Journal: Journal of Neuroinflammation

Article Title: Retention of normal glia function by an isoform-selective protein kinase inhibitor drug candidate that modulates cytokine production and cognitive outcomes

doi: 10.1186/s12974-017-0845-2

Figure Lengend Snippet: Selective attenuation of proinflammatory cytokines by MW150 administration in APP/PS1 KI mice cortex. a 11–12-month-old wild type ( WT ) or APP/PS1 knock-in ( KI ) mice were treated with saline vehicle ( veh ) or 2.5 mg/kg MW150 by intraperitoneal injection (i.p.) once daily for 14 days. b IL-1β was increased in KI + veh mice compared to WT + veh mice ( p = 0.0012), and MW150 treatment of KI mice (KI + MW150) significantly attenuated IL-1β levels compared to KI + veh ( p = 0.0243) ( F (2,38) = 6.46; p = 0.004). c TNFα was elevated in KI + veh mice compared to WT + veh and attenuated in KI + MW150 mice compared to KI + veh; however, these changes were not significant ( F (2,38) = 1.11; p = 0.34). d IL-6 was slightly elevated in KI + veh compared to WT + veh mice, and there was no effect of MW150 treatment ( n = 11 WT + veh; n = 14 KI + veh; n = 14 KI + MW150). Data are mean ± SEM. Source data is available in Additional file : Table S1

Article Snippet: Fig. 6 Effect of MW150 treatment on microglia closely associated with Aβ plaques and microglia-internalized Aβ in the cortex of APP/PS1 KI mice. a Representative images of Imaris 3D reconstruction of plaques.

Techniques: Knock-In, Saline, Injection

Journal: Journal of Neuroinflammation

Article Title: Retention of normal glia function by an isoform-selective protein kinase inhibitor drug candidate that modulates cytokine production and cognitive outcomes

doi: 10.1186/s12974-017-0845-2

Figure Lengend Snippet: No effect of MW150 on GFAP immunostaining. a Representative images of GFAP immunohistochemical (IHC) staining in cortex of WT or APP/PS1 KI mice treated with vehicle ( veh ) or MW150. b Digital quantification of GFAP in the cortex was done using the Aperio ScanScope with the entire cortex used as the region of interest. Quantification using the positive pixel algorithm showed a significant increase in GFAP staining in the KI + veh compared to WT + veh ( p < 0.0001). No significant difference was found between the KI + veh compared to the KI + MW150. (F2,41) = 34.66; p < 0.0001). ( n = 14 WT + veh; n = 14 KI + veh; n = 14 KI + MW150). Data are mean ± SEM. Source data is available in Additional file : Table S2

Article Snippet: Fig. 6 Effect of MW150 treatment on microglia closely associated with Aβ plaques and microglia-internalized Aβ in the cortex of APP/PS1 KI mice. a Representative images of Imaris 3D reconstruction of plaques.

Techniques: Immunostaining, Immunohistochemical staining, Immunohistochemistry, Staining

Journal: Journal of Neuroinflammation

Article Title: Retention of normal glia function by an isoform-selective protein kinase inhibitor drug candidate that modulates cytokine production and cognitive outcomes

doi: 10.1186/s12974-017-0845-2

Figure Lengend Snippet: No effect of MW150 on IBA1 + microglia volume. a Representative 3D surface reconstructions of IBA1 + cells generated from confocal microscopic imaging using Imaris software. b Mean volume ± standard deviation (SD) of rendered IBA1 cells is shown for the APP/PS1 KI + veh and APP/PS1 KI + MW150 groups. Data represent mean of 3–4 independent z-stacks from each mouse. ( n = 11 KI + veh; n = 14 KI + MW150)

Article Snippet: Fig. 6 Effect of MW150 treatment on microglia closely associated with Aβ plaques and microglia-internalized Aβ in the cortex of APP/PS1 KI mice. a Representative images of Imaris 3D reconstruction of plaques.

Techniques: Generated, Imaging, Software, Standard Deviation

Journal: Journal of Neuroinflammation

Article Title: Retention of normal glia function by an isoform-selective protein kinase inhibitor drug candidate that modulates cytokine production and cognitive outcomes

doi: 10.1186/s12974-017-0845-2

Figure Lengend Snippet: No effect of MW150 on CD45 and CD68 immunostaining in the cortex. a Representative images of CD45 IHC in cortex of wild type ( WT ) or APP/PS1 KI mice treated with saline vehicle ( veh ) or MW150. Digital quantification of CD45 in the cortex was done using the Aperio ScanScope, and the positive pixel algorithm. b CD45 was significantly increased in the KI + veh compared to the WT + veh treated mice ( p < 0.0001), with no effect of MW150 treatment. ( F (2,42) = 211.08; p < 0.0001). c Representative images of CD68 IHC. d CD68 was significantly increased in the KI + veh compared to the WT + veh-treated mice ( p < 0.0001), with no effect of MW150 treatment. ( F (2,42) = 28.81; p < 0.0001). ( n = 14 WT + veh; n = 14 KI + veh; n = 14 KI + MW150). Data are mean ± SEM. Source data is available in Additional file : Table S2

Article Snippet: Fig. 6 Effect of MW150 treatment on microglia closely associated with Aβ plaques and microglia-internalized Aβ in the cortex of APP/PS1 KI mice. a Representative images of Imaris 3D reconstruction of plaques.

Techniques: Immunostaining, Saline

Journal: Journal of Neuroinflammation

Article Title: Retention of normal glia function by an isoform-selective protein kinase inhibitor drug candidate that modulates cytokine production and cognitive outcomes

doi: 10.1186/s12974-017-0845-2

Figure Lengend Snippet: Effect of MW150 treatment on microglia closely associated with Aβ plaques and microglia-internalized Aβ in the cortex of APP/PS1 KI mice. a Representative images of Imaris 3D reconstruction of plaques. A region of interest ( ROI ) was generated by expanding the plaque volume by a 15 μm radius from the edge of the large plaques (larger than 10,000 voxels). This 3D ROI (shown in gray ) included the Aβ plaque, and a region near the plaque. IBA1 + cells in this ROI (shown in cyan ) were surface rendered to create a 3D volume of all IBA1 positive staining in the ROI. The IBA1 positive staining in the 3D ROI distinguishes plaque-associated microglia (shown in cyan ) compared to microglia away from plaques (shown in green ). b Volume of surface rendered IBA1 + cells within 15 μm radius around large plaques was significantly increased in KI + MW150 mice compared to KI + veh treatment ( p = 0.0397). c Representative image of microglia reconstruction with DAPI stained nuclei showing 6E10 staining within IBA1 + cell cytoplasm. d Microglia-internalized Aβ, as measured by 6E10 staining within surface rendered IBA1 + cells, was not significantly different between the KI + MW150 compared to KI + veh. ( n = 11 KI + veh; n = 14 KI + MW150). Data are mean ± SEM. Source data is available in Additional file : Table S3

Article Snippet: Fig. 6 Effect of MW150 treatment on microglia closely associated with Aβ plaques and microglia-internalized Aβ in the cortex of APP/PS1 KI mice. a Representative images of Imaris 3D reconstruction of plaques.

Techniques: Generated, Staining

Journal: Clinical and Translational Medicine

Article Title: METTL1 promotes hepatocarcinogenesis via m 7 G tRNA modification‐dependent translation control

doi: 10.1002/ctm2.661

Figure Lengend Snippet: m 7 G tRNA modification and its catalyzing enzyme components METTL1 and WDR4 are elevated in HCC. (A) The percentage of m 7 G tRNA modification and other tRNA modifications in four pairs of HCC tissues and corresponding normal liver tissues identified by liquid chromatography‐coupled mass spectrometry. Paired Student's t test was used ( n = 4). (B) Northwestern blot of m 7 G tRNA modification in four pairs of HCC tissues and corresponding normal liver tissues. U6 northern blot serves as a loading control. (C) Western blot of METTL1 and WDR4 in six pairs of HCC tissues and corresponding peritumoural tissues. GAPDH serves as a loading control. (D) Western blot of METTL1 and WDR4 in HCC cell lines. A normal liver cell line THLE‐2 was used as a normal control and GAPDH serves as a loading control. (E, F) Representative images of METTL1 (E) and WDR4 (F) IHC staining in HCC specimens. The mean density of IHC staining less than median was defined as low, while more than median was defined as high. Scale bar, 250 μm. (G, H) Quantification of METTL1 (G) and WDR4 (H) IHC staining intensity in HCC specimens. Paired Student's t test was used ( n = 48). (I, J) qRT‐PCR analysis of METTL1 (I) and WDR4 (J) mRNA expression in HCC specimens. Wilcoxon signed rank test was used ( n = 57). Data presented as mean ± SD. * p < .05, ** p < .01, *** p < .001 by Student's t test or the Mann–Whitney U test unless specified. Abbreviations: IHC, immunohistochemistry; I, inosine; i 6 A, N 6 ‐isopentenyladenosine; P, peri‐tumour tissue; Peri, peri‐tumour tissue; Q, queuosine; T, tumour tissue; t 6 A, N 6 ‐threonylcarbamoyladenosine; Um, 2′‐O‐methyluridine

Article Snippet: Briefly, 25 μg myr‐AKT1 (Addgene, Plasmid#31789) and 25 μg N‐RasV12 (Addgene, Plasmid#20205) along with 2 μg SB transposase were diluted in 2.5 ml saline, and then injected into WT or Mettl1‐KI C57BL/6 mice (8–12 weeks old) in 5–7 s via the lateral tail vein.

Techniques: Modification, Liquid Chromatography, Mass Spectrometry, Northern Blot, Western Blot, Immunohistochemistry, Quantitative RT-PCR, Expressing, MANN-WHITNEY

Journal: Clinical and Translational Medicine

Article Title: METTL1 promotes hepatocarcinogenesis via m 7 G tRNA modification‐dependent translation control

doi: 10.1002/ctm2.661

Figure Lengend Snippet: Inhibition of METTL1 impairs HCC progression in vitro and in xenograft model. (A) The knockdown effect of METTL1 in MHCC97H cells was confirmed by western blot. (B) The downregulation of m 7 G tRNA modification was confirmed by northwestern blot. (C) CCK‐8 assay of METTL1 knockdown and control MHCC97H cells. Data presented as mean ± SD (six technical replicates). (D) Representative images and quantification of clone formation in METTL1 depleted and control MHCC97H cells. Data presented as mean ± SD (three technical replicates). (E) Representative images and quantification of cell apoptosis assays in MHCC97H cells with or without METTL1 knockdown. Data presented as mean ± SD (three technical replicates). (F) Cell cycle analysis and quantification of METTL1 depleted and control MHCC97H cells. Data presented as mean ± SD (three technical replicates). (G) Representative images and quantification of migration in METTL1 depleted and control MHCC97H cells. Scale bar, 500 μm. Data presented as mean ± SD (three technical replicates). (H) Representative images and quantification of invasion in METTL1 depleted and control MHCC97H cells. Scale bar, 500 μm. Data presented as mean ± SD (three technical replicates). (I) Validation of the rescue of METTL1 by western blot in MHCC97H cells. siMETTL1‐1 was used. (J) CCK‐8 assay of METTL1 knockdown MHCC97H cells with rescue expression of wild‐type METTL1 or the mutant. (K) Growth of subcutaneous transplanted tumours in NC and shMETTL1 group. Tumour sizes were measured every 4 days. Data presented as mean ± SEM ( n = 9). shMETTL1‐2 was used. (L) Overview of subcutaneous transplanted tumours in NC and shMETTL1 group. (M) Tumour weights formed in NC and shMETTL1 group at the time of sacrifice. Data presented as mean ± SEM ( n = 9). * p < .05, ** p < .01, *** p < .001 by Student's t test, one‐way ANOVA or the Mann–Whitney U test unless specified. All the in vitro assays were biologically repeated for three times. Abbreviations: Mut, mutant METTL1; NC, negative control; sh1, shMETTL1‐1; sh2, shMETTL1‐2; shM1, shMETTL1; si1, siMETTL1‐1; WT, wild‐type METTL1

Article Snippet: Briefly, 25 μg myr‐AKT1 (Addgene, Plasmid#31789) and 25 μg N‐RasV12 (Addgene, Plasmid#20205) along with 2 μg SB transposase were diluted in 2.5 ml saline, and then injected into WT or Mettl1‐KI C57BL/6 mice (8–12 weeks old) in 5–7 s via the lateral tail vein.

Techniques: Inhibition, In Vitro, Western Blot, Modification, CCK-8 Assay, Cell Cycle Assay, Migration, Expressing, Mutagenesis, MANN-WHITNEY, Negative Control

Journal: Clinical and Translational Medicine

Article Title: METTL1 promotes hepatocarcinogenesis via m 7 G tRNA modification‐dependent translation control

doi: 10.1002/ctm2.661

Figure Lengend Snippet: METTL1 regulates m 7 G tRNA methylome, tRNA expression and global mRNA translation. (A) Flowchart of m 7 G TRAC‐Seq. (B) List of m 7 G‐modified tRNA identified in MHCC97H cells. (C) Sequence motif in the m 7 G sites identified by TRAC‐seq. (D) Representative images of cleavage scores of indicated tRNA in MHCC97H cells with or without METTL1 knockdown. (E) Global m 7 G tRNA methylation level of MHCC97H cells with or without METTL1 knockdown. Wilcoxon signed‐rank test was used. (F) Expression level of m 7 G‐modified and non‐m 7 G‐modified tRNAs revealed by TRAC‐seq. Fold change was calculated as the ratio of tRNA expression level of shMETTL1 group to the control group. (G) Validation of the downregulated expression of m 7 G‐modified tRNAs upon METTL1 depletion by m 7 G methylated tRNA immunoprecipitation qPCR. Relative expression of specific tRNA was obtained using input samples and U6 served as an internal control. The shMETTL1‐2 was used in this experiment (three technical replicates). (H) Polysome profiling of Huh7 and MHCC97H with or without METTL1 knockdown. shMETTL1‐2 was used in this experiment. (I) Global translation of SNU‐449 cells with overexpression of wild‐type or mutant METTL1. Coomassie brilliant blue staining of the gel was used as control. (J) Puromycin intake assay of siMETTL1 MHCC97H cells rescued by wild‐type METTL1 or its catalytic dead mutant. Coomassie brilliant blue staining of the gel was used as control. Quantitation of the bands was showed (three biological replicates). siMETTL1‐1 was used. Data presented as mean ± SD. * p < .05, ** p < .01, *** p < .001 by Student's t test or the Mann–Whitney U test unless specified. Abbreviations: Mut, mutant METTL1; NC, negative control; shM1, shMETTL1; si1, siMETTL1‐1; WT, wild‐type METTL1

Article Snippet: Briefly, 25 μg myr‐AKT1 (Addgene, Plasmid#31789) and 25 μg N‐RasV12 (Addgene, Plasmid#20205) along with 2 μg SB transposase were diluted in 2.5 ml saline, and then injected into WT or Mettl1‐KI C57BL/6 mice (8–12 weeks old) in 5–7 s via the lateral tail vein.

Techniques: Expressing, Modification, Sequencing, Methylation, Immunoprecipitation, Over Expression, Mutagenesis, Staining, Quantitation Assay, MANN-WHITNEY, Negative Control

Journal: Clinical and Translational Medicine

Article Title: METTL1 promotes hepatocarcinogenesis via m 7 G tRNA modification‐dependent translation control

doi: 10.1002/ctm2.661

Figure Lengend Snippet: m 7 G tRNA modification regulates HCC mRNA translation in a codon‐dependent manner. (A) Scatterplot of translation efficiency (TE) in the MHCC97 cells with or without METTL1 knockdown. TE was calculated as the ratio of the polyribosome signals to the input signals. (B) Scatterplot of mRNA expression in the MHCC97 cells with or without METTL1 knockdown. (C) Correlation of m 7 G‐related codons frequency and translation efficiency in MHCC97H. Pearson correlation analysis was used. (D) Correlation of m 7 G‐related codons frequency and translation ratio. Translation ratio was calculated as the ratio of the translation efficiency of shMETTL1 group to the control group. Pearson correlation analysis was used. (E) Translation ratio of mRNAs in low ( n = 2018) and high ( n = 2018) m 7 G‐related codon frequency groups. (F) Frequencies of m 7 G‐related codons in TE‐decreased genes ( n = 1720), TE‐increased genes ( n = 1195) and other genes ( n = 5159). (G) Pathway analysis using the TE‐decreased genes upon METTL1 knockdown. (H, I) Relative expression and translation efficiency (TE) of Cyclin A2, EGFR and VEGFA mRNA in METTL1 depleted and control MHCC97H (H) and Huh7 (I) cells. β‐Actin was used as an internal control. TE was calculated as the ratio of the polyribosome signals to input signals. RPS10 was used as a negative control. Data presented as mean ± SD (three technical replicates). (J) Western blot of Cyclin A2, EGFR, VEGFA, p‐Akt and p‐p44/42 MAPK in METTL1 depleted and control Huh7 and MHCC97H cells. GAPDH was used as a loading control. (K) Left, m 7 G tRNAs decoded‐codons frequency of TE down mRNAs identified by polyribosome‐mRNA‐seq. Right, the expression profile of m 7 G tRNAs in MHCC97H cells. (L) 5X AAG (Lys) codon sequences were inserted in the front of firefly luciferase coding region. Depletion of METTL1 resulted in decreased luciferase activity compared to that in the controls in Huh7 and MHCC97H cells. The control reporter without any insertion was used to normalize the translation differences. Data presented as mean ± SD (three technical replicates). * p < .05, ** p < .01, *** p < .001 by Student's t test, one‐way ANOVA or the Mann–Whitney U test unless specified. Polyribosome‐mRNA‐seq was biologically repeated for three times. All the in vitro assays were biologically repeated for three times. Abbreviations: NC, negative control; sh1, shMETTL1‐1; sh2, shMETTL1‐2; TE, translation efficiency

Article Snippet: Briefly, 25 μg myr‐AKT1 (Addgene, Plasmid#31789) and 25 μg N‐RasV12 (Addgene, Plasmid#20205) along with 2 μg SB transposase were diluted in 2.5 ml saline, and then injected into WT or Mettl1‐KI C57BL/6 mice (8–12 weeks old) in 5–7 s via the lateral tail vein.

Techniques: Modification, Expressing, Negative Control, Western Blot, Luciferase, Activity Assay, MANN-WHITNEY, In Vitro

Journal: Clinical and Translational Medicine

Article Title: METTL1 promotes hepatocarcinogenesis via m 7 G tRNA modification‐dependent translation control

doi: 10.1002/ctm2.661

Figure Lengend Snippet: Overexpression of LysCTT and EGFR rescues HCC malignant phenotype. (A) Validation of the LysCTT, METTL1, EGFR, Cyclin A2 expression and METTL1 depletion by northern and western blots in MHCC97H and Huh7 cells. (B) CCK‐8 assay of METTL1 knockdown MHCC97H and Huh7 cells with or without overexpression of LysCTT. Data presented as mean ± SD (six technical replicates). (C, D) Representative images and quantification of migration in METTL1‐knockdown MHCC97H (C) and Huh7 (D) cells with or without overexpression of LysCTT. Scale bar, 500 μm. Data presented as mean ± SD (three technical replicates). (E, F) Representative images and quantification of invasion in METTL1‐knockdown MHCC97H (E) and Huh7 (F) cells with or without overexpression of LysCTT. Scale bar, 500 μm. Data presented as mean ± SD (three technical replicates). (G) Validation of the EGFR overexpression and METTL1 depletion by western blot in MHCC97H cells. (H) CCK‐8 assay of METTL1 knockdown MHCC97H cells with or without overexpression of EGFR. Data presented as mean ± SD (six technical replicates). (I) Representative images and quantification of migration in METTL1‐knockdown MHCC97H cells with or without overexpression of EGFR. Scale bar, 500 μm. Data presented as mean ± SD (three technical replicates). (J) Representative images and quantification of invasion in METTL1‐knockdown MHCC97H cells with or without overexpression of EGFR. Scale bar, 500 μm. Data presented as mean ± SD (three technical replicates). * p < .05, ** p < .01, *** p < .001 by Student's t test, one‐way ANOVA or the Mann–Whitney U test unless specified. All the in vitro assays were biologically repeated for three times. Abbreviations: NC, negative control; oeEGFR, overexpression of EGFR; oeLysCTT, overexpression of LysCTT; siM1, siMETTL1‐1

Article Snippet: Briefly, 25 μg myr‐AKT1 (Addgene, Plasmid#31789) and 25 μg N‐RasV12 (Addgene, Plasmid#20205) along with 2 μg SB transposase were diluted in 2.5 ml saline, and then injected into WT or Mettl1‐KI C57BL/6 mice (8–12 weeks old) in 5–7 s via the lateral tail vein.

Techniques: Over Expression, Expressing, Northern Blot, Western Blot, CCK-8 Assay, Migration, MANN-WHITNEY, In Vitro, Negative Control

Journal: Clinical and Translational Medicine

Article Title: METTL1 promotes hepatocarcinogenesis via m 7 G tRNA modification‐dependent translation control

doi: 10.1002/ctm2.661

Figure Lengend Snippet: Liver‐specific knockout of Mettl1 inhibits HCC tumourigenesis in vivo. (A) The schematic diagram of the construction of Mettl1 conditional liver‐specific knockout mice. (B) Representative image of livers harvested from Mettl1‐cKO and control group. (C) Comparison of ratio of liver weight to body weight between Mettl1‐cKO and control group. Data presented as mean ± SD ( n = 5). (D) Representative images of H&E staining of control and Mettl1‐cKO mouse livers. Scale bar: 100 μm. (E) IHC staining of Ki‐67 in control and Mettl1‐cKO mouse livers. Arrows point to Ki67‐positive cells. Scale bar:100 μm. Data presented as mean ± SD ( n = 5). (F) General view of hydrodynamics transfection experiment in Mettl1 conditional knockout mice and control. (G) Representative image of livers harvested from Mettl1‐cKO and control group. (H) Comparison of tumour burden between Mettl1‐cKO and control group by ratio of liver weight to body weight (LW/BW). Data presented as mean ± SD ( n = 9). (I) Representative images of H&E staining of control and Mettl1‐cKO mouse livers. Arrows point to tumour lesions. Scale bar: 250 μm. The number of tumour foci in these mouse livers was evaluated. Data presented as mean ± SD ( n = 9). (J) Left panel, representative images of IHC staining of Ki‐67. Arrows point to Ki67‐positive cells. Scale bar:100 μm. Right panel, the statistical analyses. Data presented as mean ± SD ( n = 9). (K) Western blot of Mettl1, EGFR, Cyclin A2 and AFP in tumour tissues from control group and Mettl1‐cKO group. β‐Actin was used as a loading control. Downregulation of m 7 G tRNA modification and LysCTT in Mettl1‐cKO group was confirmed by northwestern blot and northern blot. U6 was used as a loading control. (L) qRT‐PCR analysis of Cyclin A2 and EGFR mRNA levels in tumour tissues from control group and Mettl1‐cKO group. Data presented as mean ± SD ( n = 3). * p < .05, ** p < .01, *** p < .001 by Student's t test, one‐way ANOVA or the Mann–Whitney U test unless specified. All the in vitro assays were biologically repeated for three times. Abbreviations: cKO, conditional knockout; Ctrl, control

Article Snippet: Briefly, 25 μg myr‐AKT1 (Addgene, Plasmid#31789) and 25 μg N‐RasV12 (Addgene, Plasmid#20205) along with 2 μg SB transposase were diluted in 2.5 ml saline, and then injected into WT or Mettl1‐KI C57BL/6 mice (8–12 weeks old) in 5–7 s via the lateral tail vein.

Techniques: Knock-Out, In Vivo, Staining, Immunohistochemistry, Transfection, Western Blot, Modification, Northern Blot, Quantitative RT-PCR, MANN-WHITNEY, In Vitro

Journal: Clinical and Translational Medicine

Article Title: METTL1 promotes hepatocarcinogenesis via m 7 G tRNA modification‐dependent translation control

doi: 10.1002/ctm2.661

Figure Lengend Snippet: Overexpression of METTL1 promotes HCC progression in vivo. (A) Representative image of livers harvested from Mettl1‐KI and control group. (B) Comparison of ratio of liver weight to body weight between Mettl1‐KI and control group. Data presented as mean ± SD ( n = 5). (C) Representative images of H&E staining of control and Mettl1‐KI mouse livers. Scale bar: 100 μm. (D) IHC staining of Ki‐67 in control and Mettl1‐KI mouse livers. Arrows point to Ki67‐positive cells. Scale bar: 50 μm. Data presented as mean ± SD ( n = 5). (E) General view of hydrodynamics transfection experiment. AKT and NRAS plasmids along with SB transposase were injected into control and Mettl1 knockin mice (Mettl1‐KI mice). The mice were sacrificed after 4 weeks. n = 6. (F) Representative image of livers harvested from Mettl1‐KI and control group. (G) Comparison of tumourigenic capacity between Mettl1‐KI and control group by ratio of liver weight to body weight (LW/BW). Data presented as mean ± SD ( n = 6). (H) Representative images of H&E staining of control and Mettl1‐KI mouse livers. Scale bar: 100 μm. The number of tumour foci in these mouse livers was evaluated. P, peri‐tumour; T, tumour. Data presented as mean ± SD ( n = 6). (I) IHC staining of Ki‐67. Scale bar: 50 μm. Arrows point to Ki67‐positive cells. Data presented as mean ± SD ( n = 6). (J) Western blot of Mettl1, EGFR, Cyclin A2 and AFP in tumour tissues from control group and Mettl1‐KI group. β‐Actin was used as a loading control. Upregulation of m 7 G tRNA modification and LysCTT in Mettl1‐KI group was confirmed by northwestern blot and northern blot. U6 was used as a loading control. (K) qRT‐PCR analysis of Cyclin A2 and EGFR mRNA levels in tumour tissues from control group and Mettl1‐KI group. Data presented as mean ± SD ( n = 3). * p < .05, ** p < .01, *** p < .001 by Student's t test, one‐way ANOVA or the Mann–Whitney U test unless specified. All the in vitro assays were biologically repeated for three times. Abbreviations: Ctrl, control; KI, knockin

Article Snippet: Briefly, 25 μg myr‐AKT1 (Addgene, Plasmid#31789) and 25 μg N‐RasV12 (Addgene, Plasmid#20205) along with 2 μg SB transposase were diluted in 2.5 ml saline, and then injected into WT or Mettl1‐KI C57BL/6 mice (8–12 weeks old) in 5–7 s via the lateral tail vein.

Techniques: Over Expression, In Vivo, Staining, Immunohistochemistry, Transfection, Injection, Knock-In, Western Blot, Modification, Northern Blot, Quantitative RT-PCR, MANN-WHITNEY, In Vitro

Journal: Stem Cells International

Article Title: METTL3-Mediated m 6 A Methylation Regulates Muscle Stem Cells and Muscle Regeneration by Notch Signaling Pathway

doi: 10.1155/2021/9955691

Figure Lengend Snippet: METTL3 is required for muscle regeneration in vivo . (a) Schematic outline of CTX injection in tamoxifen-treated CTL and cKO littermates. (b–d) Immunofluorescence staining and Western blot analysis of TA muscle after injection for 5 days for METTL3. (e) Hematoxylin and eosin (H&E) staining of TA muscles of METTL3 cKO mice and control littermates 2, 5, and 8 days after CTX injection. (f) Number of muscle fibers. (g) Area of regenerating centronuclear fibers. (h) Masson's Trichrome staining of TA muscles of METTL3 cKO and control littermates. (i) Western blot analysis of Pax7, myosin, myogenin, and METTL3 protein levels in control TA muscle at day 0, day 2, day 5, and day 8. (j) Immunofluorescence staining of TA muscles from METTL3 cKO and control mice detected by myogenin. (k) Quantifications of myogenin staining. (l) Western blot analysis of myogenin. (m) Immunohistochemical staining of myosin. (n) Quantifications of myosin staining. (o) Western blot analysis of myosin. N = 10. ∗∗∗∗ p < 0.0001; ∗∗∗ p < 0 .001; ∗∗ p < 0.01; ∗ p < .05. CTX: cardiotoxin; TAM: tamoxifen; TA: tibialis anterior; CTL: control; cKO: METTL3 conditional knockout.

Article Snippet: The METTL3 flox/wt mouse strain and the METTL3 ki/wt mouse strain were generated with CRISPR/Cas9 technology by Biocytogen Jiangsu Co., Ltd. Pax7CreERT2 mouse was purchased from The Jackson Laboratory.

Techniques: In Vivo, Injection, Immunofluorescence, Staining, Western Blot, Immunohistochemical staining, Knock-Out

Journal: Stem Cells International

Article Title: METTL3-Mediated m 6 A Methylation Regulates Muscle Stem Cells and Muscle Regeneration by Notch Signaling Pathway

doi: 10.1155/2021/9955691

Figure Lengend Snippet: METTL3 regulates muscle stem cell proliferation in vivo . (a–d) Immunohistochemical analysis of TA muscle samples from METTL3 cKO and control by Pax7 and Ki67 antibodies, respectively. Quantifications of Pax7 and Ki67 staining were shown on the right. (e) EdU of TA muscles and (f) images of cell numbers 5 days after isolation from injured muscles of METTL3cKO and control mice. ∗∗∗∗ p < 0.0001; ∗∗∗ p < 0.001. CTL: control; cKO: METTL3 conditional knockout.

Article Snippet: The METTL3 flox/wt mouse strain and the METTL3 ki/wt mouse strain were generated with CRISPR/Cas9 technology by Biocytogen Jiangsu Co., Ltd. Pax7CreERT2 mouse was purchased from The Jackson Laboratory.

Techniques: In Vivo, Immunohistochemical staining, Staining, Isolation, Knock-Out

Journal: Stem Cells International

Article Title: METTL3-Mediated m 6 A Methylation Regulates Muscle Stem Cells and Muscle Regeneration by Notch Signaling Pathway

doi: 10.1155/2021/9955691

Figure Lengend Snippet: METTL3 conditional knockin promotes muscle regeneration in vivo . (a) Schematic outline of CTX injection in TAM-treated control and METTL3 cKI littermates at the age of 8–9 weeks. (b) H&E staining of representative tibialis muscle sections from METTL3 cKI, control mice treated 2, 5, and 8 days after CTX injection. (c) Quantified of muscle fibers. (d) The upregulation effect of METTL3 was verified at protein levels by Western blot. (e, h, k) Immunohistochemical and (n) immunofluorescence staining of TA muscle from METTL3 cKI and control detected by Pax7, Ki67, myogenin, and myosin antibodies. (f, i, l, o) Quantifications of Pax7+, Ki67+, myogenin+, and myosin+ cells were shown on the right. (g, j, m, p) Western blot detected the protein expression levels of Pax7+, Ki67+, myogenin, and myosin. Data represent mean ± SEM. N = 5. ∗∗∗∗ p < 0.0001, ∗∗ p < 0.01, ∗ p < 0.05. CTX: cardiotoxin; TAM: tamoxifen; TA: tibialis anterior; CTL: control; cKI: METTL3 conditional knockin.

Article Snippet: The METTL3 flox/wt mouse strain and the METTL3 ki/wt mouse strain were generated with CRISPR/Cas9 technology by Biocytogen Jiangsu Co., Ltd. Pax7CreERT2 mouse was purchased from The Jackson Laboratory.

Techniques: Knock-In, In Vivo, Injection, Staining, Western Blot, Immunohistochemical staining, Immunofluorescence, Expressing

Journal: Stem Cells International

Article Title: METTL3-Mediated m 6 A Methylation Regulates Muscle Stem Cells and Muscle Regeneration by Notch Signaling Pathway

doi: 10.1155/2021/9955691

Figure Lengend Snippet: METTL3 regulates m 6 A modification of Notch signaling pathway components. (a) Schematic representation of m 6 A MeRIP-Seq. (b) m 6 A motif identified in the MeRIP-Seq. (c) Enriched KEGG molecular function pathways identified from differentially m 6 A-enriched genes in METTL3 cKO vs. CTL D2 samples. (d) Notch signaling pathway identified in the MeRIP-Seq. (e, f) qRT-PCR and Western blot analyzed of Notch signaling pathway. CTL: control; cKO: METTL3 conditional knockout.

Article Snippet: The METTL3 flox/wt mouse strain and the METTL3 ki/wt mouse strain were generated with CRISPR/Cas9 technology by Biocytogen Jiangsu Co., Ltd. Pax7CreERT2 mouse was purchased from The Jackson Laboratory.

Techniques: Modification, Quantitative RT-PCR, Western Blot, Knock-Out

Journal: Stem Cells International

Article Title: METTL3-Mediated m 6 A Methylation Regulates Muscle Stem Cells and Muscle Regeneration by Notch Signaling Pathway

doi: 10.1155/2021/9955691

Figure Lengend Snippet: Deletion of METTL3 affects C2C12 myoblast progenitor cell proliferation and differentiation. (a) Western blot detection of METTL3 expression. (b) Proliferation ability significantly decreased after METTL3 knockout determined by cell counting Kit-8 at indicated days. (c) Western blot analysis of the expression of early differentiation marker myogenin and late differentiation marker myosin in C2C12 cells. (d) Myosin staining (red) was performed 3 days later using an anti-myosin antibody, and the nuclei were counterstained with DAPI. (e) Differentiation index in the METTL3-depleted and control cells. Data represent mean ± SEM. ∗∗∗ p < 0.001. NC: negative control; sh-1: METTL3 shRNA-1; sh-2: METTL3 shRNA-2.

Article Snippet: The METTL3 flox/wt mouse strain and the METTL3 ki/wt mouse strain were generated with CRISPR/Cas9 technology by Biocytogen Jiangsu Co., Ltd. Pax7CreERT2 mouse was purchased from The Jackson Laboratory.

Techniques: Western Blot, Expressing, Knock-Out, Cell Counting, Marker, Staining, Negative Control, shRNA

Journal: Stem Cells International

Article Title: METTL3-Mediated m 6 A Methylation Regulates Muscle Stem Cells and Muscle Regeneration by Notch Signaling Pathway

doi: 10.1155/2021/9955691

Figure Lengend Snippet: METTL3 regulates the mRNA translation efficiency of Notch signaling pathway components. (a) The Notch pathway genes with m 6 A modification. (b) The protein levels of Notch signaling pathway in the METTL3-depleted and control cells. (c) mRNA expression of Notch signaling pathway analyzed by q-PCR. (d) Polyribosome-bound mRNA-qPCR was used to detect translation efficiency. (e) The downstream gene expression of Notch signaling pathway was detected by qRT-PCR. ∗∗∗∗ p < 0.0001; ∗∗∗ p < 0.001; ∗∗ p < 0.01. NC: negative control; sh-1: METTL3 shRNA-1; sh-2: METTL3 shRNA-2.

Article Snippet: The METTL3 flox/wt mouse strain and the METTL3 ki/wt mouse strain were generated with CRISPR/Cas9 technology by Biocytogen Jiangsu Co., Ltd. Pax7CreERT2 mouse was purchased from The Jackson Laboratory.

Techniques: Modification, Expressing, Quantitative RT-PCR, Negative Control, shRNA

Journal: Stem Cells International

Article Title: METTL3-Mediated m 6 A Methylation Regulates Muscle Stem Cells and Muscle Regeneration by Notch Signaling Pathway

doi: 10.1155/2021/9955691

Figure Lengend Snippet: YTHDF1 regulates the mRNA translation efficiency of Notch signaling pathway components. (a) The protein levels of Notch signaling pathway in the METTL3 overexpressed or (d) depleted and control cells. (b, e) mRNA expression of Notch signaling pathway analyzed by q-PCR. (c, f) Polyribosome-bound mRNA-qPCR was used to detect translation efficiency. (g) Working model of METTL3 regulates muscle stem cells by Notch signaling pathway. ∗∗∗∗ p < 0.0001; ∗∗∗ p < 0.001; ∗∗ p < 0.01. NC: negative control; OE: YTHDF1 overexpressed; si: YTHDF1 siRNA.

Article Snippet: The METTL3 flox/wt mouse strain and the METTL3 ki/wt mouse strain were generated with CRISPR/Cas9 technology by Biocytogen Jiangsu Co., Ltd. Pax7CreERT2 mouse was purchased from The Jackson Laboratory.

Techniques: Expressing, Negative Control

Journal: Dystonia

Article Title: Electrophysiological characterization of the striatal cholinergic interneurons in Dyt1 ΔGAG knock-in mice

doi: 10.3389/dyst.2022.10557

Figure Lengend Snippet: Spontaneous firing of the striatal ChIs in the acute brain slices. (A) The representative traces of the striatal ChIs. Spontaneous firing frequency (B) significantly decreased, and CV (C) significantly increased in Dyt1 KI mice. The bars represent means ± standard errors. The dots represent each data point. *p < 0.05.

Article Snippet: There was no significant difference in the inhibitory effect of muscarine between WT and Dyt1 KI mice [WT, 53 ± 3 %; KI, 54 ± 5 % ; Chi-Square(1) = 0.02, p = 0.90; ].

Techniques:

Journal: Dystonia

Article Title: Electrophysiological characterization of the striatal cholinergic interneurons in Dyt1 ΔGAG knock-in mice

doi: 10.3389/dyst.2022.10557

Figure Lengend Snippet: Responses of the striatal ChIs in the acute brain slices to quinpirole. Representative trace of the spontaneous firing and the inhibitory response to quinpirole (A). The spontaneous firings were inhibited by quinpirole in both WT (B) and Dyt1 KI mice (C). The percentage of frequency (Hz) after quinpirole (D) treatment was compared to those before treatment. The 30 s before drug application and the last 30 s during the drug application (yellow boxes) were used to analyze drug effects. There was significantly less quinpirole inhibition in the KI mice. (E) An extreme case of the paradoxical excitation of a KI ChI after quinpirole treatment was applied 13 min after the cell-attached recording. The lines under Quinpirole show the duration of drug application (E, 120 s). Overall, the spontaneous firings were not significantly inhibited by quinpirole in both WT (F) and Dyt1 KI mice (G). The frequency after quinpirole treatments was compared to before treatment (H). The bar graph show median of the percentage. The error bars show upper and lower 95% confidence intervals. The 3 minutes before drug application and the 3 minutes after 2 minutes washout (yellow boxes) were used to analyze the quinpirole effect. ns: not significant, *p < 0.05, **p < 0.01.

Article Snippet: There was no significant difference in the inhibitory effect of muscarine between WT and Dyt1 KI mice [WT, 53 ± 3 %; KI, 54 ± 5 % ; Chi-Square(1) = 0.02, p = 0.90; ].

Techniques: Inhibition

Journal: Dystonia

Article Title: Electrophysiological characterization of the striatal cholinergic interneurons in Dyt1 ΔGAG knock-in mice

doi: 10.3389/dyst.2022.10557

Figure Lengend Snippet: Responses of the striatal ChIs in the acute brain slices to muscarine and THP. The spontaneous firings were inhibited by muscarine in both WT (A) and Dyt1 KI mice (B). The percentage of frequency (Hz) after muscarine (C) was analyzed by comparing it to those before treatment. There was no significant alteration in the spontaneous firing frequencies after THP application in WT (D) and Dyt1 KI mice (E). The percentage of frequency (Hz) after THP treatment was compared to those before treatment (F). The bars represent means ± standard errors (C and F). ns: not significant, **p < 0.01.

Article Snippet: There was no significant difference in the inhibitory effect of muscarine between WT and Dyt1 KI mice [WT, 53 ± 3 %; KI, 54 ± 5 % ; Chi-Square(1) = 0.02, p = 0.90; ].

Techniques:

Journal: Dystonia

Article Title: Electrophysiological characterization of the striatal cholinergic interneurons in Dyt1 ΔGAG knock-in mice

doi: 10.3389/dyst.2022.10557

Figure Lengend Snippet: Whole-cell recording of the striatal ChIs. Representative current steps (−0.8 ~ 0.2 nA, A) and traces (B, C). There was no significant difference in the frequency-current relationship generated by the current steps (−0.8 ~ 0.6 nA) between WT and Dyt1 KI mice (D). Voltage ramp used for whole-cell voltage-clamp recording of ChIs (E). Representative traces of the electrophysiological responses of the striatal ChIs with 1 μM TTX, 1 μM TTX + 1 μM DAMGO, and 1 μM TTX + 1 μM DAMGO + 1 μM PAX (F). Measured inward currents at −140 mV of the hyperpolarized ChI membranes in WT and Dyt1 KI mice are plotted (G). The digitally subtracted currents with DAMGO and PAX treatments (H). There is no difference in the μ-opioid receptor-induced outward currents between WT and Dyt1 KI mice (I). There is no difference in the BK channel-mediated inward currents in WT and Dyt1 KI mice (J). The bar graphs in G, I, and J show means ± standard errors. ns: not significant

Article Snippet: There was no significant difference in the inhibitory effect of muscarine between WT and Dyt1 KI mice [WT, 53 ± 3 %; KI, 54 ± 5 % ; Chi-Square(1) = 0.02, p = 0.90; ].

Techniques: Generated

Journal: Dystonia

Article Title: Electrophysiological characterization of the striatal cholinergic interneurons in Dyt1 ΔGAG knock-in mice

doi: 10.3389/dyst.2022.10557

Figure Lengend Snippet: Morphological analysis of the striatal ChIs. Representative confocal microscopic images of the dorsal striatal ChIs stained with biocytin/streptavidin conjugate (A) and goat anti-ChAT antibody (B). The representative fluorescence images of the biocytin/streptavidin-stained ChIs (C, D) and their dendrites’ reconstructed traces (E, F) are shown. The intersection numbers at every Sholl ring (5 μm increment) from the center of the soma were plotted (means ± standard errors; G). There was no significant alteration in the dendritic branch numbers and soma size (H) between WT and Dyt1 KI mice. The dots and error bars in G and the bars in H show means ± standard errors. Scale bars: A and B, 10 μm; C and D, 50 μm

Article Snippet: There was no significant difference in the inhibitory effect of muscarine between WT and Dyt1 KI mice [WT, 53 ± 3 %; KI, 54 ± 5 % ; Chi-Square(1) = 0.02, p = 0.90; ].

Techniques: Staining, Fluorescence