Journal: Molecular Therapy

Article Title: Treatment with THI, an inhibitor of sphingosine-1-phosphate lyase, modulates glycosphingolipid metabolism and results therapeutically effective in experimental models of Huntington’s disease

doi: 10.1016/j.ymthe.2022.09.004

Figure Lengend Snippet: Defects in HexCer metabolism are detectable early in the disease course in different HD mouse models (A) Lipidomic analysis by LC-MS/MS of HexCer 16:0, 18:0, and 24:1 in striatal tissues of pre-manifest (4-week-old) R6/2 mice and aged-matched WT littermates. Values are represented as mean ± SD. N = 4 males for each group of mice. Unpaired t test, ∗p < 0.05. (B) qPCR analysis of Ugcg in striatal tissues of pre-manifest (4-week-old) R6/2 mice and aged-matched WT littermates. Values are represented as mean ± SD. N = 6 (females N = 3; males N = 3) for each group of mice. (C) qPCR analysis of Gba1 in striatal tissues of pre-manifest (4-week-old) R6/2 mice and aged-matched WT littermates. Values are represented as mean ± SD. N = 6 (females N = 3; males N = 3) for each group of mice. (D) qPCR analysis of Gba2 in striatal tissues of pre-manifest (4-week-old) R6/2 mice and aged-matched WT littermates. Values are represented as mean ± SD. N = 6 (females N = 3; males N = 3) for each group of mice. (E) Slot blotting and densitometric analysis of GluCer in cortical tissues from WT and heterozygous zQ175 mice at age 20 weeks. Values are represented as mean ± SD. N = 4 for WT (females N = 3; males N = 1) mice; N = 6 (females N = 3; males N = 3) for HD mice. Ponceau red was used as total protein loading control. Unpaired t test, ∗∗p < 0.01. (F) Slot blotting and densitometric analysis of GluCer in cortical tissues from WT and YAC128 mice at age 13 weeks. Values are represented as mean ± SD. N = 5 for WT (females N = 3; males N = 2) mice; N = 6 (females N = 3; males N = 3) for HD mice. Ponceau red was used as total protein loading control. Unpaired t test, ∗∗p < 0.01.

Article Snippet: Cells lysates were prepared in lysis buffer containing 20 mM Tris (pH 7.4), 1% Nonidet P-40, 1 mM EDTA, 20 mM NaF, 2 mM Na 3 VO 4 , and 1:1,000 protease inhibitor mixture (Immunological Sciences, cat. no. IS11041), sonicated with 2 ×10 s pulses and then centrifuged for 10 min at 10,000 × g . Five micrograms of total protein lysate was spotted on nitrocellulose membrane using a slot-blotting apparatus (Hoefer PR 648 Slot Blot Blotting Manifold).

Techniques: Liquid Chromatography with Mass Spectroscopy, Control

Journal: Molecular Therapy

Article Title: Treatment with THI, an inhibitor of sphingosine-1-phosphate lyase, modulates glycosphingolipid metabolism and results therapeutically effective in experimental models of Huntington’s disease

doi: 10.1016/j.ymthe.2022.09.004

Figure Lengend Snippet: Both deficiency of wtHtt and overexpression of mutant exon-1 fragments increase GluCer levels in vitro (A) Representative cropped slot blotting and densitometric analysis of GluCer in WT (STHdh 7/7 ) and HD (STHdh 111/111 ) total protein lysate. Values are represented as mean ± SD of four independent experiments. Ponceau red was used as total protein loading control. Unpaired t test, ∗p < 0.05. (B) qPCR analysis of Ugcg in STHdh 7/7 and STHdh 111/111 cells. Values are represented as mean ± SD of four independent experiments. Unpaired t test, ∗p < 0.05. (C) qPCR analysis of Gba1 in STHdh 7/7 and STHdh 111/111 cells. Values are represented as mean ± SD of four independent experiments. Unpaired t test, ∗p < 0.05. (D) qPCR analysis of Gba2 in STHdh 7/7 and STHdh 111/111 cells. Values are represented as mean ± SD of four independent experiments. (E) Representative cropped immunoblotting and densitometric analysis of wtHtt in STHdh 7/7 after 48 h of transfection with either control (CTRL) or Htt (HTT) small interfering RNA (siRNA). Values are represented as mean ± SD of four independent experiments. Unpaired t test, ∗p < 0.05. (F) Representative cropped slot blotting and densitometric analysis of GluCer in STHdh 7/7 cells after 48 h of transfection with either control (CTRL) or Htt (HTT) small interfering RNA (siRNA). Values are represented as mean ± SD of four independent experiments. Ponceau red was used as total protein loading control. Unpaired t test, ∗∗p < 0.01. (G) qPCR analysis of Ugcg in STHdh 7/7 cells after 48 h of transfection with either control (CTRL) or Htt (HTT) small interfering RNA (siRNA). Values are represented as mean ± SD of four independent experiments. (H) qPCR analysis of Gba1 in STHdh 7/7 cells after 48 h of transfection with either control (CTRL) or Htt (HTT) small interfering RNA (siRNA). Values are represented as mean ± SD of four independent experiments. (I) qPCR analysis of Gba2 in STHdh 7/7 cells after 48 h of transfection with either control (CTRL) or Htt (HTT) small interfering RNA (siRNA). Values are represented as mean ± SD of four independent experiments. (J) Representative cropped immunoblotting and densitometric analysis of GluCer in STHdh 7/7 after 48 h of transfection with either control (pcDNA3.1) or Htt (mHttex1) EGP-expressing construct. Values are represented as mean ± SD of four independent experiments. Ponceau red was used as total protein loading control. Unpaired t test, ∗p < 0.05. (K) Immunoblottings of both GFP and actin in STHdh 7/7 cells after 48 h of transfection with either control (pcDNA3.1) or Htt (mHttex1) EGP-expressing construct.

Article Snippet: Cells lysates were prepared in lysis buffer containing 20 mM Tris (pH 7.4), 1% Nonidet P-40, 1 mM EDTA, 20 mM NaF, 2 mM Na 3 VO 4 , and 1:1,000 protease inhibitor mixture (Immunological Sciences, cat. no. IS11041), sonicated with 2 ×10 s pulses and then centrifuged for 10 min at 10,000 × g . Five micrograms of total protein lysate was spotted on nitrocellulose membrane using a slot-blotting apparatus (Hoefer PR 648 Slot Blot Blotting Manifold).

Techniques: Over Expression, Mutagenesis, In Vitro, Control, Western Blot, Transfection, Small Interfering RNA, Expressing, Construct