Additional file 2 of The accumulation of modular serine protease mediated by a novel circRNA sponging miRNA increases Aedes aegypti immunity to fungus
Creators: name: Lu, Tengfei
name: Ji, Yannan
name: Chang, Mengmeng
name: Zhang, Xiaoming
name: Wang, Yanhong
name: Zou, Zhen
Publisher: name: figshare
Year: 2024
Subjects: subject: Microbiology
subject: FOS: Biological sciences
subject: Genetics
subject: Molecular Biology
subject: Biotechnology
subject: Ecology
subject: Immunology
subject: FOS: Clinical medicine
subject: Biological Sciences not elsewhere classified
subject: Mental Health
subject: Infectious Diseases
subject: FOS: Health sciences
subject: Computational Biology

description: Additional file 2: Supplementary methods and figures. Supplementary methods. Figure S1. Phylogenetic analysis of modular SPs. (A) The signs in the figures indicate ModSP. The amino acid sequences of H. armigera (Ha), D. melanogastor (Dm), Ae. aegypti (Aa), M. sexta (Ms), and An. gambiae (Ag) SPs were aligned and constructed by MEGA 7.0 and Evolview. (B) Expression analysis of multiple SPs 48 hours after fungal infection. (C) Expression analysis of ModSP at 6 and 12 hours after fungal infection. (D) The RNAi efficiency was validated after dsRNA injection by qRT-PCR. Figure S2. Identification of miRNA-novel-53 precursor and mature form. (A) Sequence confirmation of miRNA-novel-53 precursor. (B) Hairpin structure prediction of miRNA-novel-53 precursor. Predicted by RNAstructure 6.3 software. ΔG = -25.60 kcal/mol. (C) Northern blot to detect the size of miRNA to characterize the authenticity of novel miRNA in different samples. Figure S3. Temporal-spatial expression of ModSP and miRNA-novel-53 in Ae. aegypti. The relative mRNA abundance of ModSP (A) and miRNA-novel-53 abundance (B) at different stages (DS) of larvae of adult Ae. aegypti. The relative mRNA abundance of three AMPs (C, D, and E) at different stages. The relative expression of ModSP (F) and miRNA-novel-53 (G) at different tissues of adult mosquitoes. The results were performed three times and displayed in the form of mean ± SEM, and statistical difference analysis between samples was assessed using the Student’s t-test. * P &lt; 0.05, ** P &lt; 0.01. dph, days post-hatching. HD, head. FB, fat body. MG, midgut. MT, Malpighian tubules. OV, ovary. The control group in Figure A, C, E, F, and G is mosquito larvae of 1 days post-hatching. Figure S4. Verification of miRNA-novel-53 in Ae. aegypti. (A) Pairwise alignment between wild-type and the mutant-type at genome. (B) Relative quantification of miRNA in heterozygote and homozygote mosquitoes. (C) Relative quantification of ModSP mRNA in heterozygote and homozygote mosquitoes. (D) The abundance of ModSP protein in the knockout mosquito hemolymph. WT, wild-type mosquito; 53+/-, heterozygote of miRNA-novel-53 knockout; 53-/-, homozygote of miRNA-novel-53 knockout. Figure S5. Validation and overexpression of novel-circ-930 in Ae. aegypti. (A) Qualitative validation of predicted circRNAs. (B) Sanger sequencing result of the validated circRNA. (C) Relative quantification of circRNAs after B. bassiana infection. (D) Assembly and verification of circRNA overexpression vector. (N = A, T, C, and G; R = A or C; Y = U or C). pAlu, cells transfected with pAlu5.1b empty vector; pAlu-930, transfected with pAlu5.1b-novel-circ-930 cells; pAlu-1041, transfected with pAlu5.1b-novel-circ-1041 cells. pAlu and pAlu-1041 were used as negative controls.