Journal: Nature Communications

Article Title: FTO-dependent m 6 A modification of Plpp3 in circSCMH1-regulated vascular repair and functional recovery following stroke

doi: 10.1038/s41467-023-36008-y

Figure Lengend Snippet: a Total RNA was extracted from the peri-infarct cortex of PT mice, m 6 A levels were determined as the percentage of all adenosine residues in RNA. n = 6 mice/group. ** P = 0.0072 (28d), *** P < 0.0001 (3d, 14d) versus the sham; ## P = 0.0056 (3d), ## P = 0.0097 (14d), ## P = 0.0024 (28d) versus the PT + EV-Vector. b Total RNA was extracted from the primary mouse brain microvascular ECs treated with circSCMH1 plasmid at 12 h after OGD, and m 6 A levels were determined as the percentage of all adenosine residues in RNA. Data were presented by three independent experiments. * P = 0.0231 versus Con+Vector; ## P = 0.0051 versus OGD + Vector. c Interaction between circSCMH1 and FTO was detected by RNA-binding immunoprecipitation in the primary brain microvascular ECs. Data were presented by three independent experiments. *** P < 0.0001 versus FTO pull-down of circHECW2; ### P < 0.0001 versus FTO pull-down of Gapdh mRNA. d Interaction between circSCMH1 and FTO was measured by RNA pull-down assay in the primary mouse brain microvascular ECs. Data were presented by three independent experiments. ** P = 0.0085 versus the circCon probe. e Prediction of circSCMH1-FTO interaction by catRAPID algorithm. f The interaction between circSCMH1 and FTO was validated by RNA immunoprecipitation in bEnd.3 cells with WT FTO and mutant FTO. Data were presented by three independent experiments. ** P = 0.0022 versus circSCMH1 in FTO-WT. g Western blot analysis of FTO expression in lysates of bEnd.3 cells with circSCMH1 or mutated circSCMH1 (Δ426–477) overexpression following biotinylated circSCMH1 probe pull-down assay. Data were presented by three independent experiments. ** P = 0.0069 versus WT. The data in a , c , f were expressed as mean ± SEM; one-way ANOVA followed by Holm–Sidak post hoc multiple comparison test. The data in b were expressed as mean ± SEM; two-way ANOVA followed by Bonferroni’s post hoc multiple comparison tests. The data in d and g were expressed as mean ± SEM; using the Student t -test (two-sided). Source data are provided as a Source Data file. Con control, d day, IgG immunoglobulin G, IP immunoprecipitation, WT wild type. Δ151–202: lacking region 151 to 202 amino acids; Δ351–402: lacking region 351 to 402 amino acids; Δ426–477: lacking region 426 to 477 amino acids or nucleic acids.

Article Snippet: Primary human brain microvascular endothelial cells were purchased from iCell Bioscience (HUM-icell-n001, iCell Bioscience) and cultured in 5% CO 2 at 37 °C in primary endothelial cell basal medium (PriMed-iCell-002, iCell Bioscience).

Techniques: Plasmid Preparation, RNA Binding Assay, Immunoprecipitation, Pull Down Assay, RNA Immunoprecipitation, Mutagenesis, Western Blot, Expressing, Over Expression, Comparison, Control

Journal: Nature Communications

Article Title: FTO-dependent m 6 A modification of Plpp3 in circSCMH1-regulated vascular repair and functional recovery following stroke

doi: 10.1038/s41467-023-36008-y

Figure Lengend Snippet: a , b FTO expression in peri-infarct tissue’s cytoplasm ( a ) and nucleus ( b ) at day 28 after PT. Three representative immunoblots were presented from 6 mice/group. *** P < 0.0001 ( a , b ) versus sham, ### P < 0.0001 ( a ), ### P = 0.0008 ( b ) versus PT + EV-Vector. c , d Representative western blotting of FTO expression in the cytoplasm ( c ) and nucleus ( d ) of the primary mouse brain microvascular ECs at 12 h after OGD. Data were presented by three independent experiments. ** P = 0.0012 ( c ), ** P = 0.0029 ( d ) versus Con+Vector; # P = 0.0235, ( d ) ## P = 0.0039 ( c ) versus OGD + Vector. e Immunoprecipitation detected Ub-K63 modification of FTO in primary mouse brain microvascular ECs at 12 h after OGD. Data were representative of three independent experiments. f Interaction between circSCMH1 and UBC13 was detected by RNA-binding immunoprecipitation in bEnd.3 cells. Data were presented by three independent experiments. ** P = 0.0076 versus UBC13 pull-down of circHECW2; ## P = 0.0011 versus UBC13 pull-down of Gapdh mRNA. g Immunoprecipitation showed the binding of FTO with UBC13 in bEnd.3 cells. Data were presented by three independent experiments. * P = 0.0210 versus vector. h The bEnd.3 cells were transfected with siUBC13, the level of Ubc13 mRNA was measured by qPCR. Data were presented by three independent experiments. ** P = 0.0020 versus siCon. i , j Western blot analysis of FTO expression in the cytoplasm ( i ) and nucleus ( j ) of bEnd.3 cells with LV-circSCMH1 and siUBC13 at 12 h after OGD. Data were presented by three independent experiments. *** P = 0.0004 ( i ), *** P = 0.0007 ( j ) versus Con+LV-Vector+siCon; ## P = 0.0073 ( i ), ## P = 0.0019 ( j ) versus OGD + LV-Vector + siCon; † P = 0.0184 ( j ), †† P = 0.0018 ( i ) versus OGD + LV-circSCMH1+siCon. k Proposed model of the regulatory role of circSCMH1 and UBC13 for FTO translocation into the nucleus. The data in a , b , f , i , j were expressed as mean ± SEM; one-way ANOVA followed by Holm–Sidak post hoc multiple comparison test. The data in c , d were expressed as mean ± SEM; two-way ANOVA followed by Bonferroni’s post hoc multiple comparison tests. The data in g , h were expressed as mean ± SEM; using the Student t -test (two-sided). Source data are provided as a Source Data file. Con control, IB immunoblot, siUBC13 Ubc13 siRNA, Ub-K63 lysine 63-linked ubiquitination.

Article Snippet: Primary human brain microvascular endothelial cells were purchased from iCell Bioscience (HUM-icell-n001, iCell Bioscience) and cultured in 5% CO 2 at 37 °C in primary endothelial cell basal medium (PriMed-iCell-002, iCell Bioscience).

Techniques: Expressing, Western Blot, Plasmid Preparation, Immunoprecipitation, Modification, RNA Binding Assay, Binding Assay, Transfection, Translocation Assay, Comparison, Control, Ubiquitin Proteomics

Journal: Nature Communications

Article Title: FTO-dependent m 6 A modification of Plpp3 in circSCMH1-regulated vascular repair and functional recovery following stroke

doi: 10.1038/s41467-023-36008-y

Figure Lengend Snippet: a Distribution of m 6 A peaks across 5′-UTR, CDS, and 3′-UTR of mRNA at day 14 after PT. b Venn diagram showing numbers of genes with significant changes in expression (up: fold change ≥ 2, P < 0.05; down: fold change ≤ 0.5, P < 0.05, rescaled hypergeometric test). c The m 6 A level of Plpp3 transcript was regulated in PT mice after EV-circSCMH1 administration. d , e Effect of EV-circSCMH1 on Plpp3 mRNA ( d ) and LPP3 ( e ) levels in mice at day 14 after PT. n = 6 mice/group. Three representative immunoblots were presented from 6 mice/group. *** P < 0.0001 ( d ), *** P = 0.0003 ( e ) versus sham; ## P = 0.0030 ( e ), ### P < 0.0001 ( d ) versus PT + EV-Vector. f , g Effect of circSCMH1 plasmid on the expression of Plpp3 mRNA ( f ) and LPP3 ( g ) in primary mouse brain microvascular ECs after OGD. Data were presented by three independent experiments. * P = 0.0171 ( g ), *** P < 0.0001 ( f ) versus Con + Vector; # P = 0.0375 ( g ), ### P = 0.0003 ( f ) versus OGD + Vector. h Specific primers against m 6 A peak were designed to amplify m 6 A peak of Plpp3 transcript in RNA from the peri-infarct cortex of mice at day 14 after PT. n = 6/each group. *** P < 0.0001 versus the sham; ### P < 0.0001 versus the PT + EV-Vector. i Specific primers against the m 6 A peak were designed to amplify the m 6 A peak of Plpp3 transcript in bEnd.3 cells. Data were presented by three independent experiments. *** P = 0.0002 versus Con + Vector; ### P = 0.0007 versus OGD + Vector. j , k The bEnd.3 cells were transfected with LV-circSCMH1 and shFTO. The Plpp3 mRNA ( j ) and LPP3 ( k ) was detected at 12 h after OGD. Data were presented by 4 ( j ) or 3 ( k ) independent experiments. ** P = 0.0020 ( k ), *** P = 0.0003 ( j ) versus Con + LV-Vector + shCon; ## P = 0.0012 ( j ), ## P = 0.0087 ( k ) versus OGD + LV-Vector + shCon; † P = 0.0408 ( k ), †† P = 0.0083 ( j ) versus OGD + LV-circSCMH1 + shCon. The data in d , e , h , j , k were expressed as mean ± SEM; one-way ANOVA followed by Holm–Sidak post hoc multiple comparison test. The data in f , g , i were expressed as mean ± SEM; two-way ANOVA followed by Bonferroni’s post hoc multiple comparison tests. Source data are provided as a Source Data file. CDS coding region, shRNA short hairpin RNA, 3′ UTR 3′ untranslated regions, 5′ UTR 5′ untranslated regions.

Article Snippet: Primary human brain microvascular endothelial cells were purchased from iCell Bioscience (HUM-icell-n001, iCell Bioscience) and cultured in 5% CO 2 at 37 °C in primary endothelial cell basal medium (PriMed-iCell-002, iCell Bioscience).

Techniques: Expressing, Western Blot, Plasmid Preparation, Transfection, Comparison, shRNA

Journal: Frontiers in Cellular and Infection Microbiology

Article Title: Hepatitis E Virus Induces Brain Injury Probably Associated With Mitochondrial Apoptosis

doi: 10.3389/fcimb.2019.00433

Figure Lengend Snippet: Mitochondrial dysfunction was related to an increased level of NOX4 and decreased expression of ATP5A1 in HEV infected brain tissues. (A,B) in vitro , HBMVECs were inoculated with 300 MOI HEV for 48 h for western blot, and HEV-negative homogenate served as control. Data showed that expression of NOX4 in HBMVECs treated with HEV for 48 h was significantly increased compared with mock group ( * p < 0.05). Meanwhile, ATP5A1 was detected significantly attenuated in HEV inoculated cells ( ** p < 0.01). (C-H) In vivo , brain and spinal cord tissues that detected for HEV-RNA positive were selected for ultrastructural study. (C–E) Mitochondria were observed with clear cristae folded by the inner membrane in brain tissue of mock group. (F–H) Mitochondria with loss of cristae were found in tissues of HEV infected animals (arrows). The rough endoplasmic reticulum of neuron cells was also observed with mild distended cisternal space in HEV infected tissues (arrowhead). For western blot, gray value was analyzed with ImageJ to quantitatively analyze the expression levels of targeted proteins according to the level of exposure gray (resolution). Data were finally normalized to the expression of anti-β-actin.

Article Snippet: Primary human brain microvascular endothelial cells (HBMVECs) (BK-PM-010) were purchased from a company (Biopike Technology Company Ltd., Beijing, China) and cultured as described previously (Renou et al., ).

Techniques: Expressing, Infection, In Vitro, Western Blot, Control, In Vivo, Membrane

Journal: Frontiers in Cellular and Infection Microbiology

Article Title: Hepatitis E Virus Induces Brain Injury Probably Associated With Mitochondrial Apoptosis

doi: 10.3389/fcimb.2019.00433

Figure Lengend Snippet: Pro-apoptotic protein Bax but not Bcl-2 was upregulated following HEV infection. (A–D) HEV-RNA positive brain tissues collected on 14, 21, and 28 dpi were used for the immunohistochemistry study of Bax (rabbit polyclonal IgG) and Bcl-2(rabbit polyclonal IgG). Goat anti-rabbit IgG was chosen as secondary antibody. The positive signal was measured via the Motic Med 6.0 CMIAS Image Analysis System. Data showed that Bax was mainly distributed in cytosol of neuron cells, vascular endothelial cells and few microglial cells of HEV infection tissues with increased amount compared with mock group (* p < 0.05). Bcl-2 was detected in few neurons and vascular endothelial cells in both groups. (E) For western blot, HBMVECs were inoculated with 300 MOI HEV for 48 h and HEV-negative homogenate served as control. Data showed that expression level of Bax was significantly higher in HBMVECs inoculated with HEV (** p < 0.01), but induction of Bcl-2 was not significant. For western blot, gray value was analyzed with ImageJ to quantitatively analyze the expression levels of targeted proteins according to the level of exposure gray (resolution). Data were finally normalized to the expression of anti-β-actin.

Article Snippet: Primary human brain microvascular endothelial cells (HBMVECs) (BK-PM-010) were purchased from a company (Biopike Technology Company Ltd., Beijing, China) and cultured as described previously (Renou et al., ).

Techniques: Infection, Immunohistochemistry, Western Blot, Control, Expressing

Journal: Frontiers in Cellular and Infection Microbiology

Article Title: Hepatitis E Virus Induces Brain Injury Probably Associated With Mitochondrial Apoptosis

doi: 10.3389/fcimb.2019.00433

Figure Lengend Snippet: Mitochondrial apoptotic signaling was activated during HEV infection. HEV-RNA positive brain tissues collected on 14, 21, and 28 dpi were used for the immunohistochemistry study of caspase-9 (rabbit polyclonal IgG), caspase-3 (rabbit polyclonal IgG) and PCNA (rabbit polyclonal IgG). Goat anti-rabbit IgG was chosen as secondary antibody. The positive signal was measured via the Motic Med 6.0 CMIAS Image Analysis System. (A–D) Immunohistochemistry study showed that expression levels of activated caspase-9 and caspase-3 were expressed in neuronal cells and vascular endothelial cells of the brain tissue, with higher amount in HEV infected animals compared with mock animals (* p < 0.05). (E,F) For western blot, HBMVECs were inoculated with 300 MOI HEV for 48 h and HEV-negative homogenate served as control. Data showed that expression levels of cleaved caspase-9 and caspase-3 were significantly increased in HBMVECs infected with HEV compared with mock group (** p < 0.01). (G,H) Positive signal of PCNA was detected in glial cells and vascular endothelial cells, with significantly higher level in HEV infected brain sections compared with mock group (** p < 0.01). For western blot, gray value was analyzed with ImageJ to quantitatively analyze the expression levels of targeted proteins according to the level of exposure gray (resolution). Data were finally normalized to the expression of anti-β-actin.

Article Snippet: Primary human brain microvascular endothelial cells (HBMVECs) (BK-PM-010) were purchased from a company (Biopike Technology Company Ltd., Beijing, China) and cultured as described previously (Renou et al., ).

Techniques: Infection, Immunohistochemistry, Expressing, Western Blot, Control