Article Figures & Data
Figures
- Figure 1.
Efficient knock-down of hnRNP A1 in differentiated Neuro-2a cells. A, Undifferentiated Neuro-2a cells were treated with four different siA1 duplex oligonucleotides for 72 h, which showed varying degrees of hnRNP A1 knock-down. B, Quantification of A demonstrating siA1#4 was the most potent siA1 duplex oligonucleotide to significantly decrease hnRNP A1 expression compared with siNEG. Unpaired t test (ns = non-significant, *p < 0.05, **p < 0.01); n = 3 biological replicates. Data are plotted as mean ± SEM. C, Neuro-2a transfection, differentiation and data collection protocol. D, Protein from differentiated Neuro-2a cells treated with either siNEG or siA1 for 72 h were assayed by Western blotting for hnRNP A1 and β-actin. E, Band densitometry of Western blottings as in D demonstrates a significant decrease in hnRNP A1 protein expression after 72 h of treatment with siA1 as compared with siNEG. Unpaired t test (***p < 0.001); n = 3 biological replicates. Data are plotted as mean ± SEM. F, Confirmation of decreased hnRNP A1 expression (green) following treatment with siA1 using immunocytochemistry. Scale bar: 20 μm. G, Corrected total hnRNP A1 nuclear fluorescence was measured using ImageJ. Cells in the siA1 condition demonstrated significant reduction in hnRNP A1 expression as compared with siNEG-treated cells. Unpaired t test (****p < 0.0001); n = 3 biological replicates. Individual cell values (n = 30 cells per replicate) are plotted as mean ± SEM.
- Figure 2.
RNA-seq analysis of hnRNP A1 knock-down in differentiated Neuro-2a cells. A, PCA analysis of log transformed normalized RNA-seq data showing that siA1 and siNEG formed distinct clusters with strong intercluster separation. B, Heatmap of DE transcripts plotted as normalized count values for siNEG-treated (n = 3) and siA1-treated (n = 3) cells. C, Volcano plot of siA1-treated samples (siA1 vs siNEG) illustrating significantly upregulated (green dots) and downregulated (red dots) transcripts. Non-DE transcripts are represented as black dots; p threshold of 0.05 is displayed in gray. See Extended Data Figure 2-1 for list of significant DE genes. D–G, GO enrichment analysis of DE genes identified GO terms related to RNA metabolism (D), neuronal functions (E), neuronal morphology (F), cell death (G), and RNP complex (H). Values at the end of each bar represent number of DE genes in each GO process. Data are presented as -log10false discovery rate (FDR) values, which represent p-values adjusted for multiple tests by Benjamini–Hochberg procedure. See Extended Data Figure 2-2 for list of significantly enriched GO terms from biological processes.
- Figure 3.
HnRNP A1 binding to DE genes. A, Pie chart representing the subset of DE genes with human orthologs (n = 1341) that had previously been shown to be known hnRNP A1 binding targets (89.86%) and those that had not (10.14%). B, Pie chart representing subset of upregulated DE genes with human orthologs (n = 729) that had previously been shown to be known hnRNP A1 binding targets (88.89%) and those that had not (11.11%). C, Pie chart representing subset of downregulated DE genes with human orthologs (n = 612) that had previously been shown to be known hnRNP A1 binding targets (91.01%) and those that had not (8.89%).
- Figure 4.
Effect of hnRNP A1 knock-down on neuronal health. A, Immunofluorescent images of Neuro-2a cells stained for DAPI (blue), hnRNP A1 (green), and β-III-tubulin (red) to identify neurites. Cells in the siNEG condition have more neurites that appear longer as compared with the siA1 condition. Scale bar: 20 μm. B, Neurites were traced in the β-III-tubulin channel in ImageJ using the NeuronJ plugin as described in Materials and Methods. Quantification revealed that siA1-treated Neuro-2a cells have significantly fewer neurite branches as compared with the siNEG condition. Unpaired t test (***p < 0.001); n = 3 biological replicates. Individual cell values (n = 30 cells per replicate for siNEG; n = 20 cells with >50% knock-down for siA1) are plotted as the mean ± SEM. C, Corrected total hnRNP A1 nuclear fluorescence of Neuro-2a cells treated with siA1 correlates with neurite branch number. PC test (r = 0.167, r2 = 0.02,799, p = 0.0498); n = 3 biological replicates. Individual cell values (n = 30 cells per replicate) are plotted. D, Neuro-2a cells treated with siA1 have significantly shorter neurites as compared with the siNEG condition. Unpaired t test (***p < 0.001); n = 3 biological replicates. Individual cell values (n = 30 cells per replicate for siNEG; n = 20 cells with >50% knock-down for siA1) are plotted as mean ± SEM. E, Corrected total hnRNP A1 nuclear fluorescence of Neuro-2a cells treated with siA1 correlated with neurite sum length. PC test (r = 0.2959, r2 = 0.08,758, p = 0.0015); n = 3 biological replicates. Individual cell values (n = 30 cells per replicate) are plotted. F, HnRNP A1 knock-down significantly increased cellular cytotoxicity as compared with siNEG-treated cells as measured by the CYQUANT LDH cytotoxicity assay. Unpaired t test (*p < 0.05); n = 3 biological replicates. Data are plotted as mean ± SEM.
- Figure 5.
HnRNP A1 knock-down affects SG formation. A, Immunofluorescent images of differentiated Neuro-2a cells treated with siNEG or siA1 for 72 h followed by 30-min treatment with sodium arsenite. Cells are stained for β-III-tubulin (blue), hnRNP A1 (red), and G3BP (green) to identify SGs. Cells in the siNEG condition have significantly more punctate-like G3BP+ granules as compared with the siA1 condition. Scale bar: 20 μm. B, Quantification revealed that sodium arsenite-treated Neuro-2a cells in the siA1 condition form significantly fewer SGs as compared with the siNEG condition. Unpaired t test (****p < 0.0001); n = 3 biological replicates. Individual cell values (n = 90 cells per replicate for siNEG; n = 141 cells with >50% knock-down for siA1) are plotted as mean ± SEM. C, HnRNP A1 cell fluorescence of Neuro-2a cells treated with siA1 followed by sodium arsenite treatment correlates with number of SGs. PC test (r = 0.2825, r2 = 0.07,982, p < 0.0001); n = 3 biological replicates. Individual cell values (n = 90 cells per replicate) are plotted. D, Quantification revealed that sodium arsenite-treated Neuro-2a cells in the siA1 condition have significantly smaller SGs as compared with the siNEG condition. Unpaired t test (****p < 0.0001); n = 3 biological replicates. Individual cell values (n = 90 cells per replicate for siNEG; n = 141 cells with >50% knock-down for siA1) are plotted as mean ± SEM.
Tables
Figure Data structure Type of test Sample size Statistical data Figure 1B One tailed Unpaired t test siNEG: 3 experimental replicates
siA1#1: 3 experimental replicates
siA1#2: 3 experimental replicates
siA1#3: 3 experimental replicates
siA1#4: 3 experimental replicatessiNEG vs siA1#1 p = 0.1471, t = 1.206, df = 4; siNEG vs siA1#2 p = 0.0277, t = 2.678, df = 4; siNEG vs siA1#3 p = 0.0041, t = 4.871, df = 4; siNEG vs siA1#4 p = 0.0011, t = 6.929, df = 4. Figure 1E One tailed Unpaired t test siNEG: 3 experimental replicates
siA1: 3 experimental replicatesp = 0.0005, t = 8.435, df = 4, 95% CI = −1.057 to −0.5336, r2 = 0.9468, difference between means ± SEM = −0.7954 ± 0.09429 Figure 1G One tailed Unpaired t test siNEG: 3 experimental replicates with over 30 cells analyzed per replicate
siA1: 3 experimental replicates with over 30 cells analyzed per replicatep < 0.0001, t = 10.50, df = 193, 95% CI = −27493 to −18797, r2 = 0.3635, difference between means ± SEM = −23145 ± 2205 Figure 4B One tailed Unpaired t test siNEG: 3 experimental replicates with over 90 cells analyzed in total
Severe siA1: 3 experimental replicates with 20 cells analyzed in totalp = 0.0002, t = 3.644, df = 116, 95% CI = −2.890 to −0.8547, r2 = 0.1027, difference between the means ± SEM = −1.872 ± 0.5139 Figure 4D One tailed Unpaired t test siNEG: 3 experimental replicates with over 90 cells analyzed in total
Severe siA1: 3 experimental replicates with 20 cells analyzed in totalp = 0.0002, t = 3.671, df = 116, 95% CI = −62.74 to −18.76, r2 = 0.1041, difference between the means ± SEM = −40.75 ± 11.10 Figure 4F One tailed Unpaired t test siNEG: 3 experimental replicates
siA1: 3 experimental replicatesp = 0.0221, t = 2.900, df = 4,
95% CI = 0.1822–8.404, r2 = 0.6776Figure 5B One tailed Unpaired t test siNEG: 3 experimental replicates with over 290 cells analyzed in total
Severe siA1: 3 experimental replicates with 141 cells analyzed in totalp < 0.0001, t = 7.480, df = 437, 95% CI = −2.641 to −1.542, r2 = 0.1135, difference between the means ± SEM = −2.091 ± 0.2796 Figure 5C One tailed PC test siA1: analyzed 300 cells for their hnRNP A1 fluorescence and SG number; each dot represents a single cell p < 0.0001, r = 0.2825, r2 = 0.07982, 95% CI = 0.1749–0.3835 Figure 5D One tailed Unpaired t test siNEG: 3 experimental replicates with over 290 cells analyzed in total
siA1: 3 experimental replicates with 141 cells analyzed in totalp < 0.0001, t = 6.494, df = 443, 95% CI = −0.5998 to −0.3211, r2 = 0.08691, difference between the means ± SEM = −0.4605 ± 0.07091
Extended Data Figure 2-1
List of DE genes with Ensembl transcript IDs, gene symbols, fold changes for each transcript, log2(fold change), p values, adjusted p value (padj), -log(padj), change direction, and significance. Download Figure 2-1, XLS file.
Extended Data Figure 2-2
Significantly enriched GO terms from biological processes grouped into the following categories: RNA metabolism, neuronal function, neurite outgrowth, cell death, and RNP complex. Categories include number of genes (# genes), category, description, false discovery rate (FDR) values, -log(FDR value), genes, p values, and term name. Download Figure 2-2, XLS file.
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