MiRNA Exporter HuR Clears  Macrophage Internalized Pathogens by Promoting Pro-inflammatory Response (2020)
Figure 3

Figure 3 Leishmania donovani upregulates PP2A to dephosphorylate Ago2 in infected macrophage cells

A-C Expression of different cytokines and phosphatases in RAW 264.7 cells and PEC infected with Leishmania donovani (Ld) parasites. A scheme of the experiments is shown in panel A. Cells at different time points of infection were harvested and levels of various phosphatases were checked by western blot analysis using antibodies specific for different phosphatases in both RAW 264.7 ( B, left panel) and mouse peritoneal macrophage cells PEC (B, right panel). Cytokine mRNA levels like IL-10, TNF-α and IL-6 were quantified after 24 hrs of Ld infection in RAW 264.7 (C, upper panel mean +/- s.e.m., n=3) and mouse peritoneal macrophage PEC (mean+/- s.e.m., n=3) (C, Lower panel).

D-E Effect of Ld infection on Ago2 phosphorylation and its miRNA association. RAW 264.7 cells were transfected with FH-Ago2 expression construct and infection was done for various time points followed by FH-Ago2 pull down using anti-FLAG beads. Phosphorylated Ago2 levels were checked during the course of Ld infection (D). Phosphorylation levels of Ago2 were measured by doing endogenous Ago2 pull down using Ago2 specific antibody after 4 hrs of infection and quantified along with Ago2 associated let-7a level measurement in primary macrophages (mean+/- s.e.m., n=2) (E, left panel) and in RAW 264.7 cells (mean+/- s.e.m., n=3) (E, right panel) .

F-G Schematic representation of in vitro phosphatase assay (F, upper panel). PP2A, MKP1, MKP3, or SHP1 were immunoprecipitated individually from naïve or Leishmania infected macrophage cells and were incubated in vitro with FH-Ago2 isolated from FH-Ago2 stable HEK293 cells. Phosphorylated Ago2 level was detected by western blot analysis using phosphotyrosine specific 4G10 antibody (F, lower panel) and measured by densitometry. Relative intensity of 4G10 specific band against immunoprecipitated Ago2 was plotted (mean+/- s.e.m., n=3) (G). BC; Bead Control.

H-I Effect of PP2A knockdown on Ago2 phosphorylation and miRNA-Ago2 binding in RAW 264.7 cells infected with Ld. Cells were co-transfected with siRNA and FH-Ago2 expression plasmid. Phosphorylated Ago2 levels were detected after 4 hrs of infection in siCon or siPP2A transfected cells (H). Ago2 associated let-7a levels were also estimated and plotted (mean+/- s.e.m., n=4) (I).

J-K Effect of OA on Ago2 phosphorylation in Ld-infected RAW 264.7 cells and mouse PEC. Cellular levels of PP2A and Ago2 at different time points of Ld infection with or without OA treatment (100 nM) were detected. Cells were pre-treated with OA (100 nM) for 2 hrs before infection (J). β-Actin was used as loading control. Phospho-Ago2 level was measured in OA pre-treated; Ld infected RAW 264.7 cells expressing FH-Ago2 and in PEC post 24 hrs of infection with or without OA pre-treatment. Phosphorylated Ago2 levels were also checked (K).

L-N Subcellular compartmentalization of PP2A in Ld-infected RAW 264.7 cells. RAW 264.7 cells were infected with Ld for 6 hrs and cell extract were analysed on a OptiprepR density gradient to separate subcellular organelles and structures. Subcellular localization of Ago2 and PP2A in individual fractions were detected by western blots. Alix was used at MVB marker, while Calnexin was used as ER marker (L). OptiprepR fractions, positive for MVB (fraction number 2,3,4) and ER (fraction number 7,8,9) markers were pooled separately and Ago2 was immunoprecipitated using Anti-FLAG beads. Phosphorylated Ago2 levels were quantified in western blot done with phospho-tyrosine specific 4G10 antibody (M). PP2A percentage intensity plot for each fraction was done for control and infected samples (N).

Data information: In all experimental data, ns; non-significant and * represents P value of <0.05 estimated by using student's t test. HC; Heavy Chain of respective antibody used for Immunoprecipitation. 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.