MiRNA Exporter HuR Clears  Macrophage Internalized Pathogens by Promoting Pro-inflammatory Response (2020)
Figure 7

Fig 7 Leishmania donovani infection could be counteracted by ectopic expression of HuR.

A-C Effect of HA-HuR expression on Ld infection of RAW 264.7 cells. Effect of HuR expression on internalized parasite number and cytokine expression in RAW 264.7 cells. HA-HuR was transfected to RAW 264.7 cells and infection was given for 24 hrs at a host to parasite 1:10 ratio. Ld was stained with CFSE dye and parasite internalization was detected by counting the CFSE positive structures inside the cells. HA-HuR was immunostained with anti-HA antibody and detected by secondary antibody tagged with AlexaR564 (A). In the zoomed part of the merged picture are used to show the internalized parasite. Internalized parasites present in HA-HuR positive cells were counted and compared against untransfected cells without HA-HuR expression (B). Levels of expression of different cytokines are measured in control or HA-HuR expressing RAW 264.7 cells infected with Ld (C). Scale bar 20 µm. Values are mean+/- s.e.m. and, n=3.

D-E Effect of Ld infection on the HuR expression in mouse liver. BALB/c mice of 6 weeks of age were infected with Ld via cardiac puncture. Animals were sacrificed after subsequent days; protein was extracted from liver tissue followed by western blot analysis for HuR. β- Actin was used as loading control (D, left panel). Each lane have designated individual animal. Band intensities were determined by ImageJ. In each case, HuR level was normalized by its respective β- Actin bands (D, right panel mean +/- s.e.m., n=5). Animals were injected with GP63 containing liposome (gp1-3) or empty liposome (C1-2) and after 24 hrs of injection, HuR level was checked against β-Actin in liver tissue. β-Actin was used as loading control (E).

F-I Effect of expression of HA-HuR on Ld infection in mice liver. Scheme for animal experiment done with HA-HuR expression in liver is shown. Also the HA-HuR level was detected in the liver tissue by western blot analysis using anti-HA antibody (F). Parasite load in liver tissues were estimated by measuring Ld DNA in infected tissue using specific primer for Ld minicircle kDNA. Values are mean+/- s.e.m. and, n=5 (G). TNF-α (Values are mean+/- s.e.m. and, n=5) and IL-10 (Values are mean+/- s.e.m. and, n=4) mRNA levels were checked from RNA isolated from liver tissues and 18S rRNA normalized Ct values were plotted (H and I, left panels). Relative fold change was calculated by ΔΔCt method and the values plotted for Ld count, TNF-α and IL-10 mRNA levels (G mean +/- s.e.m., (n=5), H mean +/- s.e.m., (n=6) and I mean +/- s.e.m., (n=4), right panel).

J-K Effect of HA-HuR plasmid tail-vein injection on mice spleen. Levels of HA-HuR were undetectable in mice spleen after expression plasmid was injected. Sample from one liver tissue of HA-HuR injected group was used as a positive control of HA-HuR expression in moue liver after tail vein injection of HuR encoding plasmid (J). Simultaneously, TNF-α mRNA levels were checked in spleen total RNA (K). Values are mean+/- s.e.m. and, n=4.

Data information: In all experimental data, ns; non-significant and *, ** and *** represent P value of <0.05, <0.01 and <0.001 respectively calculated by using student's t test. For statistical analysis, all experiments are done three times. Exact P-values against each experimental set are presented in the Appendix Table S2. Positions of molecular weight markers are marked and shown in the western blots used in different panels.