Journal: eLife

Article Title: Pathway-specific dysregulation of striatal excitatory synapses by LRRK2 mutations

doi: 10.7554/eLife.58997

Figure Lengend Snippet:

Article Snippet: Antibody , HOMER1 polyclonal antibody rabbit polyclonal , Proteintech , RRID: AB_2295573 , WB (1:1000).

Techniques: Recombinant, Saline, Bicinchoninic Acid Protein Assay, Western Blot, Stripping, Software, Plasmid Preparation

Journal: Aging (Albany NY)

Article Title: Liraglutide preconditioning attenuates myocardial ischemia/ reperfusion injury via homer1 activation

doi: 10.18632/aging.202429

Figure Lengend Snippet: Liraglutide promoted expression of Homer1 in H9C2 cells against H/R. H9C2 cells were pretreated with 200 nmol/L liraglutide or vehicle 30 min before H/R treatment, and the expression of Homer1 mRNA ( A ), protein ( B , C ), and intracellular localization ( D , E ) were examined by RT-qPCR, western blot and immunofluorescence staining.

Article Snippet: Slides were incubated with Homer1 rabbit polyclonal primary antibody (1:200, Proteintech Group, Inc, Wuhan, Hubei, China) at 4° C in overnight.

Techniques: Expressing, Quantitative RT-PCR, Western Blot, Immunofluorescence, Staining

Journal: Aging (Albany NY)

Article Title: Liraglutide preconditioning attenuates myocardial ischemia/ reperfusion injury via homer1 activation

doi: 10.18632/aging.202429

Figure Lengend Snippet: Effect of Homer1 overexpression on liraglutide-induced cardiomyocyte protection. H9C2 cell were transfected with LV-H1 or LV-Con for 72 h before H/R treatment, and the expression of Homer1 was examined by immunofluorescence staining ( A , B ). The cell viability ( C ) and LDH release ( D ) were assayed 24 h later, and the Ca 2+ concentration in ER ([Ca 2+ ] ER ) was measured at 5, 10 and 15 min after LV-Con and LV-H1 ( E ).

Article Snippet: Slides were incubated with Homer1 rabbit polyclonal primary antibody (1:200, Proteintech Group, Inc, Wuhan, Hubei, China) at 4° C in overnight.

Techniques: Over Expression, Transfection, Expressing, Immunofluorescence, Staining, Concentration Assay

Journal: Aging (Albany NY)

Article Title: Liraglutide preconditioning attenuates myocardial ischemia/ reperfusion injury via homer1 activation

doi: 10.18632/aging.202429

Figure Lengend Snippet: Effect of Homer1 knock out on liraglutide-induced cardiomyocyte protection. H9C2 cell were transfected with si-Homer1, si-NC or vehicle for 72 h before H/R treatment, and the expression of Homer1 was examined by immunofluorescence staining ( A , B ). The cell viability ( C ) and LDH release ( D ) were assayed 24 h later, and the Ca 2+ concentration in ER ([Ca 2+ ] ER ) was measured at 5, 10 and 15 min after si-Homer1 and si-NC ( E ).

Article Snippet: Slides were incubated with Homer1 rabbit polyclonal primary antibody (1:200, Proteintech Group, Inc, Wuhan, Hubei, China) at 4° C in overnight.

Techniques: Knock-Out, Transfection, Expressing, Immunofluorescence, Staining, Concentration Assay

Journal: bioRxiv

Article Title: m6a methylation orchestrates IMP1 regulation of microtubules during human neuronal differentiation

doi: 10.1101/2023.09.12.557394

Figure Lengend Snippet: a, Schematic showing differentiation strategy for human neurogenesis from iPSC using a previously described protocol used for iCLIP. Three technical replicates from three independent iPSC lines were used for NPCs neurons. b , Representative confocal images of human iPSCs differentiated into neurons immunostained for IMP1, βIII-tubulin and nuclei labelled with Dapi at NPC and neuronal stages. Scale bar, 20 μm. c, Barplot showing the percentage of unique cDNAs mapping to each region of the transcriptome for neurons and NPC. Counts were normalised to the number of genomic nucleotides corresponding to each region, and percentage of total counts were calculated. Percentage is as follows: NPC - 5’UTR 11.44, CDS 16.13, 3’UTR 46.36, Intron 0.81, ncRNA 25.13, Intergenic 0.13; MN - 5’UTR 7.32, CDS 8.71, 3’UTR 67.12, Intron 0.37, ncRNA 16.37, Intergenic 0.11. d, Metagene plot showing IMP1 crosslink site distribution in NPC (top) and neurons (bottom). f, Mapping of IMP1 iCLIP crosslink signal (top) and RNAseq coverage signal (bottom) in neurons and NPC. For iCLIP - tracks signal for each replicate either in blue for neurons or green for NPC and the merge of all replicates in red are shown. g, Volcano plot of IMP1 bound peaks normalised by gene expression changes in NPC vs neurons. Preferential binding peaks in NPC and neurons are shown respectively in pink and orange using the following criteria: log2 FC < −1 or >1 (vertical dashed line), adjusted p-value < 0.01 (horizontal dashed line). h, Relative expression of IMP1 over GAPDH measured by RT-qPCR (left) and WB (right) at NPC and neuronal stages. Points presented are different biological replicates. Boxplot presented is the median (middle line), interquartile range and whiskers. For qPCR experiment n=4 independent iPSC lines, p-values calculated using P-values calculated using a two-sided Mann–Whitney test, * P <0.05. Data presented is the mean +/−SEM. For WB experiment n= 6 technical replicates from 4 independent iPSC lines in 2 independent experiments. Neuronal values are normalised on relative expression of corresponding clones. Data presented is the mean +/−SEM. P-values calculated using a two-sided Mann– Whitney test, * P <0.05.

Article Snippet: Rabbit anti-IMP1 (MBL, RN007P), rabbit anti-IgG (Proteintech, 30000-0-AP), mouse anti-Actb (Sigma, A2228), rabbit anti-H3 (Abcam, ab201456), chicken anti-Homer-1 (Synaptic System, 160006), mouse anti-Synaptotagmin1 (Synaptic System, 105011), mouse anti-SMI-35 (Biolegend, 835603), rabbit anti-MAP1B (Proteintech, 21633-1-AP), mouse anti-MAP2 (Abcam, ab11267), chicken anti-MAP2 (Abcam, ab5392), chicken anti-beta III tubulin (Abcam, ab41489), mouse anti-beta IV tubulin (Abcam, ab11315), rabbit anti-DCX (Proteintech, 13925-1-AP), rabbit anti-MAPRE2 (Proteintech, 10364-1-AP), rabbit anti-Stathmin1 (Proteintech,11157-1-AP), mouse anti-MAPT (Abcam, ab80579), rabbit anti-CRIPT (Proteintech,11211-1-AP), mouse anti-m6A (Abcam, ab151230), rabbit anti-METTL3 (Proteintech, 15073-1-AP).

Techniques: Expressing, Binding Assay, Quantitative RT-PCR, MANN-WHITNEY, Clone Assay

Journal: bioRxiv

Article Title: m6a methylation orchestrates IMP1 regulation of microtubules during human neuronal differentiation

doi: 10.1101/2023.09.12.557394

Figure Lengend Snippet: a, Volcano plot of proteins with differential expression in neurons treated with IMP1 siRNA (siIMP1) vs non-targeting control (siCTRL). Downregulated, unchanged and upregulated proteins are shown respectively in pink, grey and orange with the following criteria: log2 FC (z-scored) < −1 or >1 (vertical dashed line), p-value < 0.05 (horizontal dashed line), one-sample Student’s t-test; n = 3 independent iPSC lines for each condition. Embedded representative image of western blot showing expression of IMP1 and H3 as loading control from knockdown experiment. b , Barplot plot showing the number of proteins upregulated or downregulated in neurons and NPC treated with IMP1 siRNA (siIMP1) vs non-targeting control (siCTRL). For downregulated proteins selection was based on log2 FC < −1, p-value < 0.05, one-sample Student’s t-test, for upregulated proteins selection was based on log2 FC > 1, p-value < 0.05, one-sample Student’s t-test. For neurons, n = 3 independent iPSC lines for each condition; for NPCs, n= 2 independent IPSC lines for each condition. c , Top GO term enrichments of downregulated proteins in IMP1 siRNA treated neurons (log2 FC < −1, P < 0.05). Redundancy was removed using REVIGO and top 20 significantly enriched GO terms are shown. Terms are ranked based on fold enrichment. All terms presented have a false discovery rate (FDR) <0.05. Only three proteins are found in the “protein myristoylation” and “N-terminal protein myristoylation” terms; two of these are protein phosphatases. d , Volcano plot of genes with differential expression in IMP1 knockdown vs control neurons for RNAseq experiment. Transcripts which correspond to differentially expressed proteins detected by MS are shown in red. Vertical dashed lines indicate cut-off of log2 FC (1.5 or - 1.5), the horizontal dashed lines indicate cut-off of p-value (0.05); n = 3 independent iPSC lines for each condition. Embedded - representative image of western blot showing expression of IMP1, and H3 as loading control from knockdown experiment. e , Volcano plot of proteins with differential expression in neurons treated with IMP1 siRNA vs non-targeting control (detected by MS) for IMP1 bound transcripts only (detected by iCLIP). Downregulated, unchanged and upregulated proteins are shown respectively in pink, grey and orange. IMP1, CLASP1 and PEBP1 are highlighted with blue dots and blue squares. For downregulated proteins selection was based on log2 FC < −1, p-value < 0.05, one-sample Student’s t-test, for upregulated proteins selection was based on log2 FC > 1, p-value < 0.05, one-sample Student’s t-test, for transcripts containing at least one IMP1 binding site. For MS, n=3 independent iPSC lines for each condition for neurons and n=2 independent iPSC lines for each condition for NPCs; for iCLIP, n=3 technical replicates from 3 independent iPSC lines for neurons, and n=6 biological+technical replicates for NPCs f , Barplot plot showing the number of proteins upregulated or downregulated in neurons and NPC treated with IMP1 siRNA vs non-targeting control (detected by MS) for IMP1 bound transcripts only (detected by iCLIP). For downregulated proteins selection was based on log2 FC < −1, p-value < 0.05, one-sample Student’s t-test, for upregulated proteins selection was based on log2 FC > 1, p-value < 0.05, one-sample Student’s t-test, for transcripts containing at least one IMP1 binding site. For MS, n=3 independent iPSC lines for each condition for neurons and n=2 independent iPSC lines for each condition for NPCs; for iCLIP, n=3 technical replicates from 3 independent iPSC lines for neurons, and n=6 biological+technical replicates for NPCs. g , Cumulative distribution plot of log2 FC in protein expression between IMP1 KD and control in neurons detected by MS was plotted for corresponding RNA classified by the number of IMP1 peaks detected by iCLIP. For MS, n=3 independent iPSC lines for each condition; for iCLIP, n=3 technical replicates from 3 independent iPSC lines. P-values were calculated using a two-sided Kolmogorov–Smirnov test comparing 1-2 peaks, 3-5 peaks or 6 and + peaks to 0 peaks. P-values are reported on the graph. h , Number of IMP1 peaks per gene regions detected by iCLIP for GO categories related to microtubules, neuronal or other pathways classified by PANTHER - “Regulation of cytoskeleton organisation”, “Microtubule-based process”, “Neuron projection development”, “Positive regulation protein kinase activity”, “Small molecule biosynthetic process”. Number of genes in each category is in the same range (between 26 and 30). Data presented is the mean +/− SEM. Each category is represented by a number above the corresponding bar. P- values calculated using a two-sided Mann–Whitney test and added to the plot. Comparison for the same cell type was performed. i , STRING analysis of protein–protein interaction network on downregulated proteins (log2 FC < −1, P < 0.05) in IMP1 siRNA treated neurons compared to control siRNA for IMP1 bound transcripts. The GO category represented is “microtubule-based process” as classified by PANTHER.

Article Snippet: Rabbit anti-IMP1 (MBL, RN007P), rabbit anti-IgG (Proteintech, 30000-0-AP), mouse anti-Actb (Sigma, A2228), rabbit anti-H3 (Abcam, ab201456), chicken anti-Homer-1 (Synaptic System, 160006), mouse anti-Synaptotagmin1 (Synaptic System, 105011), mouse anti-SMI-35 (Biolegend, 835603), rabbit anti-MAP1B (Proteintech, 21633-1-AP), mouse anti-MAP2 (Abcam, ab11267), chicken anti-MAP2 (Abcam, ab5392), chicken anti-beta III tubulin (Abcam, ab41489), mouse anti-beta IV tubulin (Abcam, ab11315), rabbit anti-DCX (Proteintech, 13925-1-AP), rabbit anti-MAPRE2 (Proteintech, 10364-1-AP), rabbit anti-Stathmin1 (Proteintech,11157-1-AP), mouse anti-MAPT (Abcam, ab80579), rabbit anti-CRIPT (Proteintech,11211-1-AP), mouse anti-m6A (Abcam, ab151230), rabbit anti-METTL3 (Proteintech, 15073-1-AP).

Techniques: Expressing, Western Blot, Selection, Binding Assay, Activity Assay, MANN-WHITNEY, Comparison

Journal: bioRxiv

Article Title: m6a methylation orchestrates IMP1 regulation of microtubules during human neuronal differentiation

doi: 10.1101/2023.09.12.557394

Figure Lengend Snippet: a, Representative LI-Cor scanning visualisation of nitrocellulose membrane of poly(A)+ RNA crosslinked to an m6A antibody or IgG in neurons. Visualisation uses the infrared adaptor ligated to antibody-m6A RNA complex. The excised portion of the membrane used to generate miCLIP libraries is shown (dashed white masks) b , Metagene plot showing m6A sites distribution in neurons. Embedded - consensus motif from HOMER motif discovery tools. Motif with best p-value is shown. c , Venn diagram representing the number of overlapping m6A sites between neurons and NPCs. d , Number of IMP1-m6A peaks detected by overlap between miCLIP and iCLIP for downregulated, upregulated, or unchanged proteins - respectively log2 FC < −1 in pink; −1 > log2 FC< 1 in grey; log2 FC > −1 in orange - from knockdown experiments in neurons (see ). Data presented is the mean +/− SEM. For MS, n=3 independent iPSC lines for each condition, for iCLIP, n=3 technical replicates from 3 independent iPSC lines and for miCLIP, n=2 technical replicates from 4 independent iPSC lines. P-values calculated using Kruskal– Wallis with Dunn’s multiple comparisons test, *P < 0.05, ns = not significant. e, Mapping tracks of m6A sites detected by miCLIP (top) and RNAseq coverage signal (bottom) in neurons and NPC. The 3’UTR of three different genes is visualised using Integrative Genomics Viewer (IGV) software. f , Cumulative distribution plot of the log2 FC in protein expression between IMP1 knockdown and control neurons detected by MS was plotted for corresponding RNA classified by the number of IMP1-m6A peaks detected by iCLIP and miCLIP. For MS, n=3 independent iPSC lines for each condition; for iCLIP, n=3 technical replicates from 3 independent iPSC lines, for miCLIP, n=2 technical replicates from 4 independent iPSC lines. P-values calculated using two-sided Kolmogorov–Smirnov test comparing 1-2 peaks or 3-5 peaks or 6 and + peaks to 0 peaks. P-values are reported on the plot. g , Number of IMP1-m6A peaks detected by overlap between miCLIP and iCLIP for GO categories related to microtubules, neuronal or other pathways classified by PANTHER - “Regulation of cytoskeleton organisation”, “Microtubule-based process”, “Neuron projection development”, “Positive regulation protein kinase activity”, “Small molecule biosynthetic process”. Number of genes in each category is in the same range (between 26 and 30). Data presented is the mean +/− SEM. For iCLIP, n=3 technical replicates from 3 independent iPSC lines, for miCLIP, n=2 technical replicates from 4 independent iPSC lines. Data presented is the mean +/−SEM. P-values calculated using a two-sided Mann–Whitney test, * P < 0.05, *** P < 0.001. h , Western blot analysis showing expression of IMP1, METTL3, TUBB4A, DCX, MAP2 and H3 as loading control in neurons treated with non-targeting control siRNA (siCTRL) or siRNA targeting METTL3 (siMETTL3). Representative image from 2 independent experiments performed on three technical replicates from 2 independent iPSC lines. i , Barplot of the percentage of proteins belonging to the “microtubules-based process” terms as defined by PANTHER GO classification for each of the following category : downregulated proteins as defined by MS (log2 FC < −1 and p-value >0.05), downregulated proteins with RNA targets directly bound by IMP1 as defined by MS and iCLIP, downregulated proteins with RNA targets directly bound by IMP1 through an m6A site as defined by MS, iCLIP and miCLIP. j , Schematic depiction of our mechanistic working model. In summary, m6A concentration increases during human neuronal differentiation, which orchestrates IMP1 regulation of microtubules during neuronal differentiation.

Article Snippet: Rabbit anti-IMP1 (MBL, RN007P), rabbit anti-IgG (Proteintech, 30000-0-AP), mouse anti-Actb (Sigma, A2228), rabbit anti-H3 (Abcam, ab201456), chicken anti-Homer-1 (Synaptic System, 160006), mouse anti-Synaptotagmin1 (Synaptic System, 105011), mouse anti-SMI-35 (Biolegend, 835603), rabbit anti-MAP1B (Proteintech, 21633-1-AP), mouse anti-MAP2 (Abcam, ab11267), chicken anti-MAP2 (Abcam, ab5392), chicken anti-beta III tubulin (Abcam, ab41489), mouse anti-beta IV tubulin (Abcam, ab11315), rabbit anti-DCX (Proteintech, 13925-1-AP), rabbit anti-MAPRE2 (Proteintech, 10364-1-AP), rabbit anti-Stathmin1 (Proteintech,11157-1-AP), mouse anti-MAPT (Abcam, ab80579), rabbit anti-CRIPT (Proteintech,11211-1-AP), mouse anti-m6A (Abcam, ab151230), rabbit anti-METTL3 (Proteintech, 15073-1-AP).

Techniques: Membrane, Software, Expressing, Activity Assay, MANN-WHITNEY, Western Blot, Concentration Assay