Journal: Frontiers in Immunology

Article Title: NEK7-Mediated Activation of NLRP3 Inflammasome Is Coordinated by Potassium Efflux/Syk/JNK Signaling During Staphylococcus aureus Infection

doi: 10.3389/fimmu.2021.747370

Figure Lengend Snippet: Phosphorylated Syk was involved in S. aureus infection-induced NEK7-NLRP3 signaling pathway activation. J774A.1 cells (A–I) and PECs (J–M) pretreated with or without Syk inhibitor (BAY 613606) at the final concentration of 1 μmol/L for 2 h followed by S. aureus infecton (MOI of 1 or 5) for different time. (A) The protein levels of t-Syk and p-Syk were detected by western blot and analyzed by relative densitometric quantification followed by S. aureus infection (MOI of 5) for 30 min in J774A.1 cells and PECs (J) . (B) The mRNA expression levels of caspase-1 and Il-1β were quantified by RT-PCR. (C) The protein expression levels of caspase-1, p20, pro-IL-1β and mIL-1β were detected by western blot and analyzed by relative densitometric quantification. (D) The supernatants were collected at 48 h after S aureus infection. IL-1β and IL-18 in the supernatants were determined by ELISA in J774A.1 cells and PECs (M) . (E) The ASC specks formation were imaged via fluorescence microscopy. (Scale bar: 100 µm.). (F) ASC specks were quantitied in more than 200 cells from at least 5 different fields. (G) The mRNA expression level of nek7 was examined by RT-PCR. (H) The protein expression level of NEK7 was detected by western blot and analyzed by relative densitometric quantification in J774A.1 cells and PECs (K) . (I) The NEK7-NLRP3 compound were analysed by coimmunoprecipitation in J774A.1 cells and PECs (L) . Similar results were obtained in three independent experiments in data. Data shown are means ± SEMs; * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001 vs . non-infection control group, # P < 0.05, ## P < 0.01, ### P < 0.001, #### P < 0.0001 vs. infection control group.

Article Snippet: The JNK inhibitor II SP600125 (SP) was purchased from Merck KGaA, Inc. Syk inhibitor IV BAY 61-3606 was purchased from Santa Cruz Biotechnology, Inc. TRIzol ® reagent was purchased from Invitrogen Thermo Fisher Scientific, Inc.

Techniques: Infection, Activation Assay, Concentration Assay, Western Blot, Expressing, Reverse Transcription Polymerase Chain Reaction, Enzyme-linked Immunosorbent Assay, Fluorescence, Microscopy, Control

Journal: Frontiers in Immunology

Article Title: NEK7-Mediated Activation of NLRP3 Inflammasome Is Coordinated by Potassium Efflux/Syk/JNK Signaling During Staphylococcus aureus Infection

doi: 10.3389/fimmu.2021.747370

Figure Lengend Snippet: Syk/JNK signaling was critical for activation of the NLRP3 inflammasome upon S. aureus infection. Macrophages were infected with S. aureus (MOI of 5) for 2 h by pretreated with or without Syk-siRNA (A , B) for 48 h or Syk inhibitor (BAY) (C–E) for 2h. (A) The protein expression level of p-JNK was detected by western blot and analyzed by relative densitometric quantification in J774A.1 cells. (B) The mRNA expression level of jnk was quantified by RT-PCR. (C) The protein expression level of p-JNK was detected by western blot and analyzed by relative densitometric quantification in J774A.1 cells and PECs (E) . (D) The mRNA expression level of jnk was quantified by RT-PCR. Similar results were obtained in three independent experiments in data. Data shown are means ± SEMs; ** P < 0.01, **** P < 0.0001 vs . non-infection control group, # P < 0.05, ### P < 0.001, #### P < 0.0001 vs. infection control group.

Article Snippet: The JNK inhibitor II SP600125 (SP) was purchased from Merck KGaA, Inc. Syk inhibitor IV BAY 61-3606 was purchased from Santa Cruz Biotechnology, Inc. TRIzol ® reagent was purchased from Invitrogen Thermo Fisher Scientific, Inc.

Techniques: Activation Assay, Infection, Expressing, Western Blot, Reverse Transcription Polymerase Chain Reaction, Control