Journal: Cancer cell international

Article Title: The association between Salt-inducible kinase 2 (SIK2) and gamma isoform of the regulatory subunit B55 of PP2A (B55gamma) contributes to the survival of glioma cells under glucose depletion through inhibiting the phosphorylation of S6K.

doi: 10.1186/s12935-015-0164-6

Figure Lengend Snippet: Figure 3 Overexpression of B55gamma in Glioma cells inhibits the phosphorylation of S6K. A. Overexpression of B55gamma in U251 decreases phosphorylation of S6K, but phosphorylation of 4EBP1 and mTOR and AKT have no significant change. B. U251 cells were transfected with control vector or B55gamma alone or B55gamma + S6K WT or B55gamma + S6K KA for 48 hrs. Cell viability assays were performed under the treatments of Rapamycin at 15 nM and 50 nM, DMSO treatment was control. C. Transfection of S6K WT and S6K KA into U251V and U251B55gamma cells followed by the measurements of cell viabilities under the glucose withdrawal for 24 hrs. Columns, mean of three independent experiments; bars, SE. **, P < 0.01; ***, P < 0.001.

Article Snippet: Vector containing GFPtagged ORF clone of Homo sapiens protein B55gamma, WT S6K or KA S6K were purchased from Origene.

Techniques: Over Expression, Phospho-proteomics, Transfection, Control, Plasmid Preparation

Journal: Cancer cell international

Article Title: The association between Salt-inducible kinase 2 (SIK2) and gamma isoform of the regulatory subunit B55 of PP2A (B55gamma) contributes to the survival of glioma cells under glucose depletion through inhibiting the phosphorylation of S6K.

doi: 10.1186/s12935-015-0164-6

Figure Lengend Snippet: Figure 5 Knockdown of SIK in B55gamma overexpression cells recovers the phosphorylation of S6K. A. U251V, U251B55gamma, U138V and U138B55gamma cells were transfected with scramble siRNA (Ctr) or SIK2 siRNA. 48 hrs after siRNA transfection, cell lysates were prepared and Western blotting was performed to detect the phosphorylation of S6K statues. The β-actin protein was used as a loading control. B. U251B55gamma (left) and U138B55gamma (right) cells were transfected with scramble siRNA (Ctr) or SIK2 siRNA. 48 hrs after siRNA transfection, cell were plated into 6-well plate for overnight, then the regular medium was replaced by glucose free medium for 36 h-starvation followed by the cell viability assay. Columns, mean of three independent experiments; bars, SE. *, P < 0.05.

Article Snippet: Vector containing GFPtagged ORF clone of Homo sapiens protein B55gamma, WT S6K or KA S6K were purchased from Origene.

Techniques: Knockdown, Over Expression, Phospho-proteomics, Transfection, Western Blot, Control, Viability Assay

Journal: Frontiers in Nutrition

Article Title: Maternal docosahexaenoic acid supplementation during lactation improves exercise performance, enhances intestinal glucose absorption and modulates gut microbiota in weaning offspring mice

doi: 10.3389/fnut.2024.1423576

Figure Lengend Snippet: Effect of maternal DHA supplementation during lactation on the mTOR signaling pathway in weaning mice. (A) Representative Western blots of p-mTOR (S2448), total mTOR, p-p70S6 kinase (T389), total p70S6 kinase, p-S6 ribosomal protein (S240/244) and total S6 ribosomal protein, and (B, C, D) the relative band intensities of phosphorylation levels normalized to their respective total proteins in jejunal tissue of mice from different groups. Data were expressed as the mean ± SEM; n = 4 per group. Significance was set at p < 0.05 ( * p < 0.05, ** p < 0.01). L-DHA: 150 mg/(kg body weight day) DHA; H-DHA: 450 mg/(kg body weight day) DHA. DHA, docosahexaenoic acid; mTOR, mammalian target of rapamycin.

Article Snippet: After transfer, the PVDF membrane was blocked for 1 h in 5% fat-free milk and then was, respectively, incubated overnight at 4°C with dilutions of primary antibodies: GLUT2 (A12307, 1:2000, Abclonal, China), SGLT1 (bs-1128R, 1:2000, Bioss, China), S6 ribosomal protein (abs131865, 1:2000, Absin, China), p-Ser240/244-S6 ribosomal protein (ab215214, 1:2000, Abcam, UK), p70S6 kinase (14485-1-AP, 1,1,000, Proteintech, China), p-Thr389-p70S6 kinase (9,234, 1,1,000, Cell Signaling Technology, United States), mammalian target of rapamycin (mTOR, T55306, 1:2000, Abmart, China), p-S2448-mTOR (5,536, 1,1,000, Cell Signaling Technology), β-actin (T40104, 1:4000, Abmart).

Techniques: Western Blot, Phospho-proteomics

Journal: bioRxiv

Article Title: CASTOR1 : A Novel Tumor Suppressor Linking mTORC1 and KRAS Pathways in Tumorigenesis and Resistance to KRAS-Targeted Therapies in Non-Small Cell Lung Cancer

doi: 10.1101/2025.04.23.650349

Figure Lengend Snippet: (A) Representative H&E images of lungs collected from KRAS G12D ;C1 WT or KRAS G12D ;C1 KO mice 5 months after intranasal inoculation with Adeno-Cre (left panel). Tumor incidence, size and burden (tumor area/total lung area ×100) were quantified (left, middle and right panels, respectively). Data presented as mean +/- standard error of the mean (SEM). Statistical analysis was performed by nested-Anova and Student’s t-test. (B) Representative IHC staining of CASTOR1 in lung tumors from KRAS G12D ;C1 WT or KRAS G12D ;C1 KO mice. (C) Representative IHC staining images of Ki67 in lung tumors from KRAS G12D ;C1 WT or KRAS G12D ;C1 KO mice (left panel) and quantification of Ki67-positive cells (right panel). Statistical analysis was performed by nested-Anova. (D and E) Representative IHC staining images of p-4EBP1 (D) and p-S6 (E) of lung tumors from KRAS G12D ;C1 WT or KRAS G12D ;C1 KO mice (left panels) and quantification of positive cells (right panels). Statistical analysis was performed by nested-Anova. (F) Immunoblotting analysis of mTORC1 downstream targets S6K and 4EBP1, KRAS downstream target ERK and AKT, and their phosphorylated forms in normal lung tissues from KRAS WT ;C1 WT and KRAS WT ;C1 KO mice, and tumors from KRAS G12D ;C1 WT and KRAS G12D ;C1 KO mice. Three samples per group were analyzed. (G). Representative IHC staining images of p-ERK in lung tumors from KRAS G12D ;C1 WT or KRAS G12D ;C1 KO mice (left panel) and quantification of positive cells (right panel). Statistical analysis was performed by Student’s t-test.

Article Snippet: Primary antibodies include CASTOR1 (Abclonal, cat. no. A2071C, 1:50 dilution), p-ERK (Cell Signaling Technology, cat. no.4370, 1:100 dilution), p-4EBP1 (Cell Signaling Technology, cat. no.2855, 1:100), p-S6K (Abclonal, cat. no.AP0502, 1:100 dilution), p-S6 (Cell Signaling Technology, cat. no.2211, 1:100), and pCASTOR1 (1:100).

Techniques: Immunohistochemistry, Western Blot

Journal: bioRxiv

Article Title: CASTOR1 : A Novel Tumor Suppressor Linking mTORC1 and KRAS Pathways in Tumorigenesis and Resistance to KRAS-Targeted Therapies in Non-Small Cell Lung Cancer

doi: 10.1101/2025.04.23.650349

Figure Lengend Snippet: (A-C) CASTOR1 ablation enhances resistance to KRAS G12D -specific inhibitor MRTX1133 in tumors. Mice were intranasally inoculated with Adeno-Cre for 4 weeks, then treated with MRTX1133 of vehicle for 4 weeks. Representative H&E images of lungs collected from KRAS G12D ;C1 WT or KRAS G12D ;C1 KO mice (A). Tumor size (B) and burden (tumor area/total lung area ×100) (C) were quantified. Data presented as mean +/- standard error of the mean (SEM). Statistical analysis was performed by nested-Anova and Student’s t-test. (D) KRAS G12D ;C1 WT tumor organoids had heterogeneous response to MRTX1133. Representative pictures of nine KRAS G12D ;C1 WT organoids after one week of treatment with MRTX1133 or vehicle. (E) Immunoblotting analysis of KRAS G12D protein, mTORC1 downstream targets S6K and 4EBP1, and KRAS downstream target ERK in KRAS G12D ;C1 WT tumor organoids. (F) KRAS G12D ;C1 KO tumor organoids were resistant to MRTX1133. Representative pictures of four KRAS G12D ;C1 KO organoids after one week of treatment with MRTX1133 or vehicle. (G) Comparison of sensitivity to MRTX1133 between organoids derived from KRAS G12D ;C1 WT and KRAS G12D ;C1 KO tumors. (H-I) mTORC1 inhibitor Rapamycin and PI3K inhibitor BGT226 sensitized KRAS G12D ;C1 WT and KRAS G12D ;C1 KO organoids to MRTX1133. Representative pictures of organoids treated with mono- or combined therapy of MRTX1133 and Rapamycin (H) or BGT226 (I).

Article Snippet: Primary antibodies include CASTOR1 (Abclonal, cat. no. A2071C, 1:50 dilution), p-ERK (Cell Signaling Technology, cat. no.4370, 1:100 dilution), p-4EBP1 (Cell Signaling Technology, cat. no.2855, 1:100), p-S6K (Abclonal, cat. no.AP0502, 1:100 dilution), p-S6 (Cell Signaling Technology, cat. no.2211, 1:100), and pCASTOR1 (1:100).

Techniques: Western Blot, Comparison, Derivative Assay

Journal: Neuroscience letters

Article Title: The mTOR cell signaling pathway is crucial to the long-term protective effects of ischemic postconditioning against stroke

doi: 10.1016/j.neulet.2018.03.062

Figure Lengend Snippet: Antibodies, concentrations and manufacturers used

Article Snippet: The S6K1 gene was cloned from S6K1 (pcDNA3) plasmids (S6K1: 26610, Addgene, Cambridge, MA) and inserted into the lentiviral backbone plasmid, pHR’trIPostCMV-IRES-eGFP.

Techniques: