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. 2021 May 13;12:670122. doi: 10.3389/fimmu.2021.670122

Figure 2.

Figure 2

TEP1 Silencing Resulted in an Increase in Viral Load. (A) The relative mRNA levels of thioester-containing protein 1 (TEP1) in the midgut of female mosquitoes were quantified by real-time PCR after a normal or dengue virus (DENV) infectious blood meal. Midguts were collected 6, 12, 24, 48, and 72 hours after a normal or infectious blood meal. Equal amounts of total RNA from each group were used for cDNA synthesis. Ribosomal protein S7 was used for normalization of relative target gene mRNA expression. Values are mean ± S.E. (error bars) of the copy number of TEP1. At least three biological cohorts from each time point were used for analysis. (B) Midguts were collected 6, 12, 24, 48, and 72 hours after a normal or infectious blood meal. Total protein was extracted and western blot analysis was performed using the polyclonal antibody against Anopheles gambiae TEP1. Anti-β-actin antibody was used as the loading control. (C) The midguts were collected three or seven days after a naïve blood meal or infectious blood meal. Midguts of 3-day-old, non-blood-fed female mosquitoes were used as controls. Midguts from female mosquitoes were collected and treated with TEP1 double-stranded RNA (dsRNA) three or seven days after an infectious blood meal. Total RNA was extracted and quantified, followed by cDNA synthesis and subjected to quantitative real-time PCR analysis with a specific primer for DENV2. Values are mean ± S.E. (error bars) of the copy number of DENV2. At least three biological cohorts from each time point were used for analysis. Ribosomal protein S7 was used for normalization of relative target gene mRNA expression. (D) Mosquitoes pre-treated with TEP1 dsRNA were collected for infectious DENV blood feeding. Mosquito midguts were dissected and collected seven days after feeding with infectious blood for immunofluorescent analysis. The anti-DENV NS1 protein antibody was used to detect the expression of DENV in the midgut of mosquitoes. Alexa Fluor 488 goat anti-mouse IgG was used as a secondary antibody. The images were analyzed by confocal microscopy with single planes presented. Mosquitoes pre-treated with LacZ dsRNA fed infectious blood were used as controls. PE means post-eclosion.