sirnas targeting mouse alkbh1  (Revvity Signals)

 
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    Revvity Signals sirnas targeting mouse alkbh1
    (A) Representative images of dot blot assay showing that the specific antibody against DNA N6-mA can detect the levels of N6-mA in mouse DRG neurons, the differentiated neuronal cell line CAD cells, and the Hela cells, in a dose-dependent manner. (B) Representative dot blot images showing that knocking down <t>ALKBH1</t> in CAD cells led to increased level of N6-mA, whereas knocking down N6AMT1 alone had little effect. However, when ALKBH1 and N6AMT1 were knocked down together, the level of N6-mA was reversed back to the control condition. (C) Quantification of (B) (one-way ANOVA followed by Tukey’s multiple comparisons test, P = 0.0013, n = 4-5 independent experiments). (D) Representative dot blot images showing that treating the isolated DNA samples with DNase resulted in total loss of sample signals and associated N6-mA signals in either CAD or Hela cells. (E) Representative dot blot images showing that knocking down ALKBH1 in adult mouse DRG neurons led to increased level of N6-mA, whereas knocking down N6AMT1 alone had little effect. However, when ALKBH1 and N6AMT1 were knocked down together, the level of N6-mA was reversed back to the control condition. (F) Quantification of (B) (one-way ANOVA followed by Tukey’s multiple comparisons test, P = 0.002, n = 4 independent experiments). Data are represented as mean ± SEM. P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001, compared to control if not designated.
    Sirnas Targeting Mouse Alkbh1, supplied by Revvity Signals, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/sirnas targeting mouse alkbh1/product/Revvity Signals
    Average 86 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    sirnas targeting mouse alkbh1 - by Bioz Stars, 2024-09
    86/100 stars

    Images

    1) Product Images from "N6-methyladenine DNA demethylase ALKBH1 regulates mammalian axon regeneration"

    Article Title: N6-methyladenine DNA demethylase ALKBH1 regulates mammalian axon regeneration

    Journal: bioRxiv

    doi: 10.1101/2020.08.19.258038

    (A) Representative images of dot blot assay showing that the specific antibody against DNA N6-mA can detect the levels of N6-mA in mouse DRG neurons, the differentiated neuronal cell line CAD cells, and the Hela cells, in a dose-dependent manner. (B) Representative dot blot images showing that knocking down ALKBH1 in CAD cells led to increased level of N6-mA, whereas knocking down N6AMT1 alone had little effect. However, when ALKBH1 and N6AMT1 were knocked down together, the level of N6-mA was reversed back to the control condition. (C) Quantification of (B) (one-way ANOVA followed by Tukey’s multiple comparisons test, P = 0.0013, n = 4-5 independent experiments). (D) Representative dot blot images showing that treating the isolated DNA samples with DNase resulted in total loss of sample signals and associated N6-mA signals in either CAD or Hela cells. (E) Representative dot blot images showing that knocking down ALKBH1 in adult mouse DRG neurons led to increased level of N6-mA, whereas knocking down N6AMT1 alone had little effect. However, when ALKBH1 and N6AMT1 were knocked down together, the level of N6-mA was reversed back to the control condition. (F) Quantification of (B) (one-way ANOVA followed by Tukey’s multiple comparisons test, P = 0.002, n = 4 independent experiments). Data are represented as mean ± SEM. P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001, compared to control if not designated.
    Figure Legend Snippet: (A) Representative images of dot blot assay showing that the specific antibody against DNA N6-mA can detect the levels of N6-mA in mouse DRG neurons, the differentiated neuronal cell line CAD cells, and the Hela cells, in a dose-dependent manner. (B) Representative dot blot images showing that knocking down ALKBH1 in CAD cells led to increased level of N6-mA, whereas knocking down N6AMT1 alone had little effect. However, when ALKBH1 and N6AMT1 were knocked down together, the level of N6-mA was reversed back to the control condition. (C) Quantification of (B) (one-way ANOVA followed by Tukey’s multiple comparisons test, P = 0.0013, n = 4-5 independent experiments). (D) Representative dot blot images showing that treating the isolated DNA samples with DNase resulted in total loss of sample signals and associated N6-mA signals in either CAD or Hela cells. (E) Representative dot blot images showing that knocking down ALKBH1 in adult mouse DRG neurons led to increased level of N6-mA, whereas knocking down N6AMT1 alone had little effect. However, when ALKBH1 and N6AMT1 were knocked down together, the level of N6-mA was reversed back to the control condition. (F) Quantification of (B) (one-way ANOVA followed by Tukey’s multiple comparisons test, P = 0.002, n = 4 independent experiments). Data are represented as mean ± SEM. P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001, compared to control if not designated.

    Techniques Used: Dot Blot, Isolation

    (A) Real-time PCR analysis showing significantly reduced mRNA level of ALKBH1 3 days after electroporation of siRNAs against ALKBH1 (siALKBH1) or together with siRNAs against N6AMT1 (siALK.+siN6A.). One sample t test, P = 0.0315 and 0.0092 for siALKBH1 and siALK.+siN6A., respectively. n = 3 independent experiments. (B) Real-time PCR analysis showing significantly reduced mRNA level of N6AMT1 3 days after electroporation of siRNAs against ALKBH1 and N6AMT1. One-sample t test, P < 0.0001, n = 3 independent experiments. (C) Top: time line of the culture and replate experiments. Bottom: representative images of cultured sensory neurons 24 hours post-replating after knocking down ALKBH1, N6AMT1, or together with siRNAs (siALKBH1, siN6AMT1, or siALK.+siN6A.). Scale bar, 500 μm. (D) Quantification of average lengths of the longest neurites (one-way ANOVA followed by Tukey’s multiple comparisons test, P < 0.0001, n = 7-10 independent experiments). (E) Quantification of percentage of neurons with axons (one-way ANOVA followed by Tukey’s multiple comparisons test, P = 0.0827, n = 4 independent experiments). Data are represented as mean ± SEM. P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001, compared to control if not designated.
    Figure Legend Snippet: (A) Real-time PCR analysis showing significantly reduced mRNA level of ALKBH1 3 days after electroporation of siRNAs against ALKBH1 (siALKBH1) or together with siRNAs against N6AMT1 (siALK.+siN6A.). One sample t test, P = 0.0315 and 0.0092 for siALKBH1 and siALK.+siN6A., respectively. n = 3 independent experiments. (B) Real-time PCR analysis showing significantly reduced mRNA level of N6AMT1 3 days after electroporation of siRNAs against ALKBH1 and N6AMT1. One-sample t test, P < 0.0001, n = 3 independent experiments. (C) Top: time line of the culture and replate experiments. Bottom: representative images of cultured sensory neurons 24 hours post-replating after knocking down ALKBH1, N6AMT1, or together with siRNAs (siALKBH1, siN6AMT1, or siALK.+siN6A.). Scale bar, 500 μm. (D) Quantification of average lengths of the longest neurites (one-way ANOVA followed by Tukey’s multiple comparisons test, P < 0.0001, n = 7-10 independent experiments). (E) Quantification of percentage of neurons with axons (one-way ANOVA followed by Tukey’s multiple comparisons test, P = 0.0827, n = 4 independent experiments). Data are represented as mean ± SEM. P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001, compared to control if not designated.

    Techniques Used: Real-time Polymerase Chain Reaction, Electroporation, Cell Culture

    (A) Real-time PCR analysis showing significantly elevated mRNA level of ALKBH1 in sensory neurons 1-day post-nerve injury (one sample t test, P = 0.0014, n = 4 independent experiments). (B) Real-time PCR analysis showing significantly reduced mRNA level of ALKBH1 in vivo after 3 days after electroporation (one sample t test, P = 0.0076, n = 4 independent experiments). (C) Top: timeline of the experiment. Bottom: representative images showing that knocking down ALKBH1 had no effect on slower sensory axon regeneration 2 days post-nerve injury. The right column shows enlarged images of areas in the dashed yellow boxes. The red line indicates the crush sites and the read arrows label regenerating axon tips. Scale bar, 1 mm for the left panel and 0.5 mm for the right panel. (D) Quantification of average length of regenerating sensory axons showing no significant difference between the control group and the ALKBH1 knockdown group 2 days post-nerve injury (unpaired student t test, P = 0.6387, n = 7 mice in each condition). (E) Cumulative distribution curves showing similar axon regeneration between the control group and the ALKBH1 knockdown group 2 days post-nerve injury. (F) Top: timeline of the experiment. Bottom: representative images showing that knocking down N6AMT1 had no effect on slower sensory axon regeneration 2 days post-nerve injury. The right column shows enlarged images of areas in the dashed yellow boxes. The red line indicates the crush sites and the read arrows label regenerating axon tips. Scale bar, 1 mm for the left panel and 0.5 mm for the right panel. (G) Quantification of average length of regenerating sensory axons showing no significant difference between the control group and the N6AMT1 knockdown group 2 days post-nerve injury (unpaired student t test, P = 0.2715, n = 7 and 10 mice in control and siN6AMT1 group, respectively). (H) Cumulative distribution curves showing similar axon regeneration between the control group and the N6AMT1 knockdown group 2 days post-nerve injury. Data are represented as mean ± SEM. P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001, compared to control if not designated.
    Figure Legend Snippet: (A) Real-time PCR analysis showing significantly elevated mRNA level of ALKBH1 in sensory neurons 1-day post-nerve injury (one sample t test, P = 0.0014, n = 4 independent experiments). (B) Real-time PCR analysis showing significantly reduced mRNA level of ALKBH1 in vivo after 3 days after electroporation (one sample t test, P = 0.0076, n = 4 independent experiments). (C) Top: timeline of the experiment. Bottom: representative images showing that knocking down ALKBH1 had no effect on slower sensory axon regeneration 2 days post-nerve injury. The right column shows enlarged images of areas in the dashed yellow boxes. The red line indicates the crush sites and the read arrows label regenerating axon tips. Scale bar, 1 mm for the left panel and 0.5 mm for the right panel. (D) Quantification of average length of regenerating sensory axons showing no significant difference between the control group and the ALKBH1 knockdown group 2 days post-nerve injury (unpaired student t test, P = 0.6387, n = 7 mice in each condition). (E) Cumulative distribution curves showing similar axon regeneration between the control group and the ALKBH1 knockdown group 2 days post-nerve injury. (F) Top: timeline of the experiment. Bottom: representative images showing that knocking down N6AMT1 had no effect on slower sensory axon regeneration 2 days post-nerve injury. The right column shows enlarged images of areas in the dashed yellow boxes. The red line indicates the crush sites and the read arrows label regenerating axon tips. Scale bar, 1 mm for the left panel and 0.5 mm for the right panel. (G) Quantification of average length of regenerating sensory axons showing no significant difference between the control group and the N6AMT1 knockdown group 2 days post-nerve injury (unpaired student t test, P = 0.2715, n = 7 and 10 mice in control and siN6AMT1 group, respectively). (H) Cumulative distribution curves showing similar axon regeneration between the control group and the N6AMT1 knockdown group 2 days post-nerve injury. Data are represented as mean ± SEM. P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001, compared to control if not designated.

    Techniques Used: Real-time Polymerase Chain Reaction, In Vivo, Electroporation

    (A) Top: timeline of the experiment. Bottom: representative images showing that knocking down ALKBH1 significantly impaired fast sensory axon regeneration 3 days post-nerve injury. The right column shows enlarged images of areas in the dashed yellow boxes. The red line indicates the crush sites and the read arrows label regenerating axon tips. Scale bar, 1 mm for the left panel and 0.5 mm for the right panel. (B) Quantification of average length of regenerating axons showing that knocking down ALKBH1 significantly impaired sensory axon regeneration 3 days post-nerve injury (unpaired student t test, P < 0.0001, n = 8 mice in each condition). (C) Cumulative distribution curves showing that ALKBH1 knockdown resulted in significant reduced sensory axon regeneration 3 days post-nerve injury. (D) Top: timeline of the experiment. Bottom: representative images showing that knocking down ALKBH1 significantly impaired fast sensory axon regeneration 4 days post-nerve injury. The right column shows enlarged images of areas in the dashed yellow boxes. The red line indicates the crush sites and the read arrows label regenerating axon tips. Scale bar, 1 mm for the left panel and 0.5 mm for the right panel. (E) Quantification of average length of regenerating axons showing that knocking down ALKBH1 significantly impaired sensory axon regeneration 4 days post-nerve injury (unpaired student t test, P = 0.018, n = 4 mice in each condition). (F) Cumulative distribution curves showing that ALKBH1 knockdown resulted in significant reduced sensory axon regeneration 4 days post-nerve injury. Data are represented as mean ± SEM. P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001, compared to control if not designated.
    Figure Legend Snippet: (A) Top: timeline of the experiment. Bottom: representative images showing that knocking down ALKBH1 significantly impaired fast sensory axon regeneration 3 days post-nerve injury. The right column shows enlarged images of areas in the dashed yellow boxes. The red line indicates the crush sites and the read arrows label regenerating axon tips. Scale bar, 1 mm for the left panel and 0.5 mm for the right panel. (B) Quantification of average length of regenerating axons showing that knocking down ALKBH1 significantly impaired sensory axon regeneration 3 days post-nerve injury (unpaired student t test, P < 0.0001, n = 8 mice in each condition). (C) Cumulative distribution curves showing that ALKBH1 knockdown resulted in significant reduced sensory axon regeneration 3 days post-nerve injury. (D) Top: timeline of the experiment. Bottom: representative images showing that knocking down ALKBH1 significantly impaired fast sensory axon regeneration 4 days post-nerve injury. The right column shows enlarged images of areas in the dashed yellow boxes. The red line indicates the crush sites and the read arrows label regenerating axon tips. Scale bar, 1 mm for the left panel and 0.5 mm for the right panel. (E) Quantification of average length of regenerating axons showing that knocking down ALKBH1 significantly impaired sensory axon regeneration 4 days post-nerve injury (unpaired student t test, P = 0.018, n = 4 mice in each condition). (F) Cumulative distribution curves showing that ALKBH1 knockdown resulted in significant reduced sensory axon regeneration 4 days post-nerve injury. Data are represented as mean ± SEM. P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001, compared to control if not designated.

    Techniques Used:

    (A) Top: timeline of the experiment. Bottom: representative images showing that knocking down N6AMT1 reversed sensory axon regeneration impaired by down regulation of ALKBH1 3 days post-nerve injury. The right column shows enlarged images of areas in the dashed yellow boxes. The red line indicates the crush sites and the read arrows label regenerating axon tips. Scale bar, 1 mm for the left panel and 0.5 mm for the right panel. (B) Quantification of average length of regenerating axons under conditions shown in (A). One-way ANOVA followed by Tukey’s multiple comparisons test, P < 0.0001, n = 7-9 mice in each condition. (C) Cumulative distribution curves showing that knocking down N6AMT1 rescued axon regeneration impaired by ALKBH1 knockdown. Data are represented as mean ± SEM. P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001, compared to control if not designated.
    Figure Legend Snippet: (A) Top: timeline of the experiment. Bottom: representative images showing that knocking down N6AMT1 reversed sensory axon regeneration impaired by down regulation of ALKBH1 3 days post-nerve injury. The right column shows enlarged images of areas in the dashed yellow boxes. The red line indicates the crush sites and the read arrows label regenerating axon tips. Scale bar, 1 mm for the left panel and 0.5 mm for the right panel. (B) Quantification of average length of regenerating axons under conditions shown in (A). One-way ANOVA followed by Tukey’s multiple comparisons test, P < 0.0001, n = 7-9 mice in each condition. (C) Cumulative distribution curves showing that knocking down N6AMT1 rescued axon regeneration impaired by ALKBH1 knockdown. Data are represented as mean ± SEM. P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001, compared to control if not designated.

    Techniques Used:

    (A) Real-time PCR analyses of changed transcription of 18 genes in sensory neurons after knocking down ALKBH1. The 5 significantly down regulated genes were underlined in red. One sample t test, P = 0.0096, 0.0118, and 0.0202 for Efna1, Id1, and Nrn1 , respectively. P < 0.0001 for ATG9B or C1QL4 . n = 3 independent experiments. (B) Real-time PCR analysis of ATG9B mRNA levels after knocking down ALKBH1 or double knocking down ALKBH1 and N6AMT1. One-way ANOVA followed by Tukey’s multiple comparisons test, P < 0.0001, n = 3 independent experiments. (C) Real-time PCR analysis of C1QL4 mRNA levels after knocking down ALKBH1 or double knocking down ALKBH1 and N6AMT1. One-way ANOVA followed by Tukey’s multiple comparisons test, P < 0.0001, n = 3 independent experiments. (D) Real-time PCR analysis of Id1 mRNA levels after knocking down ALKBH1 or double knocking down ALKBH1 and N6AMT1. One-way ANOVA followed by Tukey’s multiple comparisons test, P = 0.0001, n = 3 independent experiments. (E) Real-time PCR analysis of Nrn1 mRNA levels after knocking down ALKBH1 or double knocking down ALKBH1 and N6AMT1. One-way ANOVA followed by Tukey’s multiple comparisons test, P = 0.0368, n = 3 independent experiments. Data are represented as mean ± SEM. P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001, compared to control if not designated.
    Figure Legend Snippet: (A) Real-time PCR analyses of changed transcription of 18 genes in sensory neurons after knocking down ALKBH1. The 5 significantly down regulated genes were underlined in red. One sample t test, P = 0.0096, 0.0118, and 0.0202 for Efna1, Id1, and Nrn1 , respectively. P < 0.0001 for ATG9B or C1QL4 . n = 3 independent experiments. (B) Real-time PCR analysis of ATG9B mRNA levels after knocking down ALKBH1 or double knocking down ALKBH1 and N6AMT1. One-way ANOVA followed by Tukey’s multiple comparisons test, P < 0.0001, n = 3 independent experiments. (C) Real-time PCR analysis of C1QL4 mRNA levels after knocking down ALKBH1 or double knocking down ALKBH1 and N6AMT1. One-way ANOVA followed by Tukey’s multiple comparisons test, P < 0.0001, n = 3 independent experiments. (D) Real-time PCR analysis of Id1 mRNA levels after knocking down ALKBH1 or double knocking down ALKBH1 and N6AMT1. One-way ANOVA followed by Tukey’s multiple comparisons test, P = 0.0001, n = 3 independent experiments. (E) Real-time PCR analysis of Nrn1 mRNA levels after knocking down ALKBH1 or double knocking down ALKBH1 and N6AMT1. One-way ANOVA followed by Tukey’s multiple comparisons test, P = 0.0368, n = 3 independent experiments. Data are represented as mean ± SEM. P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001, compared to control if not designated.

    Techniques Used: Real-time Polymerase Chain Reaction

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    Revvity Signals sirnas targeting mouse alkbh1
    (A) Representative images of dot blot assay showing that the specific antibody against DNA N6-mA can detect the levels of N6-mA in mouse DRG neurons, the differentiated neuronal cell line CAD cells, and the Hela cells, in a dose-dependent manner. (B) Representative dot blot images showing that knocking down <t>ALKBH1</t> in CAD cells led to increased level of N6-mA, whereas knocking down N6AMT1 alone had little effect. However, when ALKBH1 and N6AMT1 were knocked down together, the level of N6-mA was reversed back to the control condition. (C) Quantification of (B) (one-way ANOVA followed by Tukey’s multiple comparisons test, P = 0.0013, n = 4-5 independent experiments). (D) Representative dot blot images showing that treating the isolated DNA samples with DNase resulted in total loss of sample signals and associated N6-mA signals in either CAD or Hela cells. (E) Representative dot blot images showing that knocking down ALKBH1 in adult mouse DRG neurons led to increased level of N6-mA, whereas knocking down N6AMT1 alone had little effect. However, when ALKBH1 and N6AMT1 were knocked down together, the level of N6-mA was reversed back to the control condition. (F) Quantification of (B) (one-way ANOVA followed by Tukey’s multiple comparisons test, P = 0.002, n = 4 independent experiments). Data are represented as mean ± SEM. P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001, compared to control if not designated.
    Sirnas Targeting Mouse Alkbh1, supplied by Revvity Signals, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/sirnas targeting mouse alkbh1/product/Revvity Signals
    Average 86 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    sirnas targeting mouse alkbh1 - by Bioz Stars, 2024-09
    86/100 stars
    		  Buy from Supplier

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    (A) Representative images of dot blot assay showing that the specific antibody against DNA N6-mA can detect the levels of N6-mA in mouse DRG neurons, the differentiated neuronal cell line CAD cells, and the Hela cells, in a dose-dependent manner. (B) Representative dot blot images showing that knocking down ALKBH1 in CAD cells led to increased level of N6-mA, whereas knocking down N6AMT1 alone had little effect. However, when ALKBH1 and N6AMT1 were knocked down together, the level of N6-mA was reversed back to the control condition. (C) Quantification of (B) (one-way ANOVA followed by Tukey’s multiple comparisons test, P = 0.0013, n = 4-5 independent experiments). (D) Representative dot blot images showing that treating the isolated DNA samples with DNase resulted in total loss of sample signals and associated N6-mA signals in either CAD or Hela cells. (E) Representative dot blot images showing that knocking down ALKBH1 in adult mouse DRG neurons led to increased level of N6-mA, whereas knocking down N6AMT1 alone had little effect. However, when ALKBH1 and N6AMT1 were knocked down together, the level of N6-mA was reversed back to the control condition. (F) Quantification of (B) (one-way ANOVA followed by Tukey’s multiple comparisons test, P = 0.002, n = 4 independent experiments). Data are represented as mean ± SEM. P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001, compared to control if not designated.

    Journal: bioRxiv

    Article Title: N6-methyladenine DNA demethylase ALKBH1 regulates mammalian axon regeneration

    doi: 10.1101/2020.08.19.258038

    Figure Lengend Snippet: (A) Representative images of dot blot assay showing that the specific antibody against DNA N6-mA can detect the levels of N6-mA in mouse DRG neurons, the differentiated neuronal cell line CAD cells, and the Hela cells, in a dose-dependent manner. (B) Representative dot blot images showing that knocking down ALKBH1 in CAD cells led to increased level of N6-mA, whereas knocking down N6AMT1 alone had little effect. However, when ALKBH1 and N6AMT1 were knocked down together, the level of N6-mA was reversed back to the control condition. (C) Quantification of (B) (one-way ANOVA followed by Tukey’s multiple comparisons test, P = 0.0013, n = 4-5 independent experiments). (D) Representative dot blot images showing that treating the isolated DNA samples with DNase resulted in total loss of sample signals and associated N6-mA signals in either CAD or Hela cells. (E) Representative dot blot images showing that knocking down ALKBH1 in adult mouse DRG neurons led to increased level of N6-mA, whereas knocking down N6AMT1 alone had little effect. However, when ALKBH1 and N6AMT1 were knocked down together, the level of N6-mA was reversed back to the control condition. (F) Quantification of (B) (one-way ANOVA followed by Tukey’s multiple comparisons test, P = 0.002, n = 4 independent experiments). Data are represented as mean ± SEM. P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001, compared to control if not designated.

    Article Snippet: The siRNAs targeting mouse ALKBH1, N6AMT1 and non-targeting siRNA control were purchased from Dharmacon.

    Techniques: Dot Blot, Isolation

    (A) Real-time PCR analysis showing significantly reduced mRNA level of ALKBH1 3 days after electroporation of siRNAs against ALKBH1 (siALKBH1) or together with siRNAs against N6AMT1 (siALK.+siN6A.). One sample t test, P = 0.0315 and 0.0092 for siALKBH1 and siALK.+siN6A., respectively. n = 3 independent experiments. (B) Real-time PCR analysis showing significantly reduced mRNA level of N6AMT1 3 days after electroporation of siRNAs against ALKBH1 and N6AMT1. One-sample t test, P < 0.0001, n = 3 independent experiments. (C) Top: time line of the culture and replate experiments. Bottom: representative images of cultured sensory neurons 24 hours post-replating after knocking down ALKBH1, N6AMT1, or together with siRNAs (siALKBH1, siN6AMT1, or siALK.+siN6A.). Scale bar, 500 μm. (D) Quantification of average lengths of the longest neurites (one-way ANOVA followed by Tukey’s multiple comparisons test, P < 0.0001, n = 7-10 independent experiments). (E) Quantification of percentage of neurons with axons (one-way ANOVA followed by Tukey’s multiple comparisons test, P = 0.0827, n = 4 independent experiments). Data are represented as mean ± SEM. P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001, compared to control if not designated.

    Journal: bioRxiv

    Article Title: N6-methyladenine DNA demethylase ALKBH1 regulates mammalian axon regeneration

    doi: 10.1101/2020.08.19.258038

    Figure Lengend Snippet: (A) Real-time PCR analysis showing significantly reduced mRNA level of ALKBH1 3 days after electroporation of siRNAs against ALKBH1 (siALKBH1) or together with siRNAs against N6AMT1 (siALK.+siN6A.). One sample t test, P = 0.0315 and 0.0092 for siALKBH1 and siALK.+siN6A., respectively. n = 3 independent experiments. (B) Real-time PCR analysis showing significantly reduced mRNA level of N6AMT1 3 days after electroporation of siRNAs against ALKBH1 and N6AMT1. One-sample t test, P < 0.0001, n = 3 independent experiments. (C) Top: time line of the culture and replate experiments. Bottom: representative images of cultured sensory neurons 24 hours post-replating after knocking down ALKBH1, N6AMT1, or together with siRNAs (siALKBH1, siN6AMT1, or siALK.+siN6A.). Scale bar, 500 μm. (D) Quantification of average lengths of the longest neurites (one-way ANOVA followed by Tukey’s multiple comparisons test, P < 0.0001, n = 7-10 independent experiments). (E) Quantification of percentage of neurons with axons (one-way ANOVA followed by Tukey’s multiple comparisons test, P = 0.0827, n = 4 independent experiments). Data are represented as mean ± SEM. P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001, compared to control if not designated.

    Article Snippet: The siRNAs targeting mouse ALKBH1, N6AMT1 and non-targeting siRNA control were purchased from Dharmacon.

    Techniques: Real-time Polymerase Chain Reaction, Electroporation, Cell Culture

    (A) Real-time PCR analysis showing significantly elevated mRNA level of ALKBH1 in sensory neurons 1-day post-nerve injury (one sample t test, P = 0.0014, n = 4 independent experiments). (B) Real-time PCR analysis showing significantly reduced mRNA level of ALKBH1 in vivo after 3 days after electroporation (one sample t test, P = 0.0076, n = 4 independent experiments). (C) Top: timeline of the experiment. Bottom: representative images showing that knocking down ALKBH1 had no effect on slower sensory axon regeneration 2 days post-nerve injury. The right column shows enlarged images of areas in the dashed yellow boxes. The red line indicates the crush sites and the read arrows label regenerating axon tips. Scale bar, 1 mm for the left panel and 0.5 mm for the right panel. (D) Quantification of average length of regenerating sensory axons showing no significant difference between the control group and the ALKBH1 knockdown group 2 days post-nerve injury (unpaired student t test, P = 0.6387, n = 7 mice in each condition). (E) Cumulative distribution curves showing similar axon regeneration between the control group and the ALKBH1 knockdown group 2 days post-nerve injury. (F) Top: timeline of the experiment. Bottom: representative images showing that knocking down N6AMT1 had no effect on slower sensory axon regeneration 2 days post-nerve injury. The right column shows enlarged images of areas in the dashed yellow boxes. The red line indicates the crush sites and the read arrows label regenerating axon tips. Scale bar, 1 mm for the left panel and 0.5 mm for the right panel. (G) Quantification of average length of regenerating sensory axons showing no significant difference between the control group and the N6AMT1 knockdown group 2 days post-nerve injury (unpaired student t test, P = 0.2715, n = 7 and 10 mice in control and siN6AMT1 group, respectively). (H) Cumulative distribution curves showing similar axon regeneration between the control group and the N6AMT1 knockdown group 2 days post-nerve injury. Data are represented as mean ± SEM. P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001, compared to control if not designated.

    Journal: bioRxiv

    Article Title: N6-methyladenine DNA demethylase ALKBH1 regulates mammalian axon regeneration

    doi: 10.1101/2020.08.19.258038

    Figure Lengend Snippet: (A) Real-time PCR analysis showing significantly elevated mRNA level of ALKBH1 in sensory neurons 1-day post-nerve injury (one sample t test, P = 0.0014, n = 4 independent experiments). (B) Real-time PCR analysis showing significantly reduced mRNA level of ALKBH1 in vivo after 3 days after electroporation (one sample t test, P = 0.0076, n = 4 independent experiments). (C) Top: timeline of the experiment. Bottom: representative images showing that knocking down ALKBH1 had no effect on slower sensory axon regeneration 2 days post-nerve injury. The right column shows enlarged images of areas in the dashed yellow boxes. The red line indicates the crush sites and the read arrows label regenerating axon tips. Scale bar, 1 mm for the left panel and 0.5 mm for the right panel. (D) Quantification of average length of regenerating sensory axons showing no significant difference between the control group and the ALKBH1 knockdown group 2 days post-nerve injury (unpaired student t test, P = 0.6387, n = 7 mice in each condition). (E) Cumulative distribution curves showing similar axon regeneration between the control group and the ALKBH1 knockdown group 2 days post-nerve injury. (F) Top: timeline of the experiment. Bottom: representative images showing that knocking down N6AMT1 had no effect on slower sensory axon regeneration 2 days post-nerve injury. The right column shows enlarged images of areas in the dashed yellow boxes. The red line indicates the crush sites and the read arrows label regenerating axon tips. Scale bar, 1 mm for the left panel and 0.5 mm for the right panel. (G) Quantification of average length of regenerating sensory axons showing no significant difference between the control group and the N6AMT1 knockdown group 2 days post-nerve injury (unpaired student t test, P = 0.2715, n = 7 and 10 mice in control and siN6AMT1 group, respectively). (H) Cumulative distribution curves showing similar axon regeneration between the control group and the N6AMT1 knockdown group 2 days post-nerve injury. Data are represented as mean ± SEM. P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001, compared to control if not designated.

    Article Snippet: The siRNAs targeting mouse ALKBH1, N6AMT1 and non-targeting siRNA control were purchased from Dharmacon.

    Techniques: Real-time Polymerase Chain Reaction, In Vivo, Electroporation

    (A) Top: timeline of the experiment. Bottom: representative images showing that knocking down ALKBH1 significantly impaired fast sensory axon regeneration 3 days post-nerve injury. The right column shows enlarged images of areas in the dashed yellow boxes. The red line indicates the crush sites and the read arrows label regenerating axon tips. Scale bar, 1 mm for the left panel and 0.5 mm for the right panel. (B) Quantification of average length of regenerating axons showing that knocking down ALKBH1 significantly impaired sensory axon regeneration 3 days post-nerve injury (unpaired student t test, P < 0.0001, n = 8 mice in each condition). (C) Cumulative distribution curves showing that ALKBH1 knockdown resulted in significant reduced sensory axon regeneration 3 days post-nerve injury. (D) Top: timeline of the experiment. Bottom: representative images showing that knocking down ALKBH1 significantly impaired fast sensory axon regeneration 4 days post-nerve injury. The right column shows enlarged images of areas in the dashed yellow boxes. The red line indicates the crush sites and the read arrows label regenerating axon tips. Scale bar, 1 mm for the left panel and 0.5 mm for the right panel. (E) Quantification of average length of regenerating axons showing that knocking down ALKBH1 significantly impaired sensory axon regeneration 4 days post-nerve injury (unpaired student t test, P = 0.018, n = 4 mice in each condition). (F) Cumulative distribution curves showing that ALKBH1 knockdown resulted in significant reduced sensory axon regeneration 4 days post-nerve injury. Data are represented as mean ± SEM. P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001, compared to control if not designated.

    Journal: bioRxiv

    Article Title: N6-methyladenine DNA demethylase ALKBH1 regulates mammalian axon regeneration

    doi: 10.1101/2020.08.19.258038

    Figure Lengend Snippet: (A) Top: timeline of the experiment. Bottom: representative images showing that knocking down ALKBH1 significantly impaired fast sensory axon regeneration 3 days post-nerve injury. The right column shows enlarged images of areas in the dashed yellow boxes. The red line indicates the crush sites and the read arrows label regenerating axon tips. Scale bar, 1 mm for the left panel and 0.5 mm for the right panel. (B) Quantification of average length of regenerating axons showing that knocking down ALKBH1 significantly impaired sensory axon regeneration 3 days post-nerve injury (unpaired student t test, P < 0.0001, n = 8 mice in each condition). (C) Cumulative distribution curves showing that ALKBH1 knockdown resulted in significant reduced sensory axon regeneration 3 days post-nerve injury. (D) Top: timeline of the experiment. Bottom: representative images showing that knocking down ALKBH1 significantly impaired fast sensory axon regeneration 4 days post-nerve injury. The right column shows enlarged images of areas in the dashed yellow boxes. The red line indicates the crush sites and the read arrows label regenerating axon tips. Scale bar, 1 mm for the left panel and 0.5 mm for the right panel. (E) Quantification of average length of regenerating axons showing that knocking down ALKBH1 significantly impaired sensory axon regeneration 4 days post-nerve injury (unpaired student t test, P = 0.018, n = 4 mice in each condition). (F) Cumulative distribution curves showing that ALKBH1 knockdown resulted in significant reduced sensory axon regeneration 4 days post-nerve injury. Data are represented as mean ± SEM. P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001, compared to control if not designated.

    Article Snippet: The siRNAs targeting mouse ALKBH1, N6AMT1 and non-targeting siRNA control were purchased from Dharmacon.

    Techniques:

    (A) Top: timeline of the experiment. Bottom: representative images showing that knocking down N6AMT1 reversed sensory axon regeneration impaired by down regulation of ALKBH1 3 days post-nerve injury. The right column shows enlarged images of areas in the dashed yellow boxes. The red line indicates the crush sites and the read arrows label regenerating axon tips. Scale bar, 1 mm for the left panel and 0.5 mm for the right panel. (B) Quantification of average length of regenerating axons under conditions shown in (A). One-way ANOVA followed by Tukey’s multiple comparisons test, P < 0.0001, n = 7-9 mice in each condition. (C) Cumulative distribution curves showing that knocking down N6AMT1 rescued axon regeneration impaired by ALKBH1 knockdown. Data are represented as mean ± SEM. P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001, compared to control if not designated.

    Journal: bioRxiv

    Article Title: N6-methyladenine DNA demethylase ALKBH1 regulates mammalian axon regeneration

    doi: 10.1101/2020.08.19.258038

    Figure Lengend Snippet: (A) Top: timeline of the experiment. Bottom: representative images showing that knocking down N6AMT1 reversed sensory axon regeneration impaired by down regulation of ALKBH1 3 days post-nerve injury. The right column shows enlarged images of areas in the dashed yellow boxes. The red line indicates the crush sites and the read arrows label regenerating axon tips. Scale bar, 1 mm for the left panel and 0.5 mm for the right panel. (B) Quantification of average length of regenerating axons under conditions shown in (A). One-way ANOVA followed by Tukey’s multiple comparisons test, P < 0.0001, n = 7-9 mice in each condition. (C) Cumulative distribution curves showing that knocking down N6AMT1 rescued axon regeneration impaired by ALKBH1 knockdown. Data are represented as mean ± SEM. P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001, compared to control if not designated.

    Article Snippet: The siRNAs targeting mouse ALKBH1, N6AMT1 and non-targeting siRNA control were purchased from Dharmacon.

    Techniques:

    (A) Real-time PCR analyses of changed transcription of 18 genes in sensory neurons after knocking down ALKBH1. The 5 significantly down regulated genes were underlined in red. One sample t test, P = 0.0096, 0.0118, and 0.0202 for Efna1, Id1, and Nrn1 , respectively. P < 0.0001 for ATG9B or C1QL4 . n = 3 independent experiments. (B) Real-time PCR analysis of ATG9B mRNA levels after knocking down ALKBH1 or double knocking down ALKBH1 and N6AMT1. One-way ANOVA followed by Tukey’s multiple comparisons test, P < 0.0001, n = 3 independent experiments. (C) Real-time PCR analysis of C1QL4 mRNA levels after knocking down ALKBH1 or double knocking down ALKBH1 and N6AMT1. One-way ANOVA followed by Tukey’s multiple comparisons test, P < 0.0001, n = 3 independent experiments. (D) Real-time PCR analysis of Id1 mRNA levels after knocking down ALKBH1 or double knocking down ALKBH1 and N6AMT1. One-way ANOVA followed by Tukey’s multiple comparisons test, P = 0.0001, n = 3 independent experiments. (E) Real-time PCR analysis of Nrn1 mRNA levels after knocking down ALKBH1 or double knocking down ALKBH1 and N6AMT1. One-way ANOVA followed by Tukey’s multiple comparisons test, P = 0.0368, n = 3 independent experiments. Data are represented as mean ± SEM. P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001, compared to control if not designated.

    Journal: bioRxiv

    Article Title: N6-methyladenine DNA demethylase ALKBH1 regulates mammalian axon regeneration

    doi: 10.1101/2020.08.19.258038

    Figure Lengend Snippet: (A) Real-time PCR analyses of changed transcription of 18 genes in sensory neurons after knocking down ALKBH1. The 5 significantly down regulated genes were underlined in red. One sample t test, P = 0.0096, 0.0118, and 0.0202 for Efna1, Id1, and Nrn1 , respectively. P < 0.0001 for ATG9B or C1QL4 . n = 3 independent experiments. (B) Real-time PCR analysis of ATG9B mRNA levels after knocking down ALKBH1 or double knocking down ALKBH1 and N6AMT1. One-way ANOVA followed by Tukey’s multiple comparisons test, P < 0.0001, n = 3 independent experiments. (C) Real-time PCR analysis of C1QL4 mRNA levels after knocking down ALKBH1 or double knocking down ALKBH1 and N6AMT1. One-way ANOVA followed by Tukey’s multiple comparisons test, P < 0.0001, n = 3 independent experiments. (D) Real-time PCR analysis of Id1 mRNA levels after knocking down ALKBH1 or double knocking down ALKBH1 and N6AMT1. One-way ANOVA followed by Tukey’s multiple comparisons test, P = 0.0001, n = 3 independent experiments. (E) Real-time PCR analysis of Nrn1 mRNA levels after knocking down ALKBH1 or double knocking down ALKBH1 and N6AMT1. One-way ANOVA followed by Tukey’s multiple comparisons test, P = 0.0368, n = 3 independent experiments. Data are represented as mean ± SEM. P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001, compared to control if not designated.

    Article Snippet: The siRNAs targeting mouse ALKBH1, N6AMT1 and non-targeting siRNA control were purchased from Dharmacon.

    Techniques: Real-time Polymerase Chain Reaction