Journal: Journal of Lipid Research

Article Title: Depletion of essential isoprenoids and ER stress induction following acute liver-specific deletion of HMG-CoA reductase

doi: 10.1194/jlr.RA120001006

Figure Lengend Snippet: Hmgcr LSKO mice show hepatocyte ER stress-induced apoptosis and impaired N-glycosylation. A: Representative transmission electron microscopy of hepatocytes in liver from control and Hmgcr LSKO mice. Scale bar is 5 μM. Western blot analysis of Chop (B), Bax (C), and Aat (D) in liver lysates from control and Hmgcr LSKO mice, with β-tubulin used as a loading control. E: Western blot analysis of Aat in plasma from control and Hmgcr LSKO mice, with Apoa1 used as a loading control. F: Densitometry analysis of plasma Aat in control (blue) and Hmgcr LSKO (red) mice. Hmgcr LSKO samples in D and E)are from Hmgcrfl/fl mice injected with either 2 × 1011 (AAV-Cre normal) or 1012 (AAV-Cre high) GCs of AAV-Cre. In panels D and E, plasma was treated with an enzymatic deglycosylation kit (+) or sham (−) as control of the effect of glycosylation on protein mobility. Aat Glyc., glycosylated (mature) Aat; Aat Deglyc., deglycosylated Aat. Representative Western blots are shown. Densitometry data are normalized to Apoa1 and shown as mean ± SD with significance determined by two-tailed Student’s t-test (n = 3). *P < 0.05.

Article Snippet: As control of impaired glycosylation, plasma samples were enzymatically deglycosylated using Protein Deglycosylation Kit II (NEB).

Techniques: Transmission Assay, Electron Microscopy, Western Blot, Injection, Two Tailed Test

Journal: Journal of Lipid Research

Article Title: Depletion of essential isoprenoids and ER stress induction following acute liver-specific deletion of HMG-CoA reductase

doi: 10.1194/jlr.RA120001006

Figure Lengend Snippet: Dhdds LSKO mice show hepatocyte ER stress-induced apoptosis. A: Six-week-old C57BL/6J mice were injected with 5 × 1011 GCs of AAV-CRISPR or AAV-GFP (control) and liver and plasma samples were collected at 2 and 4 weeks postinjection for analysis. B: qPCR analysis of Dhdds mRNA levels in livers from control (blue) and Dhdds LSKO (red) mice. TUNEL staining of liver sections (C) and quantification of TUNEL-positive hepatocytes per square millimeter in control (blue) and Dhdds LSKO (red) livers at 4 weeks postinjection (D). Ki67 staining of liver sections (E) and quantification of Ki67-positive hepatocytes per square millimeter in control (blue) and Dhdds LSKO (red) livers at 4 weeks postinjection (F). Representative images from mice at 4 weeks post-AAV injection are shown (scale bar is 100 μm). Western blot analysis of Chop (G), Bax (H), and Aat (I) in liver lysates from control and Dhdds LSKO mice, with β-tubulin used as a loading control. J: Western blot analysis of Aat in plasma from control and Dhdds LSKO mice, with Apoa1 used as a loading control. Representative Western blots are shown. In panel I, plasma was treated with an enzymatic deglycosylation kit (+) or sham (−) as control of the effect of glycosylation on protein mobility. Aat Glyc., glycosylated (mature) Aat; Aat Deglyc., deglycosylated Aat. K: Densitometry analysis of plasma Aat relative to Apoa1 in control (blue) and Dhdds LSKO (red) mice. All data are presented as mean ± SD with significance determined by two-tailed Student’s t-test (n = 6 mice per group, n = 3 in densitometry analysis). *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.001.

Article Snippet: As control of impaired glycosylation, plasma samples were enzymatically deglycosylated using Protein Deglycosylation Kit II (NEB).

Techniques: Injection, CRISPR, TUNEL Assay, Staining, Western Blot, Two Tailed Test

Journal: bioRxiv

Article Title: Role of the imprinted allele of the p57 Kip2 gene in mouse neocortical development

doi: 10.1101/737692

Figure Lengend Snippet: (a) Scheme of allele specific qPCR of p57. F1 hybrid embryos of C57BL/6J (BL6) female and JF1/MS (JF1) male parents have SNPs derived from their parents. qPCR was performed by the use of specific primers for each allele. (b) The ratio of paternal to maternal p57 mRNA expression in NPCs and in neurons was determined by allele specific qPCR under the hybrid (JF1 and BL6) genetic background at E16. FACS was used to isolate NPCs (CD133 + CD24 − population) and neurons (CD133 − CD24 + population). (c)(d) Allele specific qPCR of p57 mRNA in the neocortex isolated from control ( p57 pat flox (BL6)/+(JF1)) and hybrid paternal p57 cKO (Nestin-Cre; p57 pat flox(BL6)/+(JF1) ) mice at E16. p57 mRNA expression from each allele was normalized to β -actin . Data are expressed relative to the corresponding value for control mice. (e) qPCR of total p57 mRNA expression in the neocortex isolated from control and paternal p57 cKO mice in the BL/6J background at P0. p57 mRNA expression was normalized to β -actin . Data are expressed relative to the corresponding value for control mice. Data are mean+s.e.m from three independent experiments. Paired two-tailed Student’s t-test; ** P <0.01. n.s., not significant.

Article Snippet: The obtained cDNA was subjected to real-time PCR analysis in a Roche LightCycler 480 II with THUNDERBIRD SYBR qPCR kit (TOYOBO).

Techniques: Derivative Assay, Expressing, Isolation, Control, Two Tailed Test

Journal: bioRxiv

Article Title: Role of the imprinted allele of the p57 Kip2 gene in mouse neocortical development

doi: 10.1101/737692

Figure Lengend Snippet: (a-b) Immunofluorescence staining of Ki67 at E13 ( a ) and E16 ( b ) (left panel). Quantitative analysis of cells positive for Ki67 per area within 200 μm wide bins (right panel) (n = 3-4 embryos for each genotype). Data are mean+s.e.m. Unpaired two-tailed Student’s t-test. (c) Immunostaining of cleaved caspase-3 at E13. The boxed regions in the left panels are shown at a higher magnification in the right panel. Yellow arrowheads indicate cleaved caspase-3 positive cells. (d) The number of cleaved caspase-3 positive cells per area within 500 μm wide bins were quantified at E13 and E16 (n=4-6 embryos for each genotype). Data are mean+s.e.m. Unpaired two-tailed Student’s t-test. (e) qPCR of p21 mRNA expression in the neocortex isolated from control and paternal p57 cKO mice at P0. p21 mRNA expression was normalized to β -actin (n=3 independent experiments). Data are mean+s.e.m.; expressed relative to the corresponding value for control mice. Paired two-tailed Student’s t-test. * P <0.05. n.s., not significant. Scale bars: 50 μm in ( a ) ( b ); 100 μm in ( c )

Article Snippet: The obtained cDNA was subjected to real-time PCR analysis in a Roche LightCycler 480 II with THUNDERBIRD SYBR qPCR kit (TOYOBO).

Techniques: Immunofluorescence, Staining, Two Tailed Test, Immunostaining, Expressing, Isolation, Control