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

Journal: PLOS One

Article Title: Integrated single-cell RNA-seq and bulk RNA-seq analysis to investigate key adipogenesis genes in adipose-derived stem cells

doi: 10.1371/journal.pone.0335152

Figure Lengend Snippet: (A) Venn diagram showing the key genes for inducing adipocyte differentiation (B) The differences of expression of key genes for inducing adipocyte differentiation among day 21, day 7, and day 0 (C) qPCR validation of TTC14, MBNL2, ABCD2, and SORT1 expression levels at Day 0, 7, and 21 during adipogenic differentiation. Data are presented as mean ± SEM (n = 6). P-values were calculated using unpaired two-tailed t-test (D) Venn diagram showing the key genes for maintaining the mature phenotype (E) The differences of expression of key genes for maintaining the mature phenotype among day 21, day 7, and day 0 (F) qPCR validation of UQCR11, NDUFB4, S100A10, and PRDX3 expression during adipogenesis (Day 0, 7, 21). P-values were calculated using unpaired two-tailed t-test. Data are shown as mean ± SEM (n = 6).

Article Snippet: For RNA analysis, total RNA was extracted with TRIzol (Thermo Fisher Scientific), followed by complementary DNA synthesis with Hiscript III RT SuperMix for quantitative polymerase chain reaction (qPCR) (+gDNA wiper) (Vazyme, R323-01).

Techniques: Expressing, Biomarker Discovery, Two Tailed Test