Fig. 6
RNA-seq validation by qRT-PCR in cell lines treated with AQP4 antibody E5415A and complement. (A) Volcano plot of differentially regulated genes identified by spatial transcriptomics in a MO blood perivascular lesion from rats treated with E5415A (rat E5415A MO). The area was compared to a control area using DSeq analysis, and changes with an adjusted p-value < 0.05 and a two-fold change were considered statistically significant. (B) Pie chart showing the overlap of significantly altered genes between U-87MG-AQP4-ECFP and the rat E5415A MO. (C) A heat map showing the standardized expression analyzed by RNA-Seq (log2-fold change to E5415A + AC) of the 14 differentially expressed genes overlapping between U-87MG-AQP4-ECFP and the rat E5415A MO lesion. Additionally, AQP4 was selected as an internal control. Each row shows the mean expression level for a single gene, and each column shows the mean expression level of 3 replicates. (D) A heat map showing the standardized expression (log2-fold change to E5415A + AC) analyzed by RT-qPCR as validation of the 15 genes from RNA-seq. Gene expression was first normalized to the housekeeping gene GAPDH (ΔCt), followed by normalization to E5415A + AC treatment (ΔΔCt). The data represent six biological replicates from two independent experiments. AC , active complement; AQP4, aquaporin-4; ECFP, enhanced cyan fluorescent protein; EmGFP, emerald green fluorescent protein; HA, human astrocytes; IC, inactive complement; MO, medulla oblongata.
