Journal: Lipids in health and disease

Article Title: Knockdown of RASD1 improves MASLD progression by inhibiting the PI3K/AKT/mTOR pathway.

doi: 10.1186/s12944-024-02419-z

Figure Lengend Snippet: Fig. 2 RASD1 expression was elevated in FFA-treated hepatocytes. (A) Representative ORO staining of hepatocytes that were treated with BSA or FFA and the corresponding quantitative analysis are presented on the right (scale bar: 50 μm). (B) Cellular TG content of hepatocytes that were treated with BSA or FFA. (C) WB determination and the quantification of the RASD1 protein in hepatocytes, with or without FFA or BSA treatment. (D)RASD1 mRNA levels were shown by qPCR analysis results, of hepatocytes treated with BSA or FFA. (E, F) Representative IF staining images of hepatocytes treated by either BSA or FFA (the scale bar shown as 20 μm). n = 3 in all groups. *P < 0.05, **P < 0.01, ***P < 0.001, as are compared to BSA treatment. All data presented in this figure here are means ± SDs

Article Snippet: For oil red O (ORO) staining, cultured cells and frozen liver Sects. (8–10 μm) were stained with an ORO Staining Kit (Beyotime, Shanghai, China) [28].

Techniques: Expressing, Staining

Journal: Lipids in health and disease

Article Title: Knockdown of RASD1 improves MASLD progression by inhibiting the PI3K/AKT/mTOR pathway.

doi: 10.1186/s12944-024-02419-z

Figure Lengend Snippet: Fig. 3 Overexpression of RASD1 promoted lipid deposition in FFA-treated hepatocytes. (A, B) The successful overexpression of RASD1 was confirmed by qPCR and WB (n = 3–4). The protein levels of RASD1 is quantified and shown on the right. (C) Representative ORO staining of Vector and RASD1 cell lines (scale bar: 50 μm). The corresponding quantitative analysis is presented on the right (n = 3). (D) Cellular TG contents of the Vector and RASD1 cell lines (n = 3). (E) Lipidomic analysis further demonstrated that TG synthesis was increased in the LO2 RASD1 cell line (n = 6). (F) Gene expression in lipid metabolism of the LO2 RASD1 cell line was measured and determined by qPCR. (G) WB determination of the protein levels was quantified in the Vector and RASD1 cell lines (n = 3). As are compared to Vector, *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001. All data presented in the figure are means ± SDs

Article Snippet: For oil red O (ORO) staining, cultured cells and frozen liver Sects. (8–10 μm) were stained with an ORO Staining Kit (Beyotime, Shanghai, China) [28].

Techniques: Over Expression, Staining, Plasmid Preparation, Gene Expression

Journal: Lipids in health and disease

Article Title: Knockdown of RASD1 improves MASLD progression by inhibiting the PI3K/AKT/mTOR pathway.

doi: 10.1186/s12944-024-02419-z

Figure Lengend Snippet: Fig. 4 RASD1 knockdown inhibited lipid deposition in FFA-treated hepatocytes. (A, B) The successful knockdown of RASD1 was confirmed by qPCR and WB (n = 3–4). The protein levels of RASD1 are quantified and shown on the right. (C) Representative ORO staining of the Scramble and shRASD1 cell lines with the corresponding quantification (n = 3, the scale bar shown as 50 μm). (D) Cellular TG contents derived from the Scramble and shRASD1 cell lines (n = 3). (E) Lipidomic analysis further demonstrated that TG synthesis was increased in the LO2 shRASD1 cell line (n = 6). (F) Gene expression of lipid metabolism in the LO2 shRASD1 cell line was measured by qPCR. (G) The quantification of WB detection in the Scramble and shRASD1 cell lines(n = 3). As comparing to Scramble as control, *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001. Unless otherwise noted, all data presented in the figure are means ± SDs

Article Snippet: For oil red O (ORO) staining, cultured cells and frozen liver Sects. (8–10 μm) were stained with an ORO Staining Kit (Beyotime, Shanghai, China) [28].

Techniques: Knockdown, Staining, Derivative Assay, Gene Expression, Control

Journal: Lipids in health and disease

Article Title: Knockdown of RASD1 improves MASLD progression by inhibiting the PI3K/AKT/mTOR pathway.

doi: 10.1186/s12944-024-02419-z

Figure Lengend Snippet: Fig. 5 RASD1 modulated cellular lipid metabolism via PI3K/AKT/mTOR pathway. (A,B) WB and the corresponding quantitative analysis of the expression of the pathway in stable cell lines (n = 3). (C, D) Representative ORO staining of the stable cell lines treated with 1 mM FFA with or without inhibitors for 24 h (scale bar shown as 50 μm) and the corresponding quantification (n = 3). (E-F) WB analysis and its quantification results, in the stable cell lines treated in1 mM FFA, with or without inhibitors for 24 h (n = 3). As compared to Vector, *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001 vs. Vector; As compared to Scramble, £P < 0.05, ££P < 0.01, £££P < 0.001; #P < 0.05, ##P < 0.01, ###P < 0.001, ####P < 0.0001, as compared to RASD1. Unless otherwise noted, all the data presented in the figure are means ± SDs

Article Snippet: For oil red O (ORO) staining, cultured cells and frozen liver Sects. (8–10 μm) were stained with an ORO Staining Kit (Beyotime, Shanghai, China) [28].

Techniques: Expressing, Stable Transfection, Staining, Plasmid Preparation

Journal: Lipids in health and disease

Article Title: Knockdown of RASD1 improves MASLD progression by inhibiting the PI3K/AKT/mTOR pathway.

doi: 10.1186/s12944-024-02419-z

Figure Lengend Snippet: Fig. 6 RASD1 knockdown improved MASLD development in vivo. (A) Schematic diagram of the animal study. (B) Hepatic Rasd1 mRNA levels in mice from four groups (n = 5–6). (C) Hepatic RASD1 protein levels and the corresponding quantitative analysis of mice from four groups (n = 3). (D) Representa tive images showing the gross look of the liver as well as staining of HE, IHC and ORO under microscope (scale bar: 50 μm) of mice from four groups. The quantitative analysis results of IHC and ORO staining are shown on the right (n = 3). (E) Body weights of the mice from four groups (n = 5). (F, G) results of the GTT and ITT detected in different time in the mice assigned to each group (n = 5–6). (H-K) AUC and AOC for the GTT and ITT results (n = 5–6). (L-P) Serum levels of liver function assays and insulin in the mice from four groups (n = 5–6). (Q) TG levels in the mice liver tissues from four groups (n = 5–6). With the NCD group as control comparison, *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001; With the AAV-shNC/HFD group as control comparison, #P < 0.05, ##P < 0.01, ####P < 0.0001, which are compared to the group. All data presented in the figure are means ± SDs

Article Snippet: For oil red O (ORO) staining, cultured cells and frozen liver Sects. (8–10 μm) were stained with an ORO Staining Kit (Beyotime, Shanghai, China) [28].

Techniques: Knockdown, In Vivo, Staining, Microscopy, Control, Comparison