Journal: Advanced Science

Article Title: Microglial MS4A4A Protects against Epileptic Seizures in Alzheimer's Disease

doi: 10.1002/advs.202417733

Figure Lengend Snippet: Expression of MS4A4A is upregulated in lesions of epilepsy patients. The heatmaps displaying the expression levels of MS4A family in the bulk RNA‐seq of A) FCD_2b seizure patients and B) TSC seizure patients. n = 4. The heatmaps displaying the expression levels of the genes related to phagocytosis and other processes in the bulk RNA‐seq of C) FCD_2b seizure patients and D) TSC seizure patients. n = 4. E) Relative transcript levels of M S 4A family genes in lesions and perilesional tissues of LEAT, FCD_2b, and TSC patients verified by RT‐qPCR. n ≥ 8. F) Scheme for injecting AAV9‐hSyn‐EGFP‐2A‐Cre‐WPRE (AAV9‐hSyn‐EGFP‐3×FLAG‐WPRE as control) into Tsc1 fl/fl mice. G) Relative transcript levels of Tsc1 and Ms4a4a around injection site determined by RT‐qPCR. Parenchymal stereotaxic injection was conducted two weeks before RT‐qPCR. n = 4. Data are presented as mean ± SEM. p ‐values were calculated by two‐tailed, paired Student's t ‐test (A–E and G), # p > 0.05, * p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001.

Article Snippet: APP/PS1 (JAX 004462, RRID:IMSR_JAX:004462), Ai95 (JAX 028865, RRID:IMSR_JAX:028865), Tsc1 fl/fl (JAX 038428, RRID:IMSR_JAX:038428) were purchased from the Jackson Laboratory.

Techniques: Expressing, RNA Sequencing, Quantitative RT-PCR, Control, Injection, Two Tailed Test

Journal: EMBO Reports

Article Title: mTOR controls ependymal cell differentiation by targeting the alternative cell cycle and centrosomal proteins

doi: 10.1038/s44319-025-00460-2

Figure Lengend Snippet: ( A , B ) P4 brain lateral ventricular en face, labeling Centrin (centrioles, white), p-rpS6 (red) and GT335 (cilia, green) or p21 (green) in ( A , B ), respectively. ( C ) Ependymal cells in respective phases of differentiation characterized by their staining of Centrin (centrioles, white) and GT335 (cilia, green), and p-rpS6 (red). It is worth noting that the expression of p-rpS6 is during the intermediate stages of differentiation. ( D ) Regression plot testing the correlation between apical area and centriole number in mature ependymal cells in Tsc1 cKO at P0, rapamycin injected pups at P4 and their respective controls, R 2 is the correlation coefficient; ns P = 0.0542; ** P = 0.0023; *** P = 0.0001; **** P < 0,0001. Data information: In ( D ), ** P < 0.01, *** P < 0.001, **** P < 0.0001 (Pearson’s correlation test). n = number of cells >50. Scale bars: 10 μm.

Article Snippet: Other genetic models used were Centrin2-GFP (CB6-Tg(CAG-EGFP/CETN2)3-4Jgg/J; The Jackson Laboratories) (Higginbotham et al, ), Tsc1 fl/fl (The Jackson Laboratories®), Nestin-Cre (Tronche et al, ) and S6K1/S6K2-double mutant mice (Pende et al, ).

Techniques: Labeling, Staining, Expressing, Injection

Journal: EMBO Reports

Article Title: mTOR controls ependymal cell differentiation by targeting the alternative cell cycle and centrosomal proteins

doi: 10.1038/s44319-025-00460-2

Figure Lengend Snippet: ( A ) P4 lateral walls from control and rapamycin-treated pups and their segmentation. Cells are color-coded based on their stage of differentiation. Scale bar: 10 μm. ( B , C ) Percentage ( B ) and size of the apical surface ( C ) of progenitor, differentiating and mature ependymal cells in rapamycin and control conditions; * P = 0.0429 in ( B ) and 0.0135 in ( C ); ** P = 0.0024 in ( C ) and **** P < 0.0001 in ( B , C ). ( D ) P4 ventricular wall in control and rapamycin-treated pups, showing centrioles with FOP, deuterosomes with Deup1, and cell junctions with ß-catenin. ( E , F ) Number of centrioles ( E ) and area occupied by centriolar patch in differentiated cells at P4 ( F ). ( G ) En face view showing expression of p-rpS6 and Foxj1 in Control and Tsc1 cKO. ( H ) pS6 mean gray intensity in Foxj1+ cells in control and Tsc1 cKO. ( I ) P0 brain lateral ventricular en face labeled with Fop (centrioles, red), b-Catenin (cell junction, white), and deup (deuterosome, green) of control and Tsc1 ko/lox ; Nestin-cre (Tsc1 cKO) mice. ( J , K ) Number of centrioles and size of the centriolar patch in differentiated cells in Tsc1 cKO and controls at P0. Each dot in the quantifications corresponds to one cell ( n > 3); **** P < 0.0001 in ( E , F , H , J , K ). Data information: In ( B , C , E , F , H , J , K ), data are presented as mean ± SD. **** P < 0.0001, *** P < 0.001, ** P < 0.01, * P ≤ 0.05 (Student’s t test). .

Article Snippet: Other genetic models used were Centrin2-GFP (CB6-Tg(CAG-EGFP/CETN2)3-4Jgg/J; The Jackson Laboratories) (Higginbotham et al, ), Tsc1 fl/fl (The Jackson Laboratories®), Nestin-Cre (Tronche et al, ) and S6K1/S6K2-double mutant mice (Pende et al, ).

Techniques: Control, Expressing, Labeling

Journal: EMBO Reports

Article Title: mTOR controls ependymal cell differentiation by targeting the alternative cell cycle and centrosomal proteins

doi: 10.1038/s44319-025-00460-2

Figure Lengend Snippet: ( A ) Representative images of primary cells from control and Tsc1 cKO mice at div 4 immunolabeled with FOP (centrioles), GT335, and pS6 antibodies. ( B ) Percentage of multicentriolar cells at div 4 and div 8; **** P < 0.0001; ns P = 0.8182. ( C , D ) Representative images of primary cells from Tsc1fl/fl mice infected with GFP (C) or Cre-GFP Adenovirus. Cells are immunolabeled with FOP and pS6 antibodies. Control is the same as in Fig. , as the experiments were carried out in parallel. ( E ) Percentage of multicentriolar cells at div 4 and div 8 in all conditions; * P = 0.043; ns P = 0.6991. ( F ) Representative pictures of wild-type or S6K −/− primary cells at 4 days in vitro, immunolabelled with pS6 as a marker of mTORC1 and FOP to detect multibasal bodies (MBB) cells in differentiated cells. The top panels are cells treated with vehicle (EtOH), and the bottom panels are cells treated with 20 nM Rapamycin. The arrows show examples of MBB cells. div differentiation in vitro, WT wild type. ( G ) Fold changes of differentiated ependymal cell number by counting cells with multibasal bodies. The bars represent the mean +/− SEM; each dot is one replicate, n = 6. P values were determined using a two-sided Student’s t test. ** P < 0.01 and *** P < 0.001. Data information: In ( B , E ), data are presented as mean ± SD. **** P < 0.0001, * P ≤ 0.05, ns not significant (Student’s t test). In ( G ), the bars represent the mean +/− SEM; each dot is one replicate, n = 6. P values were determined using a two-sided Student’s t test. ** P < 0.01 and *** P < 0.001. Ctrl Control.

Article Snippet: Other genetic models used were Centrin2-GFP (CB6-Tg(CAG-EGFP/CETN2)3-4Jgg/J; The Jackson Laboratories) (Higginbotham et al, ), Tsc1 fl/fl (The Jackson Laboratories®), Nestin-Cre (Tronche et al, ) and S6K1/S6K2-double mutant mice (Pende et al, ).

Techniques: Control, Immunolabeling, Infection, In Vitro, Marker

Journal: Advanced Science

Article Title: Osteocytes/Osteoblasts Produce SAA3 to Regulate Hepatic Metabolism of Cholesterol

doi: 10.1002/advs.202307818

Figure Lengend Snippet: Genetic depletion of Tsc1 in osteocytes/osteoblasts is associated with elevated serum cholesterol level in mice. A) Cre‐mediated recombination eliminates exons 17–18 of Tsc1 gene. B) Western blot of TSC1 and pS6 expression levels in cortical bone samples from Tsc1 Dmp1 mice and control littermates. C) Appearance of serum samples collected from Tsc1 Dmp1 mice and control littermates at 6 months of age. D) Serum cholesterol concentrations in Tsc1 Dmp1 mice and control littermates at 3 or 6 months of age (n = 5–14, Ctrl (3 m ): 3 females, 2 males; Tsc1 Dmp1 (3 m ): 4 females, 3 males; Ctrl (6 m ): 6 females, 8 males; Tsc1 Dmp1 (6 m ): 9 females, 5 males). E) Body weight curves of Tsc1 Dmp1 mice and control littermates from 2 to 6 months of age (n = 6, 4 females, 2 males). F) Serum LDL‐C concentrations in Tsc1 Dmp1 mice and control littermates at 6 months of age (n = 6, 4 females, 2 males). G) Serum cholesterol concentrations in Tsc1 Dmp1 mice and control littermates treated with or without rapamycin (2 mg kg −1 of body weight/day for 2 weeks) at 9 months of age (n = 3–5, Ctrl: 4 females, 1 male; Tsc1 Dmp1 without rapamycin: 2 females, 1 male; Tsc1 Dmp1 with rapamycin: 3 females, 1 male). H) Representative H&E staining images of paraffin sections of liver from 6‐month‐old Tsc1 Dmp1 mice and control littermates. I) Representative Oil red O staining images in frozen liver sections of 6‐month‐old Tsc1 Dmp1 mice and control littermates. Scale bars, 100 µm; data represent mean ± SD; each symbol represents one animal. * p < 0.05, ** p < 0.01, *** p < 0.001 compared to Ctrl; # p < 0.05 compared to Tsc1 Dmp1 , by unpaired t test (D‐F) or two‐way ANOVA (G).

Article Snippet: To generate osteocyte/osteoblast‐specific Tsc1 deletion mice, Tsc1 fl/fl mice (IMSR_JAX:005680) were crossed with Dmp1 Cre mice.

Techniques: Western Blot, Expressing, Control, Staining

Journal: Advanced Science

Article Title: Osteocytes/Osteoblasts Produce SAA3 to Regulate Hepatic Metabolism of Cholesterol

doi: 10.1002/advs.202307818

Figure Lengend Snippet: Loss of Tsc1 induces expression and secretion of SAA3 in osteocytes/osteoblasts. A) Venn diagram showing categories of upregulated genes (FDR < 0.05, RNA‐seq) in cortical bone samples collected from 3‐month‐old Tsc1 Dmp1 mice comparing to control littermates. Outlined box on the right shows the 7 genes that are known to be related to lipid metabolism. B) Volcano plot showing differences in gene expression profile between cortical bone samples collected from 3‐month‐old Tsc1 Dmp1 mice and control littermates, labeled Saa3, Lipg, Ccl8, Fgf23, Nov, Vcan, and Ang . C) Expression levels of the 7 upregulated lipid metabolism related genes identified in transcriptome analysis. D,E) Relative mRNA expression levels (qRT‐PCR) of Saa1 (E), Saa2 F), and Saa3 (D) in mouse cortical bones collected from 3‐month‐old Tsc1 Dmp1 mice or control littermates (n = 4, 3 females, 1 male). G) Quantification of serum SAA3 concentration (ELISA) in 3‐month‐old Tsc1 Dmp1 mice and control littermates (n = 6, 4 females, 2 male). H) Quantification of serum SAA3 concentration (ELISA) in 9‐month‐old Tsc1 Dmp1 mice and control littermates treated with or without rapamycin (2 mg kg −1 of body weight/day for 2 weeks) (n = 3–5, Ctrl: 4 females, 1 male; Tsc1 Dmp1 without rapamycin: 2 females, 1 male; Tsc1 Dmp1 with rapamycin: 3 females, 1 male). I,J) Saa3 /SAA3 mRNA (I) or protein (J) levels in indicated mouse osteocyte Mlo‐Y4 cell group (n = 3–4 per group). K) Representative immunofluorescence staining images of DMP1/SAA3 co‐localization in mouse femur paraffin sections and quantification of cells with different SAA3 expression levels (n = 3, 2 females, 1 male). Left panel insets show individual staining images. Scale bars, 50 µm; data represent mean ± SD; each symbol represents one animal or one well. * p < 0.05, ** p < 0.01, *** p < 0.001 compared to Ctrl; # p < 0.05, ## p < 0.01 compared to Tsc1 Dmp1 or shTsc1 , by unpaired t test (D‐G, K) or two‐way ANOVA (H‐J).

Article Snippet: To generate osteocyte/osteoblast‐specific Tsc1 deletion mice, Tsc1 fl/fl mice (IMSR_JAX:005680) were crossed with Dmp1 Cre mice.

Techniques: Expressing, RNA Sequencing, Control, Gene Expression, Labeling, Quantitative RT-PCR, Concentration Assay, Enzyme-linked Immunosorbent Assay, Immunofluorescence, Staining

Journal: Advanced Science

Article Title: Osteocytes/Osteoblasts Produce SAA3 to Regulate Hepatic Metabolism of Cholesterol

doi: 10.1002/advs.202307818

Figure Lengend Snippet: Tsc1 Dmp1 and rSAA3‐treated mice exhibit reduction in hepatic CYP7A1. A) Transcriptional analysis of liver samples collected from 3‐month‐old Tsc1 Dmp1 mice or control littermates. Diagram shows the top 20 KEGG pathways enriched. The size and color of bubbles represent the number and degree of enrichment of differentially expressed mRNA enriched in the pathway. B) Expression levels of the top 10 downregulated lipid metabolism related genes identified in transcriptome analysis. C) Relative mRNA expression levels (qRT‐PCR) of Cyp7a1 in liver samples collected from 6‐month‐old Tsc1 Dmp1 mice or control littermates (n = 6, 4 females, 2 males). D) Relative mRNA expression levels (qRT‐PCR) of Cyp7a1 in liver samples collected from mice treated with 500 ng mL −1 of rSAA3 or PBS (n = 5, 5 males). E) Western blot analysis of CYP7A1 in liver samples collected from 6‐month‐old Tsc1 Dmp1 mice or control littermates (n = 6, 4 females, 2 males). F) Representative immunohistochemical staining images of CYP7A1 in paraffin sections of liver samples collected from 6‐month‐old Tsc1 Dmp1 mice or control littermates and quantification of cells with different CYP7A1 expression levels (n = 4, 3 females, 1 male). G) Representative immunohistochemical staining of CYP7A1 in paraffin sections of liver samples collected from mice treated with 500 ng mL −1 of rSAA3 or PBS (n = 4, 4 males). H) Relative mRNA expression levels (qRT‐PCR) of Alb and Clec4f in hepatocytes and Kupffer cells (n = 6, 6 males). I) Relative mRNA expression levels (qRT‐PCR) of Cyp7a1 in hepatocytes and Kupffer cells (n = 3, 3 males). J) Western blot analysis of CYP7A1 in Hepa1‐6 cells treated with serum samples collected from 6‐month‐old Tsc1 Dmp1 mice or control littermates (n = 4, 3 females, 1 male). K) Western blot analysis of CYP7A1 in Hepa1‐6 cells treated with 50 ng mL −1 of rSAA3 (n = 4). Scale bars, 200 µm; data represent mean ± SD; each symbol represents one animal. * p < 0.05, ** p < 0.01, *** p < 0.001, by one‐way ANOVA (D, K) or unpaired t test (C, E‐J).

Article Snippet: To generate osteocyte/osteoblast‐specific Tsc1 deletion mice, Tsc1 fl/fl mice (IMSR_JAX:005680) were crossed with Dmp1 Cre mice.

Techniques: Control, Expressing, Quantitative RT-PCR, Western Blot, Immunohistochemical staining, Staining

Journal: Advanced Science

Article Title: Osteocytes/Osteoblasts Produce SAA3 to Regulate Hepatic Metabolism of Cholesterol

doi: 10.1002/advs.202307818

Figure Lengend Snippet: SAA3 binds to TLR4 on hepatocytes and phosphorylates c‐Jun to downregulate CYP7A1. A) Representative immunofluorescence staining images of TLR4/SAA3 co‐localization in Hepa1‐6 cells treated with 50 ng mL −1 of rSAA3. Left panel insets show individual staining images. B) Representative result of western blot analysis following immunoprecipitation assay detecting the binding effect of TLR4 and SAA3 in Hepa1‐6 cells treated with 50 ng mL −1 of rSAA3. C) Representative result of western blot analysis of p‐c‐Jun in liver samples collected from 6‐month‐old Tsc1 Dmp1 mice or control littermates. D) Representative result of western blot analysis of p‐c‐Jun in Hepa1‐6 cells treated with 50 ng mL −1 of rSAA3 or PBS. E) Western blot analysis of c‐Jun phosphorylation in Hepa1‐6 cells treated with 50 µ m of Sp600125 in the presence or absence of SAA3 stimulation (n = 3). Right panel shows quantification. F) Western blot analysis of CYP7A1 in liver samples collected from mice treated with 5 mg kg −1 of Sp600125 in the presence or absence of SAA3 treatment (n = 3, 3 males). Right panel shows quantification. G) Relative mRNA expression levels (qRT‐PCR) of Cyp7a1 in Hepa1‐6 cells treated with 50 µ m of Sp600125 in the presence or absence of SAA3 stimulation (n = 4). H) Representative immunohistochemical staining images of CYP7A1 in paraffin sections of liver samples collected from mice treated with 5 mg kg −1 of Sp600125 in the presence or absence of SAA3 treatment. Right panel shows quantification of cells with different CYP7A1 expression levels (n = 3, 3 males). I) Serum cholesterol concentrations in C57BL/6 mice treated with 5 mg kg −1 of Sp600125 in the presence or absence of SAA3 treatment (n = 3, 3 males). Scale bars, 50 µm (A); Scale bars, 200 µm (H); data represent mean ± SD; each symbol represents one animal. * p < 0.05, ** p < 0.01, *** p < 0.001, ***** p < 0.0001 compared to rSAA3 − /Sp600125 − , # p < 0.05, ### p < 0.001 compared to rSAA3 + /Sp600125 − , by two‐way ANOVA (E‐I).

Article Snippet: To generate osteocyte/osteoblast‐specific Tsc1 deletion mice, Tsc1 fl/fl mice (IMSR_JAX:005680) were crossed with Dmp1 Cre mice.

Techniques: Immunofluorescence, Staining, Western Blot, Immunoprecipitation, Binding Assay, Control, Phospho-proteomics, Expressing, Quantitative RT-PCR, Immunohistochemical staining

Journal: Advanced Science

Article Title: Osteocytes/Osteoblasts Produce SAA3 to Regulate Hepatic Metabolism of Cholesterol

doi: 10.1002/advs.202307818

Figure Lengend Snippet: TSC1 deficiency‐enabled crosstalk from bone to liver requires SAA3. A) Schematic illustrating the reproductive strategy in generating Tsc1 Dmp1 /Saa3 − / − mice. B) Knockout validation was monitored with the SAA3 levels by western blot analysis. C) Western blot analysis of CYP7A1, p‐c‐Jun, and c‐Jun in liver samples collected from 6‐month‐old Tsc1 Dmp1 /Saa3 − / − mice or indicated control strain (n = 3, 2 females, 1 male). D) Relative mRNA expression levels (qRT‐PCR) of Cyp7a1 in liver samples collected from 6‐month‐old Tsc1 Dmp1 /Saa3 − / − mice or indicated control strain (n = 3, 2 females, 1 male). E) Representative immunohistochemical staining images of CYP7A1 in paraffin sections of liver samples collected from 6‐month‐old Tsc1 Dmp1 /Saa3 − / − mice or indicated control strain. Right panel shows quantification of cells with different CYP7A1 expression levels (n = 3, 2 females, 1 male). F) Serum cholesterol levels in 6‐month‐old Tsc1 Dmp1 /Saa3 − / − mice or indicated control strain (n = 4, 2 females, 2 males). Bottom panel insets show magnification. Scale bars, 200 µm; data represent mean ± SD; each symbol represents one animal. * p < 0.05, ** p < 0.01, *** p < 0.001 compared to Ctrl, # p < 0.05, ## p < 0.01 compared to Tsc1 Dmp1 , by two‐way ANOVA (D‐F).

Article Snippet: To generate osteocyte/osteoblast‐specific Tsc1 deletion mice, Tsc1 fl/fl mice (IMSR_JAX:005680) were crossed with Dmp1 Cre mice.

Techniques: Knock-Out, Biomarker Discovery, Western Blot, Control, Expressing, Quantitative RT-PCR, Immunohistochemical staining, Staining

Journal: Advanced Science

Article Title: Osteocytes/Osteoblasts Produce SAA3 to Regulate Hepatic Metabolism of Cholesterol

doi: 10.1002/advs.202307818

Figure Lengend Snippet: Genetic depletion of Tsc1 in osteocytes/osteoblasts is associated with elevated serum cholesterol level in mice. A) Cre‐mediated recombination eliminates exons 17–18 of Tsc1 gene. B) Western blot of TSC1 and pS6 expression levels in cortical bone samples from Tsc1 Dmp1 mice and control littermates. C) Appearance of serum samples collected from Tsc1 Dmp1 mice and control littermates at 6 months of age. D) Serum cholesterol concentrations in Tsc1 Dmp1 mice and control littermates at 3 or 6 months of age (n = 5–14, Ctrl (3 m ): 3 females, 2 males; Tsc1 Dmp1 (3 m ): 4 females, 3 males; Ctrl (6 m ): 6 females, 8 males; Tsc1 Dmp1 (6 m ): 9 females, 5 males). E) Body weight curves of Tsc1 Dmp1 mice and control littermates from 2 to 6 months of age (n = 6, 4 females, 2 males). F) Serum LDL‐C concentrations in Tsc1 Dmp1 mice and control littermates at 6 months of age (n = 6, 4 females, 2 males). G) Serum cholesterol concentrations in Tsc1 Dmp1 mice and control littermates treated with or without rapamycin (2 mg kg −1 of body weight/day for 2 weeks) at 9 months of age (n = 3–5, Ctrl: 4 females, 1 male; Tsc1 Dmp1 without rapamycin: 2 females, 1 male; Tsc1 Dmp1 with rapamycin: 3 females, 1 male). H) Representative H&E staining images of paraffin sections of liver from 6‐month‐old Tsc1 Dmp1 mice and control littermates. I) Representative Oil red O staining images in frozen liver sections of 6‐month‐old Tsc1 Dmp1 mice and control littermates. Scale bars, 100 µm; data represent mean ± SD; each symbol represents one animal. * p < 0.05, ** p < 0.01, *** p < 0.001 compared to Ctrl; # p < 0.05 compared to Tsc1 Dmp1 , by unpaired t test (D‐F) or two‐way ANOVA (G).

Article Snippet: Tsc1 fl/fl and Dmp1‐Cre mice ( Dmp1 Cre , IMSR_JAX:023047) were purchased from The Jackson Laboratory (Bar Harbor, ME).

Techniques: Western Blot, Expressing, Control, Staining

Journal: Advanced Science

Article Title: Osteocytes/Osteoblasts Produce SAA3 to Regulate Hepatic Metabolism of Cholesterol

doi: 10.1002/advs.202307818

Figure Lengend Snippet: Loss of Tsc1 induces expression and secretion of SAA3 in osteocytes/osteoblasts. A) Venn diagram showing categories of upregulated genes (FDR < 0.05, RNA‐seq) in cortical bone samples collected from 3‐month‐old Tsc1 Dmp1 mice comparing to control littermates. Outlined box on the right shows the 7 genes that are known to be related to lipid metabolism. B) Volcano plot showing differences in gene expression profile between cortical bone samples collected from 3‐month‐old Tsc1 Dmp1 mice and control littermates, labeled Saa3, Lipg, Ccl8, Fgf23, Nov, Vcan, and Ang . C) Expression levels of the 7 upregulated lipid metabolism related genes identified in transcriptome analysis. D,E) Relative mRNA expression levels (qRT‐PCR) of Saa1 (E), Saa2 F), and Saa3 (D) in mouse cortical bones collected from 3‐month‐old Tsc1 Dmp1 mice or control littermates (n = 4, 3 females, 1 male). G) Quantification of serum SAA3 concentration (ELISA) in 3‐month‐old Tsc1 Dmp1 mice and control littermates (n = 6, 4 females, 2 male). H) Quantification of serum SAA3 concentration (ELISA) in 9‐month‐old Tsc1 Dmp1 mice and control littermates treated with or without rapamycin (2 mg kg −1 of body weight/day for 2 weeks) (n = 3–5, Ctrl: 4 females, 1 male; Tsc1 Dmp1 without rapamycin: 2 females, 1 male; Tsc1 Dmp1 with rapamycin: 3 females, 1 male). I,J) Saa3 /SAA3 mRNA (I) or protein (J) levels in indicated mouse osteocyte Mlo‐Y4 cell group (n = 3–4 per group). K) Representative immunofluorescence staining images of DMP1/SAA3 co‐localization in mouse femur paraffin sections and quantification of cells with different SAA3 expression levels (n = 3, 2 females, 1 male). Left panel insets show individual staining images. Scale bars, 50 µm; data represent mean ± SD; each symbol represents one animal or one well. * p < 0.05, ** p < 0.01, *** p < 0.001 compared to Ctrl; # p < 0.05, ## p < 0.01 compared to Tsc1 Dmp1 or shTsc1 , by unpaired t test (D‐G, K) or two‐way ANOVA (H‐J).

Article Snippet: Tsc1 fl/fl and Dmp1‐Cre mice ( Dmp1 Cre , IMSR_JAX:023047) were purchased from The Jackson Laboratory (Bar Harbor, ME).

Techniques: Expressing, RNA Sequencing, Control, Gene Expression, Labeling, Quantitative RT-PCR, Concentration Assay, Enzyme-linked Immunosorbent Assay, Immunofluorescence, Staining

Journal: Advanced Science

Article Title: Osteocytes/Osteoblasts Produce SAA3 to Regulate Hepatic Metabolism of Cholesterol

doi: 10.1002/advs.202307818

Figure Lengend Snippet: Tsc1 Dmp1 and rSAA3‐treated mice exhibit reduction in hepatic CYP7A1. A) Transcriptional analysis of liver samples collected from 3‐month‐old Tsc1 Dmp1 mice or control littermates. Diagram shows the top 20 KEGG pathways enriched. The size and color of bubbles represent the number and degree of enrichment of differentially expressed mRNA enriched in the pathway. B) Expression levels of the top 10 downregulated lipid metabolism related genes identified in transcriptome analysis. C) Relative mRNA expression levels (qRT‐PCR) of Cyp7a1 in liver samples collected from 6‐month‐old Tsc1 Dmp1 mice or control littermates (n = 6, 4 females, 2 males). D) Relative mRNA expression levels (qRT‐PCR) of Cyp7a1 in liver samples collected from mice treated with 500 ng mL −1 of rSAA3 or PBS (n = 5, 5 males). E) Western blot analysis of CYP7A1 in liver samples collected from 6‐month‐old Tsc1 Dmp1 mice or control littermates (n = 6, 4 females, 2 males). F) Representative immunohistochemical staining images of CYP7A1 in paraffin sections of liver samples collected from 6‐month‐old Tsc1 Dmp1 mice or control littermates and quantification of cells with different CYP7A1 expression levels (n = 4, 3 females, 1 male). G) Representative immunohistochemical staining of CYP7A1 in paraffin sections of liver samples collected from mice treated with 500 ng mL −1 of rSAA3 or PBS (n = 4, 4 males). H) Relative mRNA expression levels (qRT‐PCR) of Alb and Clec4f in hepatocytes and Kupffer cells (n = 6, 6 males). I) Relative mRNA expression levels (qRT‐PCR) of Cyp7a1 in hepatocytes and Kupffer cells (n = 3, 3 males). J) Western blot analysis of CYP7A1 in Hepa1‐6 cells treated with serum samples collected from 6‐month‐old Tsc1 Dmp1 mice or control littermates (n = 4, 3 females, 1 male). K) Western blot analysis of CYP7A1 in Hepa1‐6 cells treated with 50 ng mL −1 of rSAA3 (n = 4). Scale bars, 200 µm; data represent mean ± SD; each symbol represents one animal. * p < 0.05, ** p < 0.01, *** p < 0.001, by one‐way ANOVA (D, K) or unpaired t test (C, E‐J).

Article Snippet: Tsc1 fl/fl and Dmp1‐Cre mice ( Dmp1 Cre , IMSR_JAX:023047) were purchased from The Jackson Laboratory (Bar Harbor, ME).

Techniques: Control, Expressing, Quantitative RT-PCR, Western Blot, Immunohistochemical staining, Staining

Journal: Advanced Science

Article Title: Osteocytes/Osteoblasts Produce SAA3 to Regulate Hepatic Metabolism of Cholesterol

doi: 10.1002/advs.202307818

Figure Lengend Snippet: SAA3 binds to TLR4 on hepatocytes and phosphorylates c‐Jun to downregulate CYP7A1. A) Representative immunofluorescence staining images of TLR4/SAA3 co‐localization in Hepa1‐6 cells treated with 50 ng mL −1 of rSAA3. Left panel insets show individual staining images. B) Representative result of western blot analysis following immunoprecipitation assay detecting the binding effect of TLR4 and SAA3 in Hepa1‐6 cells treated with 50 ng mL −1 of rSAA3. C) Representative result of western blot analysis of p‐c‐Jun in liver samples collected from 6‐month‐old Tsc1 Dmp1 mice or control littermates. D) Representative result of western blot analysis of p‐c‐Jun in Hepa1‐6 cells treated with 50 ng mL −1 of rSAA3 or PBS. E) Western blot analysis of c‐Jun phosphorylation in Hepa1‐6 cells treated with 50 µ m of Sp600125 in the presence or absence of SAA3 stimulation (n = 3). Right panel shows quantification. F) Western blot analysis of CYP7A1 in liver samples collected from mice treated with 5 mg kg −1 of Sp600125 in the presence or absence of SAA3 treatment (n = 3, 3 males). Right panel shows quantification. G) Relative mRNA expression levels (qRT‐PCR) of Cyp7a1 in Hepa1‐6 cells treated with 50 µ m of Sp600125 in the presence or absence of SAA3 stimulation (n = 4). H) Representative immunohistochemical staining images of CYP7A1 in paraffin sections of liver samples collected from mice treated with 5 mg kg −1 of Sp600125 in the presence or absence of SAA3 treatment. Right panel shows quantification of cells with different CYP7A1 expression levels (n = 3, 3 males). I) Serum cholesterol concentrations in C57BL/6 mice treated with 5 mg kg −1 of Sp600125 in the presence or absence of SAA3 treatment (n = 3, 3 males). Scale bars, 50 µm (A); Scale bars, 200 µm (H); data represent mean ± SD; each symbol represents one animal. * p < 0.05, ** p < 0.01, *** p < 0.001, ***** p < 0.0001 compared to rSAA3 − /Sp600125 − , # p < 0.05, ### p < 0.001 compared to rSAA3 + /Sp600125 − , by two‐way ANOVA (E‐I).

Article Snippet: Tsc1 fl/fl and Dmp1‐Cre mice ( Dmp1 Cre , IMSR_JAX:023047) were purchased from The Jackson Laboratory (Bar Harbor, ME).

Techniques: Immunofluorescence, Staining, Western Blot, Immunoprecipitation, Binding Assay, Control, Phospho-proteomics, Expressing, Quantitative RT-PCR, Immunohistochemical staining

Journal: Advanced Science

Article Title: Osteocytes/Osteoblasts Produce SAA3 to Regulate Hepatic Metabolism of Cholesterol

doi: 10.1002/advs.202307818

Figure Lengend Snippet: TSC1 deficiency‐enabled crosstalk from bone to liver requires SAA3. A) Schematic illustrating the reproductive strategy in generating Tsc1 Dmp1 /Saa3 − / − mice. B) Knockout validation was monitored with the SAA3 levels by western blot analysis. C) Western blot analysis of CYP7A1, p‐c‐Jun, and c‐Jun in liver samples collected from 6‐month‐old Tsc1 Dmp1 /Saa3 − / − mice or indicated control strain (n = 3, 2 females, 1 male). D) Relative mRNA expression levels (qRT‐PCR) of Cyp7a1 in liver samples collected from 6‐month‐old Tsc1 Dmp1 /Saa3 − / − mice or indicated control strain (n = 3, 2 females, 1 male). E) Representative immunohistochemical staining images of CYP7A1 in paraffin sections of liver samples collected from 6‐month‐old Tsc1 Dmp1 /Saa3 − / − mice or indicated control strain. Right panel shows quantification of cells with different CYP7A1 expression levels (n = 3, 2 females, 1 male). F) Serum cholesterol levels in 6‐month‐old Tsc1 Dmp1 /Saa3 − / − mice or indicated control strain (n = 4, 2 females, 2 males). Bottom panel insets show magnification. Scale bars, 200 µm; data represent mean ± SD; each symbol represents one animal. * p < 0.05, ** p < 0.01, *** p < 0.001 compared to Ctrl, # p < 0.05, ## p < 0.01 compared to Tsc1 Dmp1 , by two‐way ANOVA (D‐F).

Article Snippet: Tsc1 fl/fl and Dmp1‐Cre mice ( Dmp1 Cre , IMSR_JAX:023047) were purchased from The Jackson Laboratory (Bar Harbor, ME).

Techniques: Knock-Out, Biomarker Discovery, Western Blot, Control, Expressing, Quantitative RT-PCR, Immunohistochemical staining, Staining

Journal: Theranostics

Article Title: Mutant Kras and mTOR crosstalk drives hepatocellular carcinoma development via PEG3/STAT3/BEX2 signaling

doi: 10.7150/thno.76873

Figure Lengend Snippet: Mutant Kras promotes Tsc1 insufficiency-driven HCC tumorigenesis and lung metastasis. ( A ) Representative HCC tumorigenesis images of Tsc1 fl/fl ;Alb-Cre mice (TC), Kras G12D ;Alb-cre mice (KC) and Kras G12D ;Tsc1 fl/fl ; Alb-Cre mice (KTC) at 280 days old. ( B ) Quantification of the largest tumor size, tumor number, and liver: body weight ratio in TC (n = 16), KC (n = 12) and KTC mice (n = 11). ( C ) WB showing increased expression of CCNB1 and CCNB2 proteins in KTC mice compared with TC and KC mice. ( D ) Representative proliferation curves for primary liver cells isolated from the livers of 280-day-old TC, KC and KTC mice. ( E ) Macroscopic image of lung metastases was observed in KTC mice. ( F ) The lung metastasis rate was significantly increased in KTC (9/11) compared with TC (3/16) or KC mice (5/12) (Pearson Chi-square test, p = 0.005). ( G ) Representative H&E and IHC staining images for Lipase C showing distinct lung metastatic foci expressing Lipase C. Images were obtained at 4X or 40X magnification; scale bar, 500 or 50 µm. Data are represented by the mean ± SEM. *p < 0.05; **p < 0.01; ***p < 0.001. One-way ANOVA was used in B ; two-way ANOVA was used in D .

Article Snippet: The liver-specific Cre recombinase mouse line Alb-Cre (016833) and mice containing Loxp-STOP-Loxp-Kras G12D ( LSL-Kras G12D ; 008179) or a floxed Tsc1 allele ( Tsc1 fl/fl , 005680) were obtained from the Jackson Laboratory (Bar Harbor, Maine, USA).

Techniques: Mutagenesis, Expressing, Isolation, Immunohistochemistry

Journal: Theranostics

Article Title: Mutant Kras and mTOR crosstalk drives hepatocellular carcinoma development via PEG3/STAT3/BEX2 signaling

doi: 10.7150/thno.76873

Figure Lengend Snippet: ROS generation is essential for Kras mutant- and Tsc1 insufficiency-driven mTOR hyperactivation. ( A ) Representative images of ROS measurement through dye dihydroethidium (DHE)-induced fluorescence in WT, TC, KC and KTC tumors. Cells positive for DHE staining were counted among a total of 500 cells on average from 3 independent tumors derived from 3 mice per group. Images were obtained at 10X magnification; scale bar, 250 µm. ( B ) ROS levels were measured as DCF fluorescence by flow cytometry in TC, KC and KTC tumor cells. n = 3 independent experiments. ( C ) Flow cytometry showed ROS levels (DCF intensity) of KTC primary cells after pharmacological inhibition of Mek (PD98059, 20 µM) for 48 h. n = 6 and 3 independent experiments. ( D ) WB showing the phosphorylation levels of p-Akt Ser473 , p-Erk1/2 Thr202/Tyr204 , p-mTOR Ser2448 , p-S6 Ser235/236 and p-4EBP1 Thr37/46 in KTC and TC primary cell lines after pharmacological inhibition of ROS (N-acetyl-L-cysteine, NAC) at different concentrations (0, 0.2, 1, 5, 10 mM) for 48 h. ( E-F ) The relative band intensities from WB experiments in D were normalized to the level of GAPDH. ( G ) Proliferation curves for two different KTC primary cell lines (#1145 and 1375) were generated after pharmacological inhibition of NAC at different concentrations (0, 0.2, 1, 5, 10 mM) for different time internals. Data are represented by the mean ± SEM. *p < 0.05; **p < 0.01; ***p < 0.001. ns, no significant. One-way ANOVA was used in A , B, E and F ; unpaired Student's t-test was used in C ; two-way ANOVA was used in G .

Article Snippet: The liver-specific Cre recombinase mouse line Alb-Cre (016833) and mice containing Loxp-STOP-Loxp-Kras G12D ( LSL-Kras G12D ; 008179) or a floxed Tsc1 allele ( Tsc1 fl/fl , 005680) were obtained from the Jackson Laboratory (Bar Harbor, Maine, USA).

Techniques: Mutagenesis, Fluorescence, Staining, Derivative Assay, Flow Cytometry, Inhibition, Phospho-proteomics, Generated

Journal: Theranostics

Article Title: Mutant Kras and mTOR crosstalk drives hepatocellular carcinoma development via PEG3/STAT3/BEX2 signaling

doi: 10.7150/thno.76873

Figure Lengend Snippet: mTOR inhibitors significantly block hepatocarcinogenesis triggered by Kras mutant and Tsc1 insufficiency. ( A ) Photos of whole livers with orthotopic tumors (left) and isolated tumors (right) from the vehicle group (Veh; n = 8), rapamycin group (Rapa; n = 8) and sapanisertib group (Sapa; n = 8). ( B ) The tumor volume was calculated using the formula: tumor volume = 3/4 × π × a × b 2 (a is the longer diameter of the tumor and b is the shorter diameter of the tumor). Tumor volumes and tumor weights were analyzed. ( C ) Macroscopic image of lung metastases was observed in vehicle group. ( D ) The lung metastasis rate was decreased in rapamycin group (1/8) and sapanisertib (0/8), compared with vehicle group (4/8, Pearson Chi-square test, p = 0.037). ( E ) Representative H&E and IHC staining images for Lipase C showing distinct lung metastatic mets expressing Lipase C. Images were obtained at 4X or 40X magnification; scale bar, 500 or 25 µm. ( F ) Representative consecutive IHC images of p-mTOR Ser2448 , p-4EBP1 Thr37/46 , p-S6 Ser235/236 and PEG3 in the vehicle group, rapamycin group and sapanisertib group. Cells positive for p-mTOR Ser2448 , p-4EBP1 Thr37/46 , p-S6 Ser235/236 and PEG3 signals were counted among a total of 500 cells on average from 3 independent tumors derived from 3 mice per group. Images were obtained at 40X magnification; scale bar, 50 µm. ( G ) Cell proliferation in two KTC primary cell lines (#1375 and 1145) was analyzed using the CCK-8 assay after rapamycin (100 nM) or sapanisertib (1 µM) treatment for different time intervals. *p < 0.05; **p < 0.01; ***p < 0.001. One-way ANOVA was used in B, F ; Pearson Chi-square test was used in D ; two-way ANOVA was used in G .

Article Snippet: The liver-specific Cre recombinase mouse line Alb-Cre (016833) and mice containing Loxp-STOP-Loxp-Kras G12D ( LSL-Kras G12D ; 008179) or a floxed Tsc1 allele ( Tsc1 fl/fl , 005680) were obtained from the Jackson Laboratory (Bar Harbor, Maine, USA).

Techniques: Blocking Assay, Mutagenesis, Isolation, Immunohistochemistry, Expressing, Derivative Assay, CCK-8 Assay

Journal: Theranostics

Article Title: Mutant Kras and mTOR crosstalk drives hepatocellular carcinoma development via PEG3/STAT3/BEX2 signaling

doi: 10.7150/thno.76873

Figure Lengend Snippet: Model of the role of Kras G12D in regulation of Tsc1 insufficiency-induced HCC tumorigenesis and metastasis. Under Tsc1 insufficiency conditions, mTOR is weakly activated and induces a long period of liver tumorigenesis. Under Kras mutant conditions, Kras activation initiates the Mek/Erk signaling pathway to generate high levels of ROS, which activate mTOR to induce a long period of liver tumorigenesis. Upon Tsc1 insufficiency and Kras mutant, Tsc1 insufficiency further promotes Kras-Mek-Erk-ROS-mediated mTOR activation, which upregulates PEG3 expression and promotes its interaction with STAT3. Activated STAT3 causes transcriptional activation of BEX2, leading to an accelerated liver tumorigenesis and lung metastasis with a shortened latent period and increased incidence. Targeting of mTOR (e.g., rapamycin and sapanisertib) could significantly inhibit HCC tumorigenesis and lung metastasis in HCC patients with Kras mutant and Tsc1 insufficiency.

Article Snippet: The liver-specific Cre recombinase mouse line Alb-Cre (016833) and mice containing Loxp-STOP-Loxp-Kras G12D ( LSL-Kras G12D ; 008179) or a floxed Tsc1 allele ( Tsc1 fl/fl , 005680) were obtained from the Jackson Laboratory (Bar Harbor, Maine, USA).

Techniques: Mutagenesis, Activation Assay, Expressing