Journal: Biochimica et biophysica acta. Molecular basis of disease

Article Title: Myeloid- and hepatocyte-specific deletion of group VIA calcium-independent phospholipase A2 leads to dichotomous opposing phenotypes during MCD diet-induced NASH.

doi: 10.1016/j.bbadis.2022.166590

Figure Lengend Snippet: Fig. 1. Generation and verification of conditional Pla2g6 KO mouse lines. (A) PLA2G6 locus before (Pla2g6wt) and after (Pla2g6neoflox) homologous recombination with the targeting vector insert, LoxP, and FRT sites. FLPe recombinase converts the allele Pla2g6neoflox into the allele Pla2g6floxflox (Flox) which lacks the neomycin resistance cassette. Cre recombinase converts the allele Pla2g6floxflox into the null allele Pla2g6Δex6–8, which lacks exon 6–8. (B) Upon breeding Pla2g6floxflox (Flox) with LysM-Cre mouse line, Mφ-specific Pla2g6 KO (MPla2g6−/−) mice were generated. The deletion was verified by an absence of iPLA2β protein and Pla2g6 mRNA in BMDMs of MPla2g6−/−mice when compared with Flox. Expression of Pla2g4a and PnPla8 mRNA was not altered in MPla2g6−/−BMDMs. (C) Upon breeding Pla2g6floxflox (Flox) with Alb-Cre mouse line, liver-specific Pla2g6 KO (LPla2g6−/−) mice were generated. The deletion was verified by an absence of iPLA2β protein in livers, but not in brain and BMDMs of LPla2g6−/−mice. Control mice were Flox and C57BL6/N WT mice. Data are mean ± SEM, N = 5–7 (B). *, p < 0.05, with Mann-Whitney U tests.

Article Snippet: Membranes were incubated with a primary antibody against iPLA2β (D-4, sc-376,563 or T-14, sc-14,463, Santa Cruz Biotechnology, Heidelberg, Germany), CD36 (H-300, sc9154, Santa Cruz), PPARα (H-2, sc-398,394, Santa Cruz), CHOP (B-3, sc-7351, Santa Cruz), ELOVL6 (ab69857, Abcam), SCD1 (ab19862, Abcam), αSMA (ab32575, Abcam), VIMENTIN (ab9254, Abcam), SERPINE1 (#3917-1, Epitomics), ACC (#3662, Cell Signaling), PPARγ (#2435, Cell Signaling), CEBPα (clone EP709Y, cat#1704-1, Epitomics), p-JNK (81E11, #4668, Cell Signaling), FASN (C20G5, #3180, Cell Signaling), βACTIN (AC-5, #5441, Sigma), and GAPDH (#2118, Cell Signaling).

Techniques: Homologous Recombination, Plasmid Preparation, Generated, Expressing, Control, MANN-WHITNEY

Journal: Biochimica et biophysica acta. Molecular basis of disease

Article Title: Myeloid- and hepatocyte-specific deletion of group VIA calcium-independent phospholipase A2 leads to dichotomous opposing phenotypes during MCD diet-induced NASH.

doi: 10.1016/j.bbadis.2022.166590

Figure Lengend Snippet: Fig. 5. LPla2g6−/−mice show attenuation of liver enzymes, plasma cytokines, and blood monocytes after MCDD feeding. Female control (con) and LPla2g6−/−mice were fed with chow or MCDD for 3.5 weeks. (A) Western blot analysis of hepatic iPLA2β protein (left) and quantification (right). (B) RT-qPCR analysis of hepatic Pla2g6, Pla2g4a, and Pnpla8 (left), as well as Pnpla2 and Pnpla3 (right). (C) Body and liver weights in g as well as liver weights per body weights. (D) Plasma activities of ALT (U/L) and blood glucose levels (mg/dl). (E) Plasma levels (mg/dl) of triglycerides and NEFA. (F) Plasma levels (pg/ml) of TNFα, IL6, and CCL2. (G) The levels (103/μl) of lymphocytes, granulocytes, and monocytes. (H) The levels of lipoxin A4 in plasma (mg/dl) and liver (pg/mg liver). Data are mean ± SEM, N = 4–7 (A–C), N = 3–15 (D–F), N = 5–20 (G), and N = 7–11 (H). ***, p < 0.001, **, p < 0.01, and *, p < 0.05 with Mann-Whitney U tests.

Article Snippet: Membranes were incubated with a primary antibody against iPLA2β (D-4, sc-376,563 or T-14, sc-14,463, Santa Cruz Biotechnology, Heidelberg, Germany), CD36 (H-300, sc9154, Santa Cruz), PPARα (H-2, sc-398,394, Santa Cruz), CHOP (B-3, sc-7351, Santa Cruz), ELOVL6 (ab69857, Abcam), SCD1 (ab19862, Abcam), αSMA (ab32575, Abcam), VIMENTIN (ab9254, Abcam), SERPINE1 (#3917-1, Epitomics), ACC (#3662, Cell Signaling), PPARγ (#2435, Cell Signaling), CEBPα (clone EP709Y, cat#1704-1, Epitomics), p-JNK (81E11, #4668, Cell Signaling), FASN (C20G5, #3180, Cell Signaling), βACTIN (AC-5, #5441, Sigma), and GAPDH (#2118, Cell Signaling).

Techniques: Clinical Proteomics, Control, Western Blot, Quantitative RT-PCR, MANN-WHITNEY

Journal: Biochimica et biophysica acta

Article Title: Sensitization to autoimmune hepatitis in group VIA calcium-independent phospholipase A2-null mice led to duodenal villous atrophy with apoptosis, goblet cell hyperplasia and leaked bile acids.

doi: 10.1016/j.bbadis.2015.04.025

Figure Lengend Snippet: Fig. 3. iPLA2β deficiency sensitizes ConA-induced inflammatory response in the intestine. Experimental conditions were the same as described in Fig. 1. A. qRT-PCR analysis of CD14, TNFα, IL-6 and SOCS3 mRNA expression in the jejunum of treated mice from four groups. Data are mean ± SEM (N = 6–8); *, p b 0.05; ***, p b 0.001. B. qRT-PCR analysis of CCL3, CCR5, CXCL12, and VCAM1 mRNA expression in the jejunum of treated mice from four groups. Data are mean ± SEM (N = 6–8); *, p b 0.05; ***, p b 0.001. C. Western blot analyses normalized with GADPH (left), qRT-PCR analysis (middle) of CCL2 expression, and qRT-PCR analysis of CCR2 expression (right) in the jejunum of treated mice from four groups. Data are mean ± SEM (N = 3–8); *, p b 0.05; **, p b 0.01. D. Western blot analyses normalized with GADPH (left), qRT-PCR analysis (middle) of PPARγ expression, and qRT-PCR analysis of CD36 expression (right) in the jejunum of treated mice from four groups. Data are mean ± SEM (N = 3–8); *, p b 0.05; **, p b 0.01; ***, p b 0.001.

Article Snippet: Primary antibodies used for Western blot included a rabbit antibody against iPLA2β (cat # sc-14463, Santa Cruz), cleaved caspase 3 (cat# 9664, Cell Signaling), CCL2 (cat #2027, Cell Signaling), and PPARγ (cat #2435, Cell Signaling).

Techniques: Quantitative RT-PCR, Expressing, Western Blot

Journal: Biochimica et biophysica acta

Article Title: Sensitization to autoimmune hepatitis in group VIA calcium-independent phospholipase A2-null mice led to duodenal villous atrophy with apoptosis, goblet cell hyperplasia and leaked bile acids.

doi: 10.1016/j.bbadis.2015.04.025

Figure Lengend Snippet: Fig. 3. iPLA2β deficiency sensitizes ConA-induced inflammatory response in the intestine. Experimental conditions were the same as described in Fig. 1. A. qRT-PCR analysis of CD14, TNFα, IL-6 and SOCS3 mRNA expression in the jejunum of treated mice from four groups. Data are mean ± SEM (N = 6–8); *, p b 0.05; ***, p b 0.001. B. qRT-PCR analysis of CCL3, CCR5, CXCL12, and VCAM1 mRNA expression in the jejunum of treated mice from four groups. Data are mean ± SEM (N = 6–8); *, p b 0.05; ***, p b 0.001. C. Western blot analyses normalized with GADPH (left), qRT-PCR analysis (middle) of CCL2 expression, and qRT-PCR analysis of CCR2 expression (right) in the jejunum of treated mice from four groups. Data are mean ± SEM (N = 3–8); *, p b 0.05; **, p b 0.01. D. Western blot analyses normalized with GADPH (left), qRT-PCR analysis (middle) of PPARγ expression, and qRT-PCR analysis of CD36 expression (right) in the jejunum of treated mice from four groups. Data are mean ± SEM (N = 3–8); *, p b 0.05; **, p b 0.01; ***, p b 0.001.

Article Snippet: Primary antibodies used for Western blot included a rabbit antibody against iPLA2β (cat # sc-14463, Santa Cruz), cleaved caspase 3 (cat# 9664, Cell Signaling), CCL2 (cat #2027, Cell Signaling), and PPARγ (cat #2435, Cell Signaling).

Techniques: Quantitative RT-PCR, Expressing, Western Blot