Journal: Journal of Inflammation (London, England)

Article Title: Cinnamaldehyde inhibits the NLRP3 inflammasome by preserving mitochondrial integrity and augmenting autophagy in Shigella sonnei -infected macrophages

doi: 10.1186/s12950-024-00395-w

Figure Lengend Snippet: Role of autophagy in CA-mediated IL-1β inhibition in S. sonnei -infected macrophages. ( A-C ) J774A.1 macrophages were incubated with 40 µM CA for 4–20 h or 1 µM rapamycin for 4 h. The expression levels of LC3 ( A ), ATG5 ( B ) and p62 ( C ) in the cell lysates were analysed by Western blotting. ( D ) J774A.1 macrophages were primed for 4 h with LPS in the presence or absence of 1 mM 3-MA, followed by incubation with 40 µM CA for 0.5 h before S. sonnei infection for an additional 21 h. The detectable levels of IL-1β in the supernatants were analysed by ELISA. ( E ) Wild-type or LC3-knockdown J774A.1 macrophages were incubated with 40 µM CA for 20 h. The expression levels of LC3 in the cell lysates were analysed by Western blotting. ( F ) LPS-primed wild-type or LC3-knockdown J774A.1 macrophages were incubated with 40 µM CA for 0.5 h before S. sonnei infection for an additional 21 h. The detectable levels of IL-1β in the supernatants were analysed by ELISA. The Western blotting images are representative of separate experiments. The ELISA data are expressed as the means ± SD of three separate experiments. * p < 0.05 and *** p < 0.001 as indicated

Article Snippet: Mouse LC3 CRISPR/Cas9 knockout plasmids (sc-426,563 and sc-417,828-HDR) and antibodies against IL-18 (SC-6177), ASC (SC-22,514-R), cathepsin B (SC-365,558) and actin (SC-47,778) were purchased from Santa Cruz Biotechnology (Santa Cruz, CA).

Techniques: Inhibition, Infection, Incubation, Expressing, Western Blot, Enzyme-linked Immunosorbent Assay, Knockdown

Journal: Cells

Article Title: Synthetic 4-Hydroxy Auxarconjugatin B, a Novel Autophagy Inducer, Attenuates Gouty Inflammation by Inhibiting the NLRP3 Inflammasome

doi: 10.3390/cells9020279

Figure Lengend Snippet: 4-HAB attenuated the NLRP3 inflammasome through autophagy induction. ( A ) Cells were incubated with 20 µM 4-HAB for 3–24 h. The expression levels of LC3 in the cell lysates were measured by Western blot. ( B ) THP-1 macrophages were incubated with 20 µM 4-HAB for 24 h or 100 nM rapamycin for 4 h. The fluorescent signals of acridin orange (AO) and MDC were measured by confocal microscope. ( C , D ) Cells were incubated with 1 µg/mL LPS for 5 h followed by incubated with 5 mM 3-MA or 20 µM 4-HAB for 30 min. Cells then were incubated with 100 μg/mL MSU crystals for additional 24 h. The levels of IL-1β ( C ) and caspase-1 ( D ) in the supernatants were measured by ELISA and Western blot, respectively. ( E , F ) Wild-type and LC3-knockout THP-1 macrophages were incubated with 20 µM 4-HAB or vehicle for 24 h. The levels of LC3 in the cell lysates were measured by Western blot ( E , right panel). Wild-type and LC3-knockout THP-1 macrophages were incubated with 1 µg/mL LPS for 5 h followed by incubated for 30 min with 20 µM 4-HAB. Cells then were incubated with 100 μg/mL MSU crystals for additional 24 h. The levels of IL-1β ( E , left panel) and caspase-1 ( F ) in the supernatants were measured by ELISA and Western blot, respectively. The control group was treated with vehicle control. The ELISA data are expressed as the mean ± SD of three separate experiments. *, *** and **** indicate a significant difference at the level of p < 0.05, p < 0.001 and p < 0.0001, respectively, compared to untreated cells ( B ) or as indicated. (One-way ANOVA with Dunnett’s multiple comparisons test in ( B ) or two-tailed t test in ( C ) and ( E ). “+” indicates with; “−“ indicates without.

Article Snippet: For generating gene knockout cells, cells were transfected with CRISPR/Cas9 knockout plasmids targeting LC3 (for J774A.1 macrophages: sc-426563 and sc-417828-HDR, Santa Cruz Biotechnology; for THP-1 monocytes: sc-4178288 and sc-417828-HDR, Santa Cruz Biotechnology) or Sirt1 (for J774A.1 macrophages: sc-430046 and sc-430046-HDR, Santa Cruz Biotechnology).

Techniques: Incubation, Expressing, Western Blot, Microscopy, Enzyme-linked Immunosorbent Assay, Knock-Out, Control, Two Tailed Test

Journal: Cells

Article Title: Synthetic 4-Hydroxy Auxarconjugatin B, a Novel Autophagy Inducer, Attenuates Gouty Inflammation by Inhibiting the NLRP3 Inflammasome

doi: 10.3390/cells9020279

Figure Lengend Snippet: 4-HAB inhibited the NLRP3 inflammasome by enhancing the Sirt1/autophagy axis. ( A ) Cells were incubated with 20 µM 4-HAB for 3–24 h. The expression levels of Sirt1 in the cell lysates measured by Western blot. ( B , C ) Cells were incubated with 10 µM EX527 or DMSO for 24 h followed by incubated with 20 µM 4-HAB or vehicle for 24 h. The expression levels of Sirt1 and LC3 in the cell lysates measured by Western blot ( B ). The fluorescent signals of AO and MDC were measured by confocal microscope ( C ). ( D ) Wild-type, LC3-knockout and Sirt1-knockout J774A.1 macrophages were incubated with 20 µM 4-HAB or vehicle for 24 h. The expression levels of LC3 and Sirt1 in the cell lysates were measured by Western blot. ( E ) Cells were incubated with 1 µg/mL LPS for 5 h followed by incubated with 10 µM EX527 or 20 µM 4-HAB for 30 min. Cells then were incubated with 100 μg/mL MSU crystals for additional 24 h. The expression levels of IL-1β in the supernatants were measured by ELISA. ( F ) Wild-type, LC3-knockout and Sirt1-knockout J774A.1 macrophages were incubated with 1 µg/mL LPS for 5 h followed by incubated with 20 µM 4-HAB for 30 min. Cells then were incubated with 100 μg/mL MSU crystals for additional 24 h. The expression levels of IL-1β in the supernatants were measured by ELISA. The control group was treated with vehicle control. The ELISA data are expressed as the mean ± SD of three separate experiments. *, **, *** and **** indicate a significant difference at the level of p < 0.05, p < 0.01, p < 0.001 and p < 0.0001, respectively, as indicated. (two-tailed t test). “+” indicates with; “−“ indicates without.

Article Snippet: For generating gene knockout cells, cells were transfected with CRISPR/Cas9 knockout plasmids targeting LC3 (for J774A.1 macrophages: sc-426563 and sc-417828-HDR, Santa Cruz Biotechnology; for THP-1 monocytes: sc-4178288 and sc-417828-HDR, Santa Cruz Biotechnology) or Sirt1 (for J774A.1 macrophages: sc-430046 and sc-430046-HDR, Santa Cruz Biotechnology).

Techniques: Incubation, Expressing, Western Blot, Microscopy, Knock-Out, Enzyme-linked Immunosorbent Assay, Control, Two Tailed Test