Journal: Frontiers in Cell and Developmental Biology

Article Title: Tm7sf2 Disruption Alters Radial Gene Positioning in Mouse Liver Leading to Metabolic Defects and Diabetes Characteristics

doi: 10.3389/fcell.2020.592573

Figure Lengend Snippet: Genes affecting cholesterol homeostasis are altered by Tm7sf2 function. (A) Sequential steps in the cholesterol biosynthesis pathway with genes investigated here highlighted. (B) DamID traces of Cyp51 , Msmo1 , Lbr , Nsdhl , and Acsl4 comparing cells from WT and Tm7sf2 -KO livers. Signal plotted above the line indicates association with the nuclear periphery, and signal below the line indicates internal nuclear localization. The position of the genes is shown under the DamID trace. (C) FISH confirmation of Msmo1 locus repositioning on the left and quantification of distance from the NE on the right in Tm7sf2 +/+ and Tm7sf2 –/– primary hepatocytes. *** p < 0.005 using a Mann–Whitney test. Scale bar: 10 μm. (D) qRT-PCR analysis for the expression levels of Cyp51 , Msmo1 , and Lbr mRNA relative to Hprt in livers of Tm7sf2 +/+ (n = 6) and Tm7sf2 –/– (n = 6) mice. Data are given as mean ± SD. * p < 0.05, ** p < 0.01 with respect to Tm7sf2 +/+ mice; unpaired two-tailed Student’s t -test. (E) On the left, qRT-PCR analysis for the expression levels of Srebp-2 mRNA relative to Hprt control of Srebp-2 livers of Tm7sf2 +/+ ( n = 6) and Tm7sf2 –/– ( n = 6) mice and immunoblot analysis of Srebp-2 precursor (120 kDa) and mature (68 kDa) forms in total liver homogenates of Tm7sf2 +/+ ( n = 4) and Tm7sf2 –/– ( n = 4) mice. Data are given as mean ± SD with no significant differences detected. On the right, Western blot for Srebp-2 protein levels using GAPDH as a loading control. A blot with representative samples is shown. (F) Hmgcr DamID trace as above and qRT-PCR analysis for expression levels as above with no significant differences detected. (G) The absence of Tm7sf2 and lack of a change for Srebp-2 target gene products Hmgcr and Ldlr were also confirmed at the protein level by immunoblot analysis on total membranes isolated from liver tissue of Tm7sf2 +/+ ( n = 4) and Tm7sf2 –/– ( n = 4) mice. (H) DamID traces of cholesterol uptake genes Ldlr and Vldlr and qRT-PCR analysis of Ldlr and Vldlr and statistics as above. Expression levels are relative to Hprt in livers of Tm7sf2 +/+ ( n = 6) and Tm7sf2 –/– ( n = 6) mice. (I) FISH confirmation and quantification of Vldlr in primary hepatocytes as in (C) , **** p < 0.001. Scale bar: 10 μm. (J) DamID trace of cholesterol efflux gene Abca1 and qRT-PCR analysis of Abca1. Expression levels are relative to Hprt in livers of Tm7sf2 +/+ ( n = 6) and Tm7sf2 –/– ( n = 6) mice and statistics as above.

Article Snippet: Total liver extracts (60 μg) or membrane proteins (30 μg) were separated on sodium dodecyl sulfate (SDS)-polyacrylamide gels and transferred to nitrocellulose or polyvinylidene fluoride (PVDF) membranes, respectively, at 100 V for 90 min. Filters were probed with the following primary antibodies: p-Akt Ser 473 (1:1,000, 9,271) and total Akt (1:1,000, 9,272) by Cell Signaling Technology; Gapdh (1:500, sc-32233) by Santa Cruz Biotechnology; Srebp-2 (1:1,000, ATCC ® CRL2545) was a kind gift from Prof. T. Osborne to Rita Roberti; Tm7sf2 (1:1,000) , Hmgcr (1:500, 3,952), and Ldlr (1:1,000, 3,839) by BioVision; and horseradish peroxidase (HRP)-conjugated secondary antibodies (1:5,000, Bio-Rad).

Techniques: MANN-WHITNEY, Quantitative RT-PCR, Expressing, Two Tailed Test, Control, Western Blot, Isolation

Journal: Frontiers in Cell and Developmental Biology

Article Title: Tm7sf2 Disruption Alters Radial Gene Positioning in Mouse Liver Leading to Metabolic Defects and Diabetes Characteristics

doi: 10.3389/fcell.2020.592573

Figure Lengend Snippet: Insulin signaling pathways are defective in Tm7sf2 –/– mice. (A) DamID traces of genes Irs1 , Irs2 , Ces1g , and Atp6v0d2 as in . (B) FISH confirmation of locus mispositioning for Irs1 in Tm7sf2 +/+ and Tm7sf2 –/– primary hepatocytes; quantification and statistics as in , * p < 0.05. Scale bar: 10 μm. (C) Irs1/2 genes downregulated. qRT-PCR analysis for the expression levels of Irs1 and Irs2 mRNA relative to Hprt in livers of Tm7sf2 +/+ ( n = 5) and Tm7sf2 –/– ( n = 5) mice fed with a chow diet. Data are given as mean ± SD. ** p < 0.01 with respect to Tm7sf2 +/+ mice; unpaired two-tailed Student’s t -test. (D) Insulin tolerance tests, resting. Plasma glucose concentration during intraperitoneal insulin tolerance test (ITT) of Tm7sf2 +/+ ( n = 9) and Tm7sf2 –/– ( n = 11) mice. Data are given as mean ± SEM. * p < 0.05, ** p < 0.01 with respect to Tm7sf2 +/+ mice; unpaired two-tailed Student’s t -test. (E) Glucose tolerance tests, resting. Plasma glucose concentration during intraperitoneal glucose tolerance test (GTT) in fed condition of Tm7sf2 +/+ ( n = 10) and Tm7sf2 –/– ( n = 12) mice. Statistics as in (D) . (F) Glucose tolerance test after fasting. Plasma glucose concentration during intraperitoneal glucose tolerance test (GTT) in fasted (O/N) condition of Tm7sf2 +/+ ( n = 14) and Tm7sf2 –/– ( n = 9) mice. Statistics applied as in (D) yielded no significant differences. (G) Body weight of 1-year-old WT ( n = 8) and Tm7sf2 –/– ( n = 8) mice (left) and daily food intake of WT ( n = 19) and Tm7sf2 –/– ( n = 13) mice (right). Data are given as mean ± SEM. * p < 0.05, ** p < 0.01 with respect to Tm7sf2 +/+ mice; unpaired two-tailed Student’s t -test. (H) Akt phosphorylation. Immunoblot analysis for phosphorylated form of Akt (Ser473) in liver lysates obtained from Tm7sf2 +/+ ( n = 6) and Tm7sf2 –/– ( n = 6) mice fed on a chow diet. GAPDH was used as loading control. A blot with representative samples is shown. Graph on the right represents the mean ratio (%) ± SD of Akt Ser473 phosphorylation over total Akt levels. * p < 0.05, unpaired two-tailed Student’s t -test.

Article Snippet: Total liver extracts (60 μg) or membrane proteins (30 μg) were separated on sodium dodecyl sulfate (SDS)-polyacrylamide gels and transferred to nitrocellulose or polyvinylidene fluoride (PVDF) membranes, respectively, at 100 V for 90 min. Filters were probed with the following primary antibodies: p-Akt Ser 473 (1:1,000, 9,271) and total Akt (1:1,000, 9,272) by Cell Signaling Technology; Gapdh (1:500, sc-32233) by Santa Cruz Biotechnology; Srebp-2 (1:1,000, ATCC ® CRL2545) was a kind gift from Prof. T. Osborne to Rita Roberti; Tm7sf2 (1:1,000) , Hmgcr (1:500, 3,952), and Ldlr (1:1,000, 3,839) by BioVision; and horseradish peroxidase (HRP)-conjugated secondary antibodies (1:5,000, Bio-Rad).

Techniques: Protein-Protein interactions, Quantitative RT-PCR, Expressing, Two Tailed Test, Clinical Proteomics, Concentration Assay, Phospho-proteomics, Western Blot, Control