Journal: Nature Communications
Article Title: Post-transcriptional control of a stemness signature by RNA-binding protein MEX3A regulates murine adult neurogenesis
doi: 10.1038/s41467-023-36054-6
Figure Lengend Snippet: a MEX3A RNA-immunoprecipitation (RIP) diagram. IP: immunoprecipitation; C: crosslinked samples; NC: non-crosslinked samples. b Protein-protein interaction network generated from MEX3A-bound coding RNAs (STRING PPI enrichment p -value = 3.67 × 10 −11 ). Line thickness indicates strength and confidence of the connection ( https://string-db.org/ ). See Supplementary Fig. and Supplementary Data for detailed target lists. c Representative Gene Ontology (GO, Biological Process) terms enriched in the subset of MEX3A-bound coding RNAs (see Supplementary Data for a complete GO list). Significance values are reported after FDR correction. d Scatterplot comparing differential expression (DE) data from DFFDA high vs. DFFDA low populations with MEX3A RIP-seq enrichment (FDR < 0.05). Red dots represent up-regulated, and blue dots down-regulated genes in DFFDA high cells. Dark red and blue dots label DE genes also bound by MEX3A. e Overlap between MEX3A-bound genes (orange) and genes enriched in DFFDA high (red, top) and DFFDA low (blue, bottom) populations. DFFDA high and MEX3A-bound genes show more overlap than expected ( p -value = 8.5 × 10 −8 , by hypergeometric test). f DE data from isolated pNSC vs. aNSC populations with MEX3A RIP-seq enrichment data (FDR < 0.05). Red dots represent up-regulated, and blue dots down-regulated genes in pNSCs. Dark red and blue dots label DE genes also bound by MEX3A. g Overlap between MEX3A-bound genes (orange) and genes enriched in pNSCs (red, top) and aNSCs (blue, bottom). pNSC and MEX3A-bound genes show more overlap than expected ( p -value=0.003, by hypergeometric test). Quantification of Aqp4 ( h ) and Sdc4 ( i ) gene expression by RT-qPCR in Mex3a +/+ and Mex3a KI/KI NSCs (left panels) ( * p -value < 0.05 and NS = 0.317 by unpaired two-tailed Student’s t -test). AQP4 ( h ) and SDC4 ( i ) protein levels (median fluorescence intensity) by FACS in wild-type and KI homozygous NSC cultures (right panels) (p-values: AQP4 = 0.032, SDC4 = 0.011, by unpaired two-tailed Student’s t -test). Box plots show median± interquartile range and whiskers define minimum to maximum. Exact p -values and the number of biologically independent samples used are indicated. Source data are provided as a Source Data file.
Article Snippet: Individual cells were incubated with 100 μl of the specific fluorescent-labeled primary antibodies (human anti-SDC4-APC (Miltenyi, cat. no. 130-109-831, 1:200); rabbit anti-AQP4 (Sigma, cat. no. HPA014784, 1: 150)) in flow cytometry blocking buffer at 4 °C for 30 min. Immunostained samples were analyzed in an LSR-Fortessa cytometer (350, 405, 488, 561 and 640 nm lasers, Becton Dickinson) with FACSDiva (v8.0.2, BD).
Techniques: Immunoprecipitation, Generated, Expressing, Isolation, Quantitative RT-PCR, Two Tailed Test, Fluorescence
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