.Pmel/.RAB38-NFhgp100 (F) and HC.Pmel/.CDK4R24C-NFhgp100 (G) melanomas. See also Figure S4.

Article Snippet: After 1 h of blocking with 5% bovine serum albumin (GE Healthcare) in TBS with 0.5% Tween-20, membranes were incubated with primary antibodies overnight at 4 C. Blots were immunostained with b-Actin (C4, Santa Cruz sc-47778, 1:2,000, mouse mAb), CDK4 (DCS-35, Santa Cruz sc-23896, 1:100, mouse mAb), FLAG (L5, Novus Biologicals NBP2-06712, 1:500, rat mAb), MITF (HPA003259, Atlas Antibodies, Sigma-Aldrich, 1:250; rabbit poAb), RAB38 (A-8, Santa Cruz sc-390176, 1:100, mouse mAb) and TRP1 (G-9, Santa Cruz sc-166857, 1:100, mouse mAb).

Techniques: Expressing, RNA Sequencing, Cytometry, Western Blot

Journal: CNS Neuroscience & Therapeutics

Article Title: LncRNA INPP5F ameliorates stress‐induced hypertension via the miR‐335/Cttn axis in rostral ventrolateral medulla

doi: 10.1111/cns.14142

Figure Lengend Snippet: Primers used in quantitative reverse transcription polymerase chain reaction (qRT‐PCR) analysis.

Article Snippet: The lambda and bregma skull points were laid in the same horizontal plane. pLV‐EF1A > lncRNA INPP5F (pLV‐lncRNA INPP5F, lentiviral vector‐mediated lncRNA INPP5F overexpression, >10 9 TU/mL) and control pLV‐EGFP:T2A:Puro‐EF1A > mCherry (pLV‐NC, >10 9 TU/mL) plasmids were synthesized by VectorBuilder (China).

Techniques: Reverse Transcription Polymerase Chain Reaction, Sequencing

Journal: CNS Neuroscience & Therapeutics

Article Title: LncRNA INPP5F ameliorates stress‐induced hypertension via the miR‐335/Cttn axis in rostral ventrolateral medulla

doi: 10.1111/cns.14142

Figure Lengend Snippet: LncRNA INPP5F was downexpressed in RVLM after 15 days of stress exposure in rats. (A) Chronic stress increased the levels of SBP, MAP, and HR in rats. (B) Cluster analysis of the top five most increased and decreased lncRNAs identified by RNA sequencing in the control and SIH rats. (C) Relative expression of the four indicated lncRNAs listed in (B) measured by qRT‐PCR. (D) Relative expression of lncRNA INPP5F in 11 tissues of SIH rats, as determined by qRT‐PCR. (E) Bioinformatics prediction of the subcellular location of lncRNA INPP5F by lncLocator and iLoc‐LncRNA software. (F) Subcellular localization of lncRNA INPP5F in B104 cells using FISH. NC means Cy3‐labeled negative control probe. (G) qRT‐PCR was used to measure the expression of lncRNA INPP5F in either the nucleus or cytoplasm. Data were presented as mean ± SEM. Statistical significance was determined by two‐tailed unpaired Student's t ‐test (A, C) and one‐way ANOVA, followed by post hoc Bonferroni test (D). n = 6 rats per group (A, C, D). n = 6 of independent cell culture preparations (G). * p < 0.05, ** p < 0.01, and *** p < 0.001 versus control group. ### p < 0.001 versus heart group. DAPI, 4′,6‐diamidino‐2‐phenylindole; FISH, fluorescence in situ hybridization; HR, heart rate; MAP, mean arterial pressure; NC, negative control; qRT‐PCR, quantitative reverse transcription polymerase chain reaction; RVLM, rostral ventrolateral medulla; SBP, systolic blood pressure; SEM, standard error of the mean; SIH, stress‐induced hypertension.

Article Snippet: The lambda and bregma skull points were laid in the same horizontal plane. pLV‐EF1A > lncRNA INPP5F (pLV‐lncRNA INPP5F, lentiviral vector‐mediated lncRNA INPP5F overexpression, >10 9 TU/mL) and control pLV‐EGFP:T2A:Puro‐EF1A > mCherry (pLV‐NC, >10 9 TU/mL) plasmids were synthesized by VectorBuilder (China).

Techniques: RNA Sequencing Assay, Expressing, Quantitative RT-PCR, Software, Labeling, Negative Control, Two Tailed Test, Cell Culture, Fluorescence, In Situ Hybridization, Reverse Transcription Polymerase Chain Reaction

Journal: CNS Neuroscience & Therapeutics

Article Title: LncRNA INPP5F ameliorates stress‐induced hypertension via the miR‐335/Cttn axis in rostral ventrolateral medulla

doi: 10.1111/cns.14142

Figure Lengend Snippet: Upregulated lncRNA INPP5F expression in RVLM contributed to improve SIH. (A) Tail‐cuff method was employed to record SBP, MAP, and HR in rats after lncRNA INPP5F overexpression. (B, C) RSNA recording and plasma NE ELISA test of SIH rats microinjected with pLV‐lncRNA INPP5F to evaluate sympathetic nerve activity. (D) Representative immunofluorescent image of co‐localization of c‐fos with TH and percentage of c‐Fos‐positive TH+ neurons in RVLM. Data were presented as mean ± SEM. Statistical significance was determined by one‐way ANOVA, followed by post hoc Bonferroni test (A–D). n = 6 rats per group (A–C). n = 12 slices from 6 rats, two slices per rat (D). * p < 0.05, ** p < 0.01, and *** p < 0.001 versus SIH group. ns means non‐significant versus SIH group. HR, heart rate; MAP, mean arterial pressure; NC, negative control; NE, norepinephrine; RVLM, rostral ventrolateral medulla; RSNA, renal sympathetic nerve activity; SBP, systolic blood pressure; SEM, standard error of the mean; SIH, stress‐induced hypertension; TH, tyrosine hydroxylase.

Article Snippet: The lambda and bregma skull points were laid in the same horizontal plane. pLV‐EF1A > lncRNA INPP5F (pLV‐lncRNA INPP5F, lentiviral vector‐mediated lncRNA INPP5F overexpression, >10 9 TU/mL) and control pLV‐EGFP:T2A:Puro‐EF1A > mCherry (pLV‐NC, >10 9 TU/mL) plasmids were synthesized by VectorBuilder (China).

Techniques: Expressing, Over Expression, Enzyme-linked Immunosorbent Assay, Activity Assay, Negative Control

Journal: CNS Neuroscience & Therapeutics

Article Title: LncRNA INPP5F ameliorates stress‐induced hypertension via the miR‐335/Cttn axis in rostral ventrolateral medulla

doi: 10.1111/cns.14142

Figure Lengend Snippet: LncRNA INPP5F attenuated apoptosis in SIH via activating Cttn/PI3K‐AKT signal pathway. (A) Western blot analysis of Cttn, p‐PI3K, and p‐AKT expression in RVLM of SIH and control rats. (B–D) After microinjection of pLV‐lncRNA INPP5F plasmid in RVLM of SIH rats, Cttn, p‐PI3K, p‐AKT, BCL2, BAX, and cleaved Caspase 3 protein levels were detected by Western blot, cleaved Caspase 3 protein was determined by immunofluorescence, and apoptosis level was tested by TUNEL staining. Data were presented as mean ± SEM. Statistical significance was determined by two‐tailed unpaired Student's t ‐test (A) and one‐way ANOVA, followed by post hoc Bonferroni test (B–D). n = 3 rats per group (A, B). n = 12 slices from 6 rats, two slices per rat (C, D). * p < 0.05, ** p < 0.01, and *** p < 0.001 versus SIH group. ns means non‐significant versus SIH group. DAPI, 4′,6‐diamidino‐2‐phenylindole; NC, negative control; RVLM, rostral ventrolateral medulla; SEM, standard error of the mean; SIH, stress‐induced hypertension; TUNEL, TdT‐mediated dUTP‐biotin nick end labeling.

Article Snippet: The lambda and bregma skull points were laid in the same horizontal plane. pLV‐EF1A > lncRNA INPP5F (pLV‐lncRNA INPP5F, lentiviral vector‐mediated lncRNA INPP5F overexpression, >10 9 TU/mL) and control pLV‐EGFP:T2A:Puro‐EF1A > mCherry (pLV‐NC, >10 9 TU/mL) plasmids were synthesized by VectorBuilder (China).

Techniques: Western Blot, Expressing, Plasmid Preparation, Immunofluorescence, TUNEL Assay, Staining, Two Tailed Test, Negative Control, End Labeling

Journal: CNS Neuroscience & Therapeutics

Article Title: LncRNA INPP5F ameliorates stress‐induced hypertension via the miR‐335/Cttn axis in rostral ventrolateral medulla

doi: 10.1111/cns.14142

Figure Lengend Snippet: LncRNA INPP5F knockdown induced apoptosis by regulating Cttn/PI3K‐AKT signal pathway in B104 cells. (A–C) After transfection of lncRNA INPP5F ASO for 48 h in B104 cells, Cttn, p‐PI3K, p‐AKT, BCL2, BAX, and cleaved Caspase 3 protein levels were assessed by Western blot, cleaved Caspase 3 protein was explored by immunofluorescence, and apoptosis level was investigated by TUNEL staining. (D) Cell viability was surveyed by Calcein/PI staining in B104 cells after transfection of lncRNA INPP5F ASO for 48 h. (E) CCK‐8 assay for examining cell viability in B104 cells after transfection of lncRNA INPP5F ASO for 24, 48, and 72 h. (F) Apoptosis rate was evaluated by flow cytometry in B104 cells after transfection of lncRNA INPP5F ASO for 48 h. Data were presented as mean ± SEM. Statistical significance was determined by two‐tailed unpaired Student's t ‐test (A–F). n = 3 of independent cell culture preparations (A). n = 6 of independent cell culture preparations (E, F). n = 12 slices from 6 of independent cell culture preparations, 2 slices per independent cell culture preparation (B–D). # p < 0.05, ## p < 0.01, and ### p < 0.001 versus ASO NC group. ASO, antisense oligonucleotide; CCK‐8, cell counting kit‐8; DAPI, 4′,6‐diamidino‐2‐phenylindole; NC, negative control; PI, propidium iodide; SEM, standard error of the mean; TUNEL, TdT‐mediated dUTP‐biotin nick end labeling.

Article Snippet: The lambda and bregma skull points were laid in the same horizontal plane. pLV‐EF1A > lncRNA INPP5F (pLV‐lncRNA INPP5F, lentiviral vector‐mediated lncRNA INPP5F overexpression, >10 9 TU/mL) and control pLV‐EGFP:T2A:Puro‐EF1A > mCherry (pLV‐NC, >10 9 TU/mL) plasmids were synthesized by VectorBuilder (China).

Techniques: Transfection, Western Blot, Immunofluorescence, TUNEL Assay, Staining, CCK-8 Assay, Flow Cytometry, Two Tailed Test, Cell Culture, Cell Counting, Negative Control, End Labeling

Journal: CNS Neuroscience & Therapeutics

Article Title: LncRNA INPP5F ameliorates stress‐induced hypertension via the miR‐335/Cttn axis in rostral ventrolateral medulla

doi: 10.1111/cns.14142

Figure Lengend Snippet: LncRNA INPP5F interacted with and negatively regulated miR‐335. (A) qRT‐PCR analysis of miR‐335 expression in RVLM of SIH and control rats. (B) Schematic of lncRNA INPP5F‐WT and lncRNA INPP5F‐MUT binding sites for miR‐335. (C) Biotinylated miR‐335 transfected into B104 cells with lncRNA INPP5F overexpression. LncRNA INPP5F expression was examined by qRT‐PCR after streptavidin capture. (D) Luciferase assay of B104 cells co‐transfected with lncRNA INPP5F‐WT reporter or lncRNA INPP5F‐MUT reporter and miR‐335 agomir or agomir NC. (E, F) qRT‐PCR analysis of miR‐335 expression in B104 cells with lncRNA INPP5F overexpression or silencing. (G) MiR‐335 expression was evaluated using qRT‐PCR after microinjection of pLV‐lncRNA INPP5F plasmid in RVLM of SIH rats. Data were presented as mean ± SEM. Statistical significance was determined by two‐tailed unpaired Student's t ‐test (A, C–F) and one‐way ANOVA, followed by post hoc Bonferroni test (G). n = 6 rats per group (A, G). n = 6 of independent cell culture preparations (C–F). ** p < 0.01, ns means non‐significant versus SIH group. ### p < 0.001, ns means non‐significant versus agomir NC group. ΔΔ p < 0.01 versus pLV‐NC group. && p < 0.01 versus ASO NC group. ASO, antisense oligonucleotide; MUT, mutant; NC, negative control; qRT‐PCR, quantitative reverse transcription polymerase chain reaction; RVLM, rostral ventrolateral medulla; SEM, standard error of the mean; SIH, stress‐induced hypertension; WT, wild type.

Article Snippet: The lambda and bregma skull points were laid in the same horizontal plane. pLV‐EF1A > lncRNA INPP5F (pLV‐lncRNA INPP5F, lentiviral vector‐mediated lncRNA INPP5F overexpression, >10 9 TU/mL) and control pLV‐EGFP:T2A:Puro‐EF1A > mCherry (pLV‐NC, >10 9 TU/mL) plasmids were synthesized by VectorBuilder (China).

Techniques: Quantitative RT-PCR, Expressing, Binding Assay, Transfection, Over Expression, Luciferase, Plasmid Preparation, Two Tailed Test, Cell Culture, Mutagenesis, Negative Control, Reverse Transcription Polymerase Chain Reaction

Journal: CNS Neuroscience & Therapeutics

Article Title: LncRNA INPP5F ameliorates stress‐induced hypertension via the miR‐335/Cttn axis in rostral ventrolateral medulla

doi: 10.1111/cns.14142

Figure Lengend Snippet: LncRNA INPP5F modulated Cttn expression by absorbing miR‐335. (A–D) qRT‐PCR and Western blot analyses of the effects of lncRNA INPP5F overexpression and miR‐335 agomir or lncRNA INPP5F silencing and miR‐335 antagomir on the mRNA and protein levels of Cttn in B104 cells. (E, F) The mRNA and protein expression levels of Cttn were detected by qRT‐PCR and Western blot in SIH rats after microinjection of pLV‐lncRNA INPP5F plasmid and miR‐335 agomir. Data were presented as mean ± SEM. Statistical significance was determined by one‐way ANOVA, followed by post hoc Bonferroni test (A–F). n = 6 of independent cell culture preparations (A, C). n = 3 of independent cell culture preparations (B, D). n = 6 rats per group (E). n = 3 rats per group (F). # p < 0.05, ### p < 0.001, ns means non‐significant versus pLV‐NC group. ΔΔ p < 0.01, ns means non‐significant versus ASO NC group. * p < 0.05, *** p < 0.001, ns means non‐significant versus SIH+pLV‐NC group. ASO, antisense oligonucleotide; NC, negative control; qRT‐PCR, quantitative reverse transcription polymerase chain reaction; SEM, standard error of the mean; SIH, stress‐induced hypertension.

Article Snippet: The lambda and bregma skull points were laid in the same horizontal plane. pLV‐EF1A > lncRNA INPP5F (pLV‐lncRNA INPP5F, lentiviral vector‐mediated lncRNA INPP5F overexpression, >10 9 TU/mL) and control pLV‐EGFP:T2A:Puro‐EF1A > mCherry (pLV‐NC, >10 9 TU/mL) plasmids were synthesized by VectorBuilder (China).

Techniques: Expressing, Quantitative RT-PCR, Western Blot, Over Expression, Plasmid Preparation, Cell Culture, Negative Control, Reverse Transcription Polymerase Chain Reaction