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SENP6 promotes autophagic degradation of NLRP3. (A to C) HEK293T cells were transfected with Flag-NLRP3 and increasing amounts of Myc-SENP6 for 24 h. Lysates were analyzed by immunoblotting (A), with densitometric quantification of NLRP3 protein levels (B). NLRP3 mRNA expression was measured by RT-qPCR (C) ( n = 3). (D and E) HEK293T cells were transfected with Flag-CASP1 or Flag-PYCARD together with increasing amounts of Myc-SENP6. Lysates were subjected to immunoblotting to assess CASP1 and PYCARD expression. (F and G) MH-S cells transfected with Myc-SENP6 were stimulated with LPS (200 ng/ml) for 0, 4, or 8 h. Lysates were collected for immunoblotting (F), followed by densitometric analysis to quantify protein expression (G) ( n = 3). (H to K) SENP6-knockdown MH-S cells and SENP6-deficient PMs cells were stimulated with LPS for the indicated times. Lysates were analyzed by immunoblotting (H and I) and densitometric quantification (J and K) ( n = 3). (L and M) MH-S and SENP6-knockdown MH-S cells were treated with LPS (200 ng/ml) for 4 h, followed by CHX (100 μg/ml) for the indicated times. Lysates were analyzed by immunoblotting (L), and NLRP3 protein levels were quantified (M) ( n = 3). (N to R) HEK293T cells transfected with Flag-NLRP3 and Myc-SENP6 were treated for 6 h with DMSO (vehicle), chloroquine (CQ; 50 μM) (N), 3-methyladenine (3-MA, 10 mM) (O), MG132 (10 μM) (P), or carfilzomib (100 nM) (Q) followed by immunoblot analysis. Quantitative statistical analysis of Flag-NLRP3 expression levels in each group was performed (R). Data are presented as mean ± SD. Statistical analysis was performed using one-way ANOVA (B and C) or two-way ANOVA with Bonferroni test based on n = 3 independent biological experiments. * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001.

Journal: Research

Article Title: SENP6 Restrains NLRP3 Inflammasome Activation via DeSUMOylation-Driven K48-Linked Ubiquitination of NLRP3 in Acute Lung Injury

doi: 10.34133/research.1069

Figure Lengend Snippet: SENP6 promotes autophagic degradation of NLRP3. (A to C) HEK293T cells were transfected with Flag-NLRP3 and increasing amounts of Myc-SENP6 for 24 h. Lysates were analyzed by immunoblotting (A), with densitometric quantification of NLRP3 protein levels (B). NLRP3 mRNA expression was measured by RT-qPCR (C) ( n = 3). (D and E) HEK293T cells were transfected with Flag-CASP1 or Flag-PYCARD together with increasing amounts of Myc-SENP6. Lysates were subjected to immunoblotting to assess CASP1 and PYCARD expression. (F and G) MH-S cells transfected with Myc-SENP6 were stimulated with LPS (200 ng/ml) for 0, 4, or 8 h. Lysates were collected for immunoblotting (F), followed by densitometric analysis to quantify protein expression (G) ( n = 3). (H to K) SENP6-knockdown MH-S cells and SENP6-deficient PMs cells were stimulated with LPS for the indicated times. Lysates were analyzed by immunoblotting (H and I) and densitometric quantification (J and K) ( n = 3). (L and M) MH-S and SENP6-knockdown MH-S cells were treated with LPS (200 ng/ml) for 4 h, followed by CHX (100 μg/ml) for the indicated times. Lysates were analyzed by immunoblotting (L), and NLRP3 protein levels were quantified (M) ( n = 3). (N to R) HEK293T cells transfected with Flag-NLRP3 and Myc-SENP6 were treated for 6 h with DMSO (vehicle), chloroquine (CQ; 50 μM) (N), 3-methyladenine (3-MA, 10 mM) (O), MG132 (10 μM) (P), or carfilzomib (100 nM) (Q) followed by immunoblot analysis. Quantitative statistical analysis of Flag-NLRP3 expression levels in each group was performed (R). Data are presented as mean ± SD. Statistical analysis was performed using one-way ANOVA (B and C) or two-way ANOVA with Bonferroni test based on n = 3 independent biological experiments. * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001.

Article Snippet: Total RNA was extracted using the Fastagen RNA Extraction Kit (Shanghai, China), and cDNA was synthesized with the HiScript III RT SuperMix for quantitative polymerase chain reaction (qPCR) (+gDNA wiper) kit from Vazyme (R323, Nanjing, China), following the manufacturer’s protocol.

Techniques: Transfection, Western Blot, Expressing, Quantitative RT-PCR, Knockdown