Metformin inhibits EV71‑induced pyroptosis by upregulating DEP domain‑containing mTOR‑interacting protein

Figures

• 

  Figure 1. Shift in cell viability and cell damage caused by enterovirus 71. (A) Cell viability was detected using a Cell Counting Kit-8 assay. (B) Concentration of LDH in the culture supernatant as detected by an LDH release assay. **P<0.01 compared with the mock group. LDH, lactate dehydrogenase; MOI, multiplicity of infection.

• 

  Figure 2. EV71 induces cell pyroptosis. Western blotting was performed to detect the levels of (A) NLRP3 and caspase-1, (B) GSDMD and (C) Pro-IL-1β, with β-actin or GAPDH as an internal reference. Western blot analysis of the levels of (D) caspase-1 and GSDMD and (E) Pro-IL-1β, with β-actin or GAPDH as internal references. (F) NLRP3 staining in GES-1 cells was detected using an immunofluorescence assay (magnification, x200). *P<0.05 and **P<0.01 compared with the mock group. EV71, enterovirus 71; GSDMD, gasdermin D; NLRP3, NLR family, pyrin domain-containing 3; MOI, multiplicity of infection; hpi, hours post inoculation.

• 

  Figure 3. Effect of metformin on the cell viability inhibition and cell damage in EV71-infected GES-1 cells. (A) Cell viability was measured using a Cell Counting Kit-8 assay. (B) Cell damage was assessed using an LDH release assay. (C) Cells were stained using PI (red fluorescence) and observed under a fluorescence microscope (magnification, x200). *P<0.05 and **P<0.01 compared with the mock group; #P<0.05 and ##P<0.01 compared with the EV71-infected group (without metformin treatment). EV71, enterovirus 71; MOI, multiplicity of infection; LDH, lactate dehydrogenase; PI, propidium iodide.

• 

  Figure 4. Effect of metformin on EV71-induced cell pyroptosis. (A) Western blot analysis was performed to investigate the levels of caspase-1 P20. GAPDH was used as an internal reference. (C) Western blot analysis was performed to investigate the levels of caspase-1 and caspase-1 P20. GAPDH was used as an internal reference. (B, E and F) Western blot analysis was performed to determine Pro-IL-1β protein expression in GES-1 cells. β-actin was used as an internal reference. (D) Protein levels of NLRP3 and GSDMD in GES-1 cells were examined by western blotting. GAPDH was used as an internal reference. **P<0.01 compared with the mock group. ##P<0.01 compared with the EV71-infected group (without metformin treatment). EV71, enterovirus 71; GSDMD, gasdermin D; NLRP3, NLR family, pyrin domain-containing 3; MET, metformin MOI, multiplicity of infection.

• 

  Figure 5. Shift in the levels of DEPTOR caused by EV71 and metformin in GES-1 cells. (A) Western blotting was performed to detect the changes in the protein levels of DEPTOR in GES-1 cells infected with different MOI for EV71. GAPDH was used an internal reference. (B) Western blotting was performed to detect the changes in the protein levels of DEPTOR in GES-1 cells infected with EV71 for different times. GAPDH was used an internal reference. (C) Western blot analysis of DEPTOR protein expression in GES-1 cells treated with different concentrations of metformin for 24 h. GAPDH was used as an internal reference. (D) Western blot analysis of the changes in DEPTOR protein expression in EV71-infected GES-1 cells treated with different concentrations (0, 1, 2, 3, 4 and 5 µM) of metformin. GAPDH was used as an internal reference. *P<0.05 and **P<0.01 compared with the mock group or without metformin treatment group; ##P<0.01 compared with the EV71-infected group (without metformin treatment). DEPTOR, DEP domain-containing mTOR interacting protein; EV71, enterovirus 71; MOI, multiplicity of infection; hpi, hours post inoculation.

• 

  Figure 6. Possible involvement of DEPTOR in the regulatory effect of metformin on pyroptosis. Western blot analysis of the changes in the levels of (A) GSDMD, (B) caspase-1 and caspase-1 P20, (C) NLRP3 and (D) Pro-IL-1β in GES-1 cells overexpressing DEPTOR. β-actin or GAPDH was used as a loading control. #P<0.05; ##P<0.01 compared with the NC with EV71-infected group. DEPTOR, DEP domain-containing mTOR-interacting protein; EV71, enterovirus 71; GSDMD, gasdermin D; NLRP3, NLR family, pyrin domain-containing 3; MOI, multiplicity of infection; NC, negative control (empty vector).