PMC

Body weight development of Balb/C mice treated with PBS or 1.5 × 10⁶ colon 26 (C26) cells over 3 weeks (means ± SEM, n ≥ 6, Two Way Repeated Measures ANOVA, Holm–Sidak post hoc).Gastrocnemius (GC) skeletal muscle weight in the same mice as in A.Abdominal and inguinal fat weight in the same mice as in A.Serum triglyceride levels in the same mice as in A. Statistical test B–D: Student's t-test.

Western blot of liver extracts from representative control or TSC22D4 shRNA adenovirus–injected C57Bl/6 mice 7 days after injection using TSC22D4 and VCP antibodies.Liver TG levels of random fed, 16 h fasted and 6 h refed control or TSC22D4 shRNA adenovirus–injected C57Bl/6 mice 7 days after injection (means ± SEM, n ≥ 6).Total serum TG levels of the same mice as in B.Lipoprotein-associated serum TG levels as measured by fast protein liquid chromatography (FPLC) in random fed control or TSC22D4 shRNA adenovirus-injected C57Bl/6 mice 7 days after injection (means ± SEM, n ≥ 6). Statistical test B, C: Two Way ANOVA; Holm–Sidak post hoc.

Quantitative PCR analysis of ATP citrate lyase (Acly), acetyl-coenzyme A carboxylase 1 (Acc1), fatty acid synthase (Fasn), stearoyl-CoA desaturase-1 (Scd1), sterol regulatory element-binding protein-1c (Srebp1) and Lipin1 RNA levels in livers of control or TSC22D4 shRNA adenovirus-injected wild-type C57Bl/6 mice (means ± SEM, n > 7).Quantitative PCR analysis of TSC22D4, Acly and Scd1 mRNA levels in primary (1°) mouse hepatocytes treated with control or TSC22D4 shRNA adenovirus (means ± SEM, n = 3).Quantitative PCR analysis of Acly, Acc1, Fasn, Scd1, Srebp1 and Lipin1 RNA levels in livers of control or TSC22D4 cDNA adenovirus-injected wild-type C57Bl/6 mice (means ± SEM, n > 4).Quantitative PCR analysis of Tsc22d4, Acly, Fasn, Scd1, Srebp1 and Lipin1 RNA levels in primary (1°) human hepatocytes treated with control or TSC22D4 shRNA adenovirus (means ± SEM, n = 3). Statistical test A–D: Student's t-test.

Western blot of liver extracts from representative empty control or Flag-TSC22D4 cDNA adenovirus-injected C57Bl/6 mice 7 days after injection using FLAG and VCP antibodies (means ± SEM, n ≥ 4).Lipoprotein-associated serum TG levels as measured by fast protein liquid chromatography (FPLC) in mice injected with empty control or TSC22D4 cDNA adenovirus in the fed or 16 h fasted state (means ± SEM, n ≥ 4).Lipoprotein-associated serum TG levels as measured by fast protein liquid chromatography (FPLC) in high fat diet (HFD) fed mice injected with empty control or TSC22D4 cDNA adenovirus (means ± SEM, n ≥ 3).Liver TG levels of HFD or control diet (LFD) fed mice (means ± SEM, n ≥ 3, Two Way ANOVA; Holm–Sidak post hoc).

Correlation of TSC22D4 RNA expression and the degree of weight loss due to tumour implantation in Balb/C mice treated with PBS or 1.5 × 10⁶ colon 26 (C26) cells over 3 weeks (means ± SEM, n ≥ 6, Pearson correlation coefficient, F-test to determine significance).Correlation of TSC22D4 RNA expression and VLDL-TG release in the same mice as in A. (Pearson correlation coefficient, F-test to determine significance)Quantitative PCR analysis of Acly, Scd1 and Fasn RNA levels in primary mouse hepatocytes treated with conditioned media from C26 cells for 24 h and control or TSC22D4 shRNA adenovirus for 48 h (means ± SEM, n = 3, Student's t-test for DMEM NC shRNA vs. C26 NC shRNA; C26 NC shRNA vs. C26 TSC22D4 shRNA).Quantitative PCR analysis of Acly, Scd1 and Fasn RNA levels in primary mouse hepatocytes treated with conditioned media from B16 cells for 24 h and control or TSC22D4 shRNA adenovirus for 48 h (means ± SEM, n = 3, Student's t-test for DMEM NC shRNA vs. B16 NC shRNA; B16 NC shRNA vs. B16 TSC22D4 shRNA).

Hepatic VLDL release in Balb/C mice injected either PBS or 1.5 × 10⁶ C26 cells (means ± SEM, n ≥ 6, Two Way Repeated Measures ANOVA, Holm-Sidak post hoc).Clearance of human ApoB from serum of Balb/C mice injected with either PBS or 1.5 × 10⁶ C26 cells. 20 µg of human VLDL were injected into each animal and serum samples were taken at the indicated time points. Human ApoB levels were determined by human-specific ELISA (means ± SEM, n = 2–4).Quantitative PCR analysis of hepatic ATP citrate lyase (Acly), fatty acid synthase (Fasn) and stearoyl-CoA desaturase-1 (Scd1) RNA levels in the same mice as in A (Student's t-test).Lipoprotein-associated serum TG levels as measured by fast protein liquid chromatography (FPLC) in the same mice as in A.Lipoprotein-associated serum cholesterol levels as measured by fast protein liquid chromatography (FPLC) in the same mice as in A.

Hepatic VLDL release in control or TSC22D4 shRNA-injected wild-type C57Bl/6 mice. Time after tyloxapol injection indicated (means ± SEM, n = 6). Statistical test: Two Way Repeated Measures ANOVA, Holm–Sidak post hoc.Apolipoprotein B protein expression is elevated in the livers of representative TSC22D4 shRNA adenovirus-injected C57Bl/6 mice. Western blots were performed 7 days after injection using ApoB and VCP antibodies.Hepatic VLDL release in empty control or TSC22D4 cDNA-injected wild-type C57Bl/6 mice fed a high fat diet (HFD) for 11 weeks. Time after tyloxapol injection indicated (means ± SEM, n = 4). Statistical test: Two Way Repeated Measures ANOVA, Holm–Sidak post hoc.White adipose tissue (WAT) lipoprotein lipase (LPL) activity in the same mice as in A. (student's t-test)Clearance of human ApoB from serum of control or TSC22D4 shRNA adenovirus–injected C57Bl/6 mice 7 days after virus injection. 20 µg of human VLDL were injected into each animal and serum samples were taken at the indicated time points. Human ApoB levels were determined by human-specific ELISA (means ± SEM, n = 6). Statistical test: Two Way Repeated Measures ANOVA, Holm–Sidak post hoc.

Quantitative PCR analysis of Peroxisome proliferator-activated receptor gamma coactivator 1-alpha (Pgc1a), Peroxisome proliferator-activated receptor gamma coactivator 1-beta (Pgc1b), Nuclear receptor coactivator 1 (Src1), nuclear receptor coactivator 3 (Src3), Transducin beta like 1 (Tbl1), Creb binding protein (Cbp) and Tsc22 domain family member 3 (Tsc22d3) in Balb/C mice injected with either PBS or 1.5 × 10⁶ C26 cells (means ± SEM, n ≥ 6). Statistical test: Student's t-test.Western blot analysis of TSC22D4 and valosine-containing protein (VCP) expression in representative mice from A.Serum TG levels in C57Bl/6 mice fed a methionine and choline deficient diet (MCD) or a corresponding control diet for 4 weeks (means ± SEM, n ≥ 5).Quantitative PCR analysis of fatty acid synthase (Fasn) and sterol regulatory element-binding protein-1c (Srebp1) RNA levels in the same mice as in C.Quantitative PCR analysis of Tsc22 domain family member 4 (Tsc22d4) RNA levels in the same mice as in C.Western blot analysis of TSC22D4 and VCP expression in representative mice from C. Statistical test C–E: Student's t-test.