Fig 1: HDAC3 knockdown affects acetylation levels of histone H3 in T. castaneum larvae. A The knockdown of HDAC1 and HDAC3 increased acetylation levels of histone H3. Total protein extracted from dsHDAC1, dsHDAC3 or dsmalE injected larvae were separated on SDS-PAGE gels, transferred to western blots, and hybridized with antibodies recognizing Acetyl-Histone H3 (Antibody Sampler Kit # 9927-Cell Signaling. ß-actin served as a loading control. The HRP-linked IgG (#7074-Cell Signaling) was used as a secondary antibody. The lysine acetylation status of histone H3K9 and H3K27 increased in HDAC3 knockdown larvae. The images of complete Western blots are shown in Additional file 5. B Loading control protein, ß-Actin, was used to normalize the levels of H3K9 and H3K27. Band intensities were determined by Image-J software. The fold change in treatments compared to control was represented in the graph
Fig 2: HDAC11 knockdown affects acetylation levels of histone H3 in T. castaneum. (A) The total protein extracted from dsHDAC11 or dsmalE injected larvae were resolved on SDS-PAGE gels, transferred to western blots, and probed with antibodies recognizing Acetyl-Histone H3 (Antibody Sampler Kit # 9927-Cell Signaling (H3K9, H3K14, H3K18, H3K27, and H3K56). ß-actin served as a loading control. The HRP-linked IgG (#7074-Cell Signaling) was used as a secondary antibody. Band densities were determined by Image-J software and normalized with loading control protein-ß-Actin. The Mean + SE (n = 3) band densities are shown. Means marked with different letters are significantly different from each other, P ≤ 0.05 by ANOVA. (B) Acetylated-Lysine (Ac-K2-100) MultiMabTM Rabbit mAb mix #9814 was used to detect acetylation levels of proteins extracted from TcA cells exposed to dsHDAC11 or dsmalE. The acetylation levels of histone H3K9, H3K18 and H3K27 increased in HDAC11 knockdown cells were detected as described in Figure 7A. The band densities were quantified and plotted as described in Figure 7A. The Mean + SE (n = 3) band densities are shown. Means marked with different letters are significantly different from each other, P ≤ 0.05 by ANOVA.
Fig 3: Expression and function of glucose transporters in hCMEC/D3 cells–self inhibition.A) GLUT1 expression in hCMEC/D3 cells. Three passages of hCMEC/D3 cells (P30, P31 and P33) (30 μg of protein per well) were tested for GLUT1 (40–60 kDa) expression. The figure is an example membrane of three technical repeats. Caco-2 cell lysate was used as a positive control; HEK-293 cell lysate was used as a negative control. GAPDH (37 kDa) was used as a loading control. Antibodies used: anti-GLUT1 antibody– 1:100 000, #ab115730; anti-GAPDH antibody– 1:2500, #ab9485, Abcam; secondary anti-rabbit IgG, HRP-linked antibody– 1:2000, #7074, Cell Signalling Technology. B) Vd of [3H]mannitol was not significantly different between the control and the experimental conditions. C) Non-labelled glucose significantly decreased the accumulation of [14C]D-glucose ([3H]mannitol corrected) in hCMEC/D3 cells after 1 h of incubation. Results are expressed as mean ± SEM, n = 3–4 plates, passages (P33, P34 and P35) with five well replicates per treatment in each plate. Data were analysed with an unpaired one-tailed Student’s t-test, using GraphPad Prism 9, each point represents a plate *p<0.05.
Fig 4: HCoV-OC43 nLuc replication kinetics in Huh7.5 IFITM3 over-expressing cells. A. Western blot analysis of HCoV-OC43 replication in Huh7.5 cells expressing FLAG-tagged IFITM3. HCoV-OC43 nucleocapsid was detected by mouse anti-coronavirus primary antibody (Millipore Sigma: MAB9012). FLAG-tagged human IFITM3 was detected by anti-FLAG M2 antibody (CST: 14793A). GAPDH was detected by anti-GAPDH (CST: 2118S). Following primary antibody incubation, blots were incubated with either anti-mouse (SeraCare: 5220–0341) or anti-rabbit (CST: 7074S) HRP secondary antibodies. B. Representative example of HCoV-OC43 nLuc foci observed upon infection of Huh7.5 IFITM3 cells with serially diluted virus sample after 48hpi at 37 °C. For foci visualization, cells were fixed with 2.0% PFA for 1 hour at room temperature, stained with mouse anti-coronavirus primary antibody (Millipore Sigma: MAB9012) followed by goat anti-mouse HRP secondary antibody (SeraCare: 5220–0341), and then incubated with KPL TrueBlue Substrate (SeraCare: 5510–0030). Cells were washed 3 times with PBS between antibody incubations, and antibodies were diluted in blocking buffer (PBS, 0.1% BSA, 0.1% saponin). C. Growth of infectious HCoV-OC43 nLuc over time in Huh7.5 EV or Huh7.5 IFITM3 cells at 32 °C or 37 °C. Huh7.5 EV or Huh7.5 IFITM3 cells were infected with HCoV-OC43 nLuc at an MOI of 0.01 at 32 °C or 37 °C, and virus supernatants were titered by focus-forming assay (FFA) at 0, 24, 48, & 72hpi. D. Growth of HCoV-OC43 nLuc as determined by nano luciferase luminescence over time in Huh7.5 EV or Huh7.5 IFITM3 cells at 32 °C or 37 °C. Huh7.5 EV or Huh7.5 IFITM3 cells were infected with HCoV-OC43 nLuc at an MOI of 0.01 at 32 °C or 37 °C, and nano luciferase activity in the virus supernatants was measured at 0, 24, 48, & 72 h.p.i. using the Promega NanoGlo Assay. Data is from two independent, biological replicates with two or three technical replicates each. Focus forming units (FFU). Relative light units (RLU).
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