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: Advanced Science

Article Title: CGRP Enhances the Regeneration of Bone Defects by Regulating Bone Marrow Mesenchymal Stem Cells Through Promoting ANGPTL4 Secretion by Bone Blood Vessels

doi: 10.1002/advs.202522295

Figure Lengend Snippet: CGRP promotes endothelial angiogenesis by activating the FAK–AKT–VEGF signaling pathway. (A) The activity score for 14 signaling pathways (androgen, estrogen, EGFR, hypoxia, JAK‐STAT, MAPK, NFkβ, PI3K, p53, TGFβ, TNFα, TRAIL, VEGF, and WNT) in scRNA‐seq data. (B) PI3K pathway activity in high‐ and low‐CGRP groups. (C) VEGF pathway activity in high‐ and low‐CGRP groups. (D) Enrichment analysis of differentially expressed genes between high and low CGRP groups. (E) GSEA results of the PI3K‐AKT‐mTOR and VEGF signaling pathways. (F–I) Correlation analysis between CGRP expression and Akt1/Akt2/Akt3/Vegfa expression. (J) Experimental workflow for TMT labeling‐based phosphoproteomic analysis of HMEC‐1. (K) Quantitative analysis of phosphorylation sites and phosphoproteins identified by liquid chromatography–mass spectrometry. (L) GO enrichment analysis of common up‐regulated and down‐regulated phosphoproteins using the package clusterProfiler. The top 20 GO terms are displayed. (M) Kinase enrichment analysis was performed. The enriched kinases are depicted as a bubble plot. (N) Western blotting of p‐FAK 397, p‐FAK 576, p‐FAK 925, FAK, p‐AKT, AKT, p‐ERK, ERK, VEGFA, and tubulin in HMEC‐1 cells treated with different concentrations of CGRP. (O) Western blotting of p‐FAK 397, p‐FAK 576, p‐FAK 925, FAK, p‐AKT, AKT, p‐ERK, ERK, VEGFA, and tubulin at different time points in HMEC‐1 cells treated with the same concentration of CGRP. (P) Western blotting of p‐FAK 397, FAK, p‐ERK, ERK, p‐AKT, AKT, VEGFA, and tubulin in HMEC‐1 cells treated with Control group, CGRP group, and FAK inhibitors (PF‐562271) group.

Article Snippet: Staining was carried out using primary antibodies against CD31 (Abcam, ab182981, 1:500), EMCN (Abcam, ab106100, 1:200), VEGFA (Boster Bio, BA0407, 1:200), Runx2 (Abcam, ab192256, 1:500), CGRP (Cell Signaling Technology, #14 959, 1:800), PGP9.5 (Proteintech, 14730‐1‐AP, 1:800), LEPR (Proteintech, 20966‐1‐AP, 1:50), and ANGPTL4 (Proteintech, 18374‐1‐AP, 1:500).

Techniques: Activity Assay, Protein-Protein interactions, Expressing, Labeling, Phospho-proteomics, Liquid Chromatography, Mass Spectrometry, Western Blot, Concentration Assay, Control

Journal: Advanced Science

Article Title: CGRP Enhances the Regeneration of Bone Defects by Regulating Bone Marrow Mesenchymal Stem Cells Through Promoting ANGPTL4 Secretion by Bone Blood Vessels

doi: 10.1002/advs.202522295

Figure Lengend Snippet: Cgrp − / − mice exhibited reduced bone angiogenesis and lower bone formation, and bone mass than WT controls. (A) Agarose gel electrophoresis images of samples from wild type (WT) and Cgrp knockout (Cgrp−/−) mice. (B) Western blotting of CGRP, p‐FAK 397, p‐FAK 576, p‐FAK 925, FAK, p‐AKT, AKT, p‐ERK, ERK, VEGFA, and tubulin in HMEC‐1 cells with WT mice and CGRP−/− knockout mice. (C and D) Flow cytometry dot plot and quantification of CD31highEmcnhigh endothelial cells from WT and Cgrp−/− mice. (E and F) Representative images and quantification of CD31 (green)‐ and Emcn (red)‐stained femora from WT and Cgrp−/− mice. Scale bar, 100 µm. (G and H) Representative images and quantitation of Vefga (green) immunostaining in the femora of WT and Cgrp−/− mice. Scale bar, 100 µm. n = 3 mice in each group. (I–M) Representative micro‐CT images and quantitative micro‐CT analysis of the trabecular bone microarchitecture of WT and Cgrp−/− mice. Scale bar, 1 mm. (N and O) Representative images of hematoxylin–eosin staining in distal femora and quantification of the number of adipocytes related to the tissue area. Scale bar, 100 µm. (P and Q) Representative images and quantitation of Runx2 (green) immunostaining in femora of WT and Cgrp−/− mice. Scale bar, 100 µm. (R and S) Representative images of TRAP staining and quantification of TRAP‐positive cells on the trabecular bone surfaces of WT and Cgrp−/− mice. Scale bar, 100 µm. n = 3 mice in each group. The data are shown as the mean ± standard deviation. * p < 0.05; ** p < 0.01; *** , and p < 0.001 by Student's t ‐test and one‐way analysis of variance (J–M). BV/TV, trabecular bone volume per tissue volume; Tb. N, trabecular number; Tb. Sp, trabecular separation; Tb. Th, trabecular thickness.

Article Snippet: Staining was carried out using primary antibodies against CD31 (Abcam, ab182981, 1:500), EMCN (Abcam, ab106100, 1:200), VEGFA (Boster Bio, BA0407, 1:200), Runx2 (Abcam, ab192256, 1:500), CGRP (Cell Signaling Technology, #14 959, 1:800), PGP9.5 (Proteintech, 14730‐1‐AP, 1:800), LEPR (Proteintech, 20966‐1‐AP, 1:50), and ANGPTL4 (Proteintech, 18374‐1‐AP, 1:500).

Techniques: Agarose Gel Electrophoresis, Knock-Out, Western Blot, Flow Cytometry, Staining, Quantitation Assay, Immunostaining, Micro-CT, Standard Deviation

Journal: Scientific Reports

Article Title: Integrative bioinformatics and molecular analysis revealed the roles of mTOR/S6K Axis, CASC15 , and miR-30a-3p in laryngeal squamous cell carcinoma

doi: 10.1038/s41598-026-39618-w

Figure Lengend Snippet: Genes/miRNAs/lncRNAs network. The red arrow indicates the direction of the mTOR/S6K axis. Green nodes denote common miRNAs that target mTOR and S6K . The lncRNAs linked to miR-30a-3p . Nodes represent miRNAs and lncRNAs associated with the mTOR/S6K axis. The edges represent predicted regulatory interactions based on TCGA, GEO, and other databases.

Article Snippet: The primary antibodies included mTOR (Cat No: 2971 S, Cell Signaling, 1:1000), S6K (Cat No: M01475-1, Boster Bio, 1:1000), and GAPDH (Cat No: ab8245, Abcam, 1:2500).

Techniques:

Journal: Scientific Reports

Article Title: Integrative bioinformatics and molecular analysis revealed the roles of mTOR/S6K Axis, CASC15 , and miR-30a-3p in laryngeal squamous cell carcinoma

doi: 10.1038/s41598-026-39618-w

Figure Lengend Snippet: The gene expression levels of mTOR , S6K , lncRNA CASC15 , and miR-30a-3p . mTOR (A), S6K (B), and CASC15 (C) genes are upregulated in LSCC tissues as compared to non-tumor tissues. miR-30a-3p (D) expression decreased in LSCC tissues. All data are expressed as mean ± SEM. (* p < 0.05 and **** p < 0.0001).

Article Snippet: The primary antibodies included mTOR (Cat No: 2971 S, Cell Signaling, 1:1000), S6K (Cat No: M01475-1, Boster Bio, 1:1000), and GAPDH (Cat No: ab8245, Abcam, 1:2500).

Techniques: Gene Expression, Expressing

Journal: Scientific Reports

Article Title: Integrative bioinformatics and molecular analysis revealed the roles of mTOR/S6K Axis, CASC15 , and miR-30a-3p in laryngeal squamous cell carcinoma

doi: 10.1038/s41598-026-39618-w

Figure Lengend Snippet: The protein expression levels of mTOR and S6K in the study population. Western blot image, N: normal tissues; T: Tumor ( A ). The mTOR ( B ) and S6K ( C ) protein levels are upregulated in LSCC tissues compared with non-tumor tissues. All data are expressed as mean ± SEM. (**** P < 0.0001).

Article Snippet: The primary antibodies included mTOR (Cat No: 2971 S, Cell Signaling, 1:1000), S6K (Cat No: M01475-1, Boster Bio, 1:1000), and GAPDH (Cat No: ab8245, Abcam, 1:2500).

Techniques: Expressing, Western Blot

Journal: Frontiers in Molecular Neuroscience

Article Title: Amyloid β1-42 (Aβ1-42) Induces the CDK2-Mediated Phosphorylation of Tau through the Activation of the mTORC1 Signaling Pathway While Promoting Neuronal Cell Death

doi: 10.3389/fnmol.2017.00229

Figure Lengend Snippet: Aβ facilitates HIF1α synthesis and autophagy inhibition via mTOR activation. (A) SK-N-MC cells were exposed to Aβ (5 μM) for 0–48 h. HIF1α and β-actin expression was analyzed by western blot. n = 3. (B) Cells were pretreated with NAC (1 mM) for 30 min prior to Aβ treatment for 24 h. HIF1α and β-actin expression were analyzed by western blot. n = 3. (C,E) Cells were incubated with rapamycin (10 nM) for 30 min prior to Aβ treatment for 24 h. Phosphorylation of 4EBP1 (Thr 37/46) and 4EBP1, phosphorylation of p70S6K1 (Thr 389), HIF1α and β-actin were analyzed by western blot. n = 6. (D) Protein samples were immunoprecipitated by eukaryotic translation initiation factor 4E (eIF4E) antibody-conjugated protein A/G agarose beads. Samples were blotted with 4EBP1 and eIF4E-specific antibodies. n = 3. (F) Cells were exposed to PF4708671 (10 μM) for 30 min prior to Aβ treatment for 24 h. HIF1α and β-actin expression was detected by western blot. n = 6. (G) Cells were exposed to cycloheximide (4 μM) for 30 min prior to Aβ treatment for 24 h. HIF1α and β-actin expressions were detected by western blot. n = 6. (H) Cells were pretreated with rapamycin (10 nM) for 30 min, incubated with Aβ for 24 h and analyzed by western blotting with LC3, p62 and β-actin specific antibodies. n = 3–6. (I) LC3 puncta was visualized by confocal microscopy. Presented results are merged images. Green and red fluorescents indicate LC3 and PI respectively. Scale bars, 50 μm (magnification × 600). (J) Cells were pretreated with trehalose (10 μM) for 30 min prior to Aβ treatment for 24 h. Cytotoxicity was measured by MTT assay at an absorbance of 545 nm using a microplate reader. Data present the mean ± SE. n = 6. (K) Cell viability was measured by trypan blue exclusion assay. Data are presented as a mean ± SE. n = 6. Each blot image was presented as representative image. * p < 0.05 vs. control, # p < 0.05 vs. Aβ treatment.

Article Snippet: The antibodies of hypoxia inducible factor (HIF1α; NB100-105), p70S6K1 (NB600-1049), LC3 (NB100-2220) and p62 (NBP1-48320) were obtained from Novus Biologicals (Littleton, CO, USA) and the HRP-conjugated goat anti-rabbit IgG was purchased from Santa Cruz Biotechnology.

Techniques: Inhibition, Activation Assay, Expressing, Western Blot, Incubation, Phospho-proteomics, Immunoprecipitation, Confocal Microscopy, MTT Assay, Trypan Blue Exclusion Assay, Control

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: A viral kinase mimics S6 kinase to enhance cell proliferation

doi: 10.1073/pnas.1600587113

Figure Lengend Snippet: KSHV vPK displays limited homology to cellular S6KB1. (A) In silico model of vPK based on the partially activated state of S6KB1 is rendered in spheres. Residues highly conserved between S6KB1 and vPK are colored yellow, and the rest are teal. Most of the conserved residues between the two kinases occur in three motifs that cross the active site pocket. The first motif—KrLGRGaFG (uppercase residues are conserved and in yellow)—consists of residues K88 to G96. The second and third motifs—DvsPDNI and LTDFG—consist of residues D201 to I207 and L223 to G227. (B) A PepChip array was used to identify targets of both vPK and S6KB1. Recombinant kinases were incubated with radiolabeled ATP on a glass slide arrayed with >1,000 kinase substrate peptides. (C) Twenty-four peptides are phosphorylated by both vPK and S6KB1. vPK and S6KB1 were found to uniquely phosphorylate an additional 56 and 53 peptides, respectively. (D) Scatter plot analysis of spot intensities of peptides phosphorylated either uniquely by vPK (y axis; closed circles) or S6KB1 (x axis; closed squares), dually phosphorylated by both (closed triangles), or spots phosphorylated by neither kinase (open circles).

Article Snippet: S6KB1 construct (RC217324) was purchased from Origene; the C-terminal FLAG and Myc tags were deleted, and an N-terminal FLAG tag was inserted using standard PCR.

Techniques: In Silico, Recombinant, Incubation

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: A viral kinase mimics S6 kinase to enhance cell proliferation

doi: 10.1073/pnas.1600587113

Figure Lengend Snippet: KSHV vPK phosphorylates several S6KB1 substrates. (A) An in vitro kinase assay was performed with recombinant vPK and S6K using a synthetic S6 substrate. Data are representative of four independent experiments; error bars denote SEM. (B) Impact of S6KB1-specific and nonspecific kinase inhibitors on recombinant vPK and S6KB1 phosphorylation of S6 peptide substrate in an in vitro kinase assay. Data are representative of three independent experiments; error bars are ±SEM. (C) Ectopic expression of vPK in 293 cells increases phosphorylated levels of S6. Total S6 and tubulin are shown as loading controls. Construct expression is verified using a vPK-specific antibody. These images are representative of three independent experiments. ℗, phospho. (D) Stable HUVECs were first plated in normal media; subsequently, serum was withdrawn for 16 h, and immunoblots were performed with harvested lysates for the indicated phosphorylated and corresponding total proteins. Tubulin is shown as loading control. These images are representative of four independent experiments. (E) Metabolic labeling of de novo protein synthesis using 35S-labeled methionine and cysteine was quantified in 293 cells transiently transfected with vector, vPK, or S6KB1. Counts are normalized to total protein content. Data are representative of three independent experiments; error bars are ±SEM. (F) Metabolic labeling of de novo protein synthesis using 35S-labeled methionine and cysteine was quantified in stable HUVECs expressing vPK, S6KB1, or matched vector control. Counts are normalized to total protein content. Data are representative of three independent experiments; error bars are ±SEM, **P < 0.01, ****P < 0.0001.

Article Snippet: S6KB1 construct (RC217324) was purchased from Origene; the C-terminal FLAG and Myc tags were deleted, and an N-terminal FLAG tag was inserted using standard PCR.

Techniques: In Vitro, Kinase Assay, Recombinant, Phospho-proteomics, Expressing, Construct, Western Blot, Control, Labeling, Transfection, Plasmid Preparation

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: A viral kinase mimics S6 kinase to enhance cell proliferation

doi: 10.1073/pnas.1600587113

Figure Lengend Snippet: Immunoblot of HUVECs transfected for 24 h with indicated siRNAs and then infected with KSHV for 90 min. Infection was carried out as described by West and Damania (24). One hour postinfection (HPI), cells were harvested, and immunoblots were performed. KSHV infection induces S6 phosphorylation in an S6KB1-independent manner.

Article Snippet: S6KB1 construct (RC217324) was purchased from Origene; the C-terminal FLAG and Myc tags were deleted, and an N-terminal FLAG tag was inserted using standard PCR.

Techniques: Western Blot, Transfection, Infection, Phospho-proteomics

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: A viral kinase mimics S6 kinase to enhance cell proliferation

doi: 10.1073/pnas.1600587113

Figure Lengend Snippet: KSHV vPK enhances cellular proliferation. (A) Stable HUVECs transfected with indicated siRNAs were plated in a 96-well plate, and their basal metabolic rate was measured 72 h afterward using the Cell Titer Aqueous One Assay. Data are representative of three independent experiments; error bars are ±SEM, ****P < 0.001. NTC, nontargeting control. (B) Immunoblot analyses of indicated proteins with lysates prepared from 293 cells transiently transfected with empty vector, vPK, or S6KB1 and then treated with either LY294002 or rapamycin (10 nM), each for 1 h. Transgene expression was confirmed using vPK- and FLAG-specific antibodies. Tubulin is shown as a loading control. Images represent one of three independent experiments. (C) Stable HUVECs were treated with increasing doses of rapamycin (10, 50 nM) for 1 h, and harvested lysates were subject to immunoblotting with indicated phospho-specific and total antibodies. Although phospho-S6 remains elevated after rapamycin treatment in HUVEC-vPK, it is diminished in vector- and S6KB1-expressing HUVECs. Images are representative of three independent experiments. (D) Stable 293 cells were transfected with indicated siRNAs for 48 h, and immunoblots were performed after 12-h serum withdrawal. mT, pooled Raptor- and mTOR-specific siRNA; Scr, scrambled. (E) Immunoblot analysis of both latent (No Dox) and reactivated (Dox) iSLK.219 cells transfected with either vPK-directed or a nonspecific (NS) siRNA. Representative of four independent experiments. (F) Infectivity of 293 cells infected with KSHV derived from the experiment described in E; **** P < 0.0001.

Article Snippet: S6KB1 construct (RC217324) was purchased from Origene; the C-terminal FLAG and Myc tags were deleted, and an N-terminal FLAG tag was inserted using standard PCR.

Techniques: Transfection, Control, Western Blot, Plasmid Preparation, Expressing, Infection, Derivative Assay

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: A viral kinase mimics S6 kinase to enhance cell proliferation

doi: 10.1073/pnas.1600587113

Figure Lengend Snippet: KSHV vPK augments tubule formation. (A) Rat1 fibroblasts stably expressing the indicated transgenes were plated in soft agar for 3 wk. Colonies were stained with crystal violet and quantified to measure anchorage independence. Data are representative of four independent experiments; error bars are ±SEM; ****P < 0.0001. (B) When placed in growth factor-reduced Matrigel, vPK-stable HUVECs form tubular networks within 4 h. Also shown are EV- and S6KB1-expressing cells. (C) Significantly increased branching was observed at 4 h (Left) and 8 h (Right) in vPK-expressing HUVECs, compared with EV- and S6KB1-HUVEC. Tubule formation data are representative of three independent experiments. (D) Proposed model for vPK’s mechanism of action.

Article Snippet: S6KB1 construct (RC217324) was purchased from Origene; the C-terminal FLAG and Myc tags were deleted, and an N-terminal FLAG tag was inserted using standard PCR.

Techniques: Stable Transfection, Expressing, Staining