Fig 1: Enforced NEK7 reversed the inhibitory effect of WHSC1 silencing on LPS-induced alveolar macrophage pyroptosis. (a) The expression level of NEK7 was determined using Western blot in alveolar macrophages co-transfected with NEK7A overexpression vector and siRNAs targeting WHSC1 and treated with 10 mg/kg LPS. (b) The protein levels of WHSC1, NEK7, NLRP3, ASC, caspase-1 p20, IL-18, IL-1ß and GSDMD CL were determined using Western blot in alveolar macrophages co-transfected with NEK7A overexpression vector and siRNAs targeting WHSC1 and treated with 10 mg/kg LPS. (c) The activity of caspase-1 in alveolar macrophage was determined in alveolar macrophages co-transfected with NEK7A overexpression vector and siRNAs targeting WHSC1 and treated with 10 mg/kg LPS. (d) The expressions of caspase-1 and GSDMD CL in alveolar macrophages co-transfected with NEK7A overexpression vector and siRNAs targeting WHSC1 and treated with 10 mg/kg LPS were detected using immunofluorescence. Ns: P > 0.05.
Fig 2: Knockdown of WHSC1 attenuated LPS-induced pyroptosis. (a) The protein levels of NLRP3, ASC, caspase-1 p20, IL-18, IL-1ß, GSDMD FL and GSDMD CL in alveolar macrophages of mice introduced into Ad-shWHSC1 and treated with 10 mg/kg LPS were detected using Western blot. (b) The activity of caspase-1 in the alveolar macrophage of mice introduced into Ad-shWHSC1 and treated with 10 mg/kg LPS was determined. Ns: P > 0.05.
Fig 3: The effect of WHSC1 and NEK7 on alveolar macrophage pyroptosis. (a) The expressions of caspase-1 and GSDMD CL in alveolar macrophages transfected with siRNAs targeting WHSC1 and NEK7 and treated with 1 µg/ml LPS were detected using immunofluorescence. (b) The activity of caspase-1 in alveolar macrophages was determined in alveolar macrophages transfected with siRNAs targeting WHSC1 and NEK7 and treated with 1 µg/ml LPS. (c) The pyroptosis of macrophages in alveolar macrophages transfected with siRNAs targeting WHSC1 and NEK7 and treated with 1 µg/ml LPS was assessed by flow cytometry. (d) The pyroptosis proportion of macrophages in alveolar macrophages transfected with siRNAs targeting WHSC1 and NEK7 and treated with 1 µg/ml LPS is presented.
Fig 4: Wolf–Hirschhorn syndrome candidate 1 (WHSC1) was up-regulated in LPS-induced acute lung injury (ALI). (a) Hematoxylin and eosin (HE) stain was conducted to assess lung tissue damage in mice treated with 5 or 10 mg/kg LPS. (b) The lung injury score was determined to evaluate lung injury in mice treated with LPS. (c) Levels of IL-18 and IL-1ß in bronchoalveolar lavage fluid (BALF) were detected by ELISA assay. (d) The lung wet/dry mass ratio of lung tissue was measured in mice treated with LPS. (e) The level of BALF total protein was determined in mice treated with LPS. (f) The mRNA and protein levels of WHSC1 in alveolar macrophages of mice treated with LPS were detected by quantitative RT-PCR and Western blot.
Fig 5: Knockdown of WHSC1 attenuated LPS-induced ALI. (a) The mRNA and protein levels of WHSC1 were detected by quantitative RT-PCR and Western blot in alveolar macrophages of mice introduced into Ad-shWHSC1 and treated with 10 mg/kg LPS. (b) The lung tissue damage in mice introduced into Ad-shWHSC1 and treated with 10 mg/kg LPS was determined by HE stain. (c) The lung injury score was determined to evaluate lung injury in mice introduced into Ad-shWHSC1 and treated with 10 mg/kg LPS. (d) The levels of IL-18 and IL-1ß in BALF of mice introduced into Ad-shWHSC1 and treated with 10 mg/kg LPS were detected by ELISA assay. (e) The lung wet/dry mass ratio of lung tissues in mice introduced into Ad-shWHSC1 and treated with 10 mg/kg LPS was determined. (f) The BALF total protein level was determined in mice introduced into Ad-shWHSC1 and treated with 10 mg/kg LPS.
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