Fig 1: Representative miRNA overrepresented in WT vs RNase2-KO samples (log2fold > 2 and p < 0.05) and predicted cleavage sites on precursor miRNA to release mature miRNAs. Cleavage sites in the precursor miRNA were predicted based on the 3'-terminal positions of each miRNA in bam files
Fig 2: RSV activates the expression of RNase2 in THP1-induced macrophages. 106 THP-1 cells/well were seeded in 6-well tissue culture plate and induced by 50 nM of PMA treatment. After induction, macrophages were exposed to RSV under MOI = 1 for 2 h and then cells were washed and replaced with fresh RPMI + 10%FBS (0 h time point post of inoculation). At each time point post of inoculation, the supernatant and cells were collected to quantify the expression of RNase2. A qPCR detection of relative expression of RNase2 gene; B concentration of the cells was controlled as 106 cells/mL, secreted RNase2 in culture supernatant was measured by ELISA and normalized with alive cell number detected by MTT assay; C intracellular RNase2 protein in macrophage was detected by WB; “ + ” and “ - ” indicate with or without RSV, respectively; * and ** indicate the significance of p < 0.05 and p < 0.01, respectively
Fig 3: Overall cleavage preference of RNase2 on tRNAs in THP-1 macrophage cells of WT vs RNase2-KO identified by cP-RNA-seq. A Estimated base preference at 5' and 3' of cleavage site (B1 and B2, respectively) deduced from analysis of differential sequence coverage. B Percentage of predicted cleavage location is depicted from low (white) to high (blue) values
Fig 4: Knocking out of RNase2 reduced the macrophages cell viability upon RSV inoculation. Macrophage cell viability was monitored by registering the absorbance at 570 nm using the MTT assay from 0 to 72 h post of RSV inoculation; “ + ” and “ - ” indicate in the presence or absence of RSV, respectively; the star refers to the significance between KO + and WT + (*p < 0.05)
Fig 5: CRISPR/Cas9 mediated knock out of RNase2 gene in THP1-derived macrophages. A Scheme of the mutation of RNase2 caused by sgRNA1, the sequence was validated by Sanger sequencing; replacement is indicated: red labelled sequence in wild type was replaced by the green labelled sequence, resulting in the coding frame change and stop codon insertion; B western blot assay was applied to detect RNase2 protein; C secreted RNase2 in supernatant was measured by ELISA, the RPMI + 10%FBS complete culture medium was used as a negative control, the supernatant was concentrated 50 × ; D ribonuclease activity staining assay was used to confirm the removal of catalytic function. Cells were collected and resuspended in water and sonicated, cell lysates were loaded in each well at the indicated quantity
Supplier Page from Abcam for Anti-Eosinophil derived neurotoxin antibody