kinase dead s6k  (Addgene inc)

Journal: Nature Communications

Article Title: mTORC1 upregulates B7-H3/CD276 to inhibit antitumor T cells and drive tumor immune evasion

doi: 10.1038/s41467-023-36881-7

Figure Lengend Snippet: Kaplan-Meier plot of overall patient survival stratified by high ( n = 2076) and low ( n = 2185) CD276 expression ( a ), high ( n = 2001) and low ( n = 2268) mTORC1 score ( b ), and high CD276 expression plus high mTORC1 score ( n = 1296) and low CD276 expression plus low mTORC1 score ( n = 1160) ( c ). Log-rank test. Forest plot of hazard ratios by tumor type and overall (with 95% confidence intervals) stratified by high and low CD276 expression ( d ), high and low mTORC1 score ( e ), and high and low CD276 expression plus high and low mTORC1 score ( f ). Hazard ratio greater than 1 (red) indicates an association with worse outcome. COX proportional hazards regression with Efron’s approximation. g , h Representative images of strong and weak immunohistochemical staining of phospho-S6 (Ser240/244) (pS6) and B7-H3 in tumor tissue microarrays, which include 12 cancer types: meningothelial meningioma, breast infiltrating ductal adenocarcinoma, colon adenocarcinoma, stomach adenocarcinoma, prostate adenocarcinoma, uterus adenocarcinoma, ovarian adenocarcinoma, esophageal squamous cell carcinomas, kidney clear cell carcinomas, hepatocellular carcinomas, lung adenocarcinoma/squamous cell carcinomas, and bladder transitional/papillary cell carcinomas. Scale bar = 50 μm ( g ). Correlation analysis between the phospho-S6 immunoreactivity score and B7-H3 expression score in the tumors in g (Pearson correlation coefficient test) ( h ). i , j Representative images of strong and weak immunohistochemical staining of phospho-S6K (Thr389) (pS6K) and B7-H3 in tumor tissue microarrays, which include 12 cancer types indicated in ( g ). Scale bar = 50 μm ( i ). Correlation analysis between the phospho-S6K immunoreactivity score and B7-H3 expression score in the tumors in i (Pearson correlation coefficient test) ( j ). mTORC1 score ( k ) and CD276 expression (l ) among the six immune subtypes. Bars indicate median line, boxes denote interquartile range, whiskers denote 1–99 percentile and outliers are shown. Nonparametric Kruskal-Wallis test with Dunn’s multiple comparisons test. n = 5429, **** p < 0.0001. m Heatmap indicates mean scores of immune-cell of all patients in high CD276 and high mTORC1 tumors (left) and low CD276 - low mTORC1 tumors (right). Source data is provided in the Source data file.

Article Snippet: Plasmids encoding wild-type YY2 (Myc-DDK-YY2, Cat#MR218884) was purchased from Origene, USA, wild-type S6K (pRK7-HA-S6K1-WT, Cat# 8984), kinase-dead S6K (pRK7-HA-S6K1-KR, Cat# 8985), wild-type Smurf1 (pCMV5B-Flag-Smurf1 wt, Cat# 11752), wild-type mTOR (pcDNA3-FLAG-MTOR, Cat#26603), L1460P mutant mTOR (pcDNA3-FLAG-MTOR-L1460P, Cat#69006), C1483F mutant mTOR (pcDNA3-FLAG-MTOR-C1483F, Cat#69008), S2215Y mutant mTOR (pcDNA3-FLAG-MTOR-S2215Y, Cat#69013), and R2505P mutant mTOR (pcDNA3-FLAG-MTOR-R2505P, Cat#69015) were purchased from Addgene, USA.

Techniques: Expressing, Immunohistochemical staining, Staining

Journal: Nature Communications

Article Title: mTORC1 upregulates B7-H3/CD276 to inhibit antitumor T cells and drive tumor immune evasion

doi: 10.1038/s41467-023-36881-7

Figure Lengend Snippet: B7-H3 protein expression in Tsc2−/− 105K cells with stable reconstitution of TSC2 or empty vector (EV) ( a ), TSC2−/− 621-101 angiomyolipoma-derived cells with reconstitution of TSC2 or empty vector (EV) ( b ), and Tsc2 -WT and Tsc2 KO MEFs ( c ). For all figure legends, n = 3 indicates representative of 3 biologic samples ( n = 3). B7-H3 mRNA expression in Tsc2−/− 105K cells with stable reconstitution of TSC2 or empty vector (EV) ( d ), TSC2−/− 621-101 angiomyolipoma tumor cells with reconstitution of TSC2 or empty vector (EV) ( e ), Tsc2 -WT and Tsc2 KO MEFs ( f ). Means ± SD, two-tailed unpaired Student’s t -test ( d , f ) or one-way ANOVA with Dunnett’s multiple comparisons test ( e ), * p < 0.05, ** p < 0.01. n = 3. B7-H3 protein expression in Tsc2−/− 105K cells ( g ), TSC2−/− 621-101 angiomyolipoma tumor cells ( h ), and Tsc2 KO MEFs ( i ) treated with 20 nM rapamycin (Rapa), 500 nM Torin 1 or vehicle for 24 hr ( n = 3). B7-H3 mRNA expression in Tsc2−/− 105K cells ( j ), TSC2−/− 621-101 angiomyolipoma tumor cells ( k ), and Tsc2 KO MEFs ( l ) treated with 20 nM rapamycin (Rapa), 500 nM Torin 1, or vehicle for 24 hr. n = 3, means ± SD, one-way ANOVA with Dunnett’s multiple comparisons test, * p < 0.05, ** p < 0.01, *** p < 0.001. m Immunoblotting analysis of Tsc2 -WT and Tsc2 KO MEFs transfected with non-targeting control siRNA (Ctrl) or SMARTpool siRNAs targeting Raptor, Rictor, mTOR, S6K, or 4E-BP1 for 48 h hr ( n = 3). n qRT-PCR analysis of Tsc2 -WT and Tsc2 KO MEFs transfected with non-targeting control siRNA (Ctrl) or SMARTpool siRNAs targeting either Raptor, Rictor, mTOR, S6K, or 4E-BP1 for 48 hr. n = 3, means ± SD, two-way ANOVA with Dunnett’s multiple comparisons test, *** p < 0.001, **** p < 0.0001. Source data and exact p values are provided in the Source data file.

Article Snippet: Plasmids encoding wild-type YY2 (Myc-DDK-YY2, Cat#MR218884) was purchased from Origene, USA, wild-type S6K (pRK7-HA-S6K1-WT, Cat# 8984), kinase-dead S6K (pRK7-HA-S6K1-KR, Cat# 8985), wild-type Smurf1 (pCMV5B-Flag-Smurf1 wt, Cat# 11752), wild-type mTOR (pcDNA3-FLAG-MTOR, Cat#26603), L1460P mutant mTOR (pcDNA3-FLAG-MTOR-L1460P, Cat#69006), C1483F mutant mTOR (pcDNA3-FLAG-MTOR-C1483F, Cat#69008), S2215Y mutant mTOR (pcDNA3-FLAG-MTOR-S2215Y, Cat#69013), and R2505P mutant mTOR (pcDNA3-FLAG-MTOR-R2505P, Cat#69015) were purchased from Addgene, USA.

Techniques: Expressing, Plasmid Preparation, Derivative Assay, Two Tailed Test, Western Blot, Transfection, Control, Quantitative RT-PCR

Journal: Nature Communications

Article Title: mTORC1 upregulates B7-H3/CD276 to inhibit antitumor T cells and drive tumor immune evasion

doi: 10.1038/s41467-023-36881-7

Figure Lengend Snippet: Enhanced Cd276 promoter activity in Tsc2−/− 105K cells expressing empty vector (EV) compared to reconstitution of TSC2 ( a ) and in Tsc2 KO MEFs compared to Tsc2 -WT MEFs ( b ). Relative luciferase activity was determined by a dual-luciferase assay system. psiCHECK2-Cd276 encodes the Cd276 promoter. Empty psiCHECK2 vector was used as the negative control. n = 6, means ± SD, two-way ANOVA with Holm-Sidak’s multiple comparisons test, * p < 0.05, *** p < 0.001. Raptor, mTOR or S6K knockdown suppresses Cd276 promoter activity in Tsc2−/− 105K cells ( c ) and Tsc2 KO MEFs ( d ). n = 6, means ± SD, two-way ANOVA with Holm-Sidak’s multiple comparisons test, * p < 0.05, ** p < 0.01, **** p < 0.0001. siRNA knockdown of YY2 reduces B7-H3 protein expression in Tsc2−/− 105K cells ( e ) and Tsc2 KO MEFs ( f ) ( n = 3). Knockdown of YY2 reduces Cd276 mRNA expression in Tsc2−/− 105K cells ( g ) and Tsc2 KO MEFs ( h ). n = 3, means ± SD, two-tailed unpaired Student’s t -test, * p < 0.05, *** p < 0.001. i Promoter region of Cd276 displaying the location of the YY2 binding site. YY2 ChIP-qPCR analysis showing increased YY2 occupancy on the Cd276 promoter in Tsc2−/− 105K cells ( j ) and Tsc2 KO MEFs ( k ). n = 4, means ± SEM, two-way ANOVA with Holm-Sidak’s multiple comparisons test, * p < 0.05, ** p < 0.01, **** p < 0.0001. Cd276 promoter activity is suppressed by YY2 knockdown in Tsc2−/− 105K cells ( l ) and Tsc2 KO MEFs ( m ). n = 6, means ± SD, two-way ANOVA with Holm-Sidak’s multiple comparisons test, * p < 0.05, *** p < 0.001. Source data and exact p values are provided in the Source data file.

Article Snippet: Plasmids encoding wild-type YY2 (Myc-DDK-YY2, Cat#MR218884) was purchased from Origene, USA, wild-type S6K (pRK7-HA-S6K1-WT, Cat# 8984), kinase-dead S6K (pRK7-HA-S6K1-KR, Cat# 8985), wild-type Smurf1 (pCMV5B-Flag-Smurf1 wt, Cat# 11752), wild-type mTOR (pcDNA3-FLAG-MTOR, Cat#26603), L1460P mutant mTOR (pcDNA3-FLAG-MTOR-L1460P, Cat#69006), C1483F mutant mTOR (pcDNA3-FLAG-MTOR-C1483F, Cat#69008), S2215Y mutant mTOR (pcDNA3-FLAG-MTOR-S2215Y, Cat#69013), and R2505P mutant mTOR (pcDNA3-FLAG-MTOR-R2505P, Cat#69015) were purchased from Addgene, USA.

Techniques: Activity Assay, Expressing, Plasmid Preparation, Luciferase, Negative Control, Knockdown, Two Tailed Test, Binding Assay, ChIP-qPCR

Journal: Nature Communications

Article Title: mTORC1 upregulates B7-H3/CD276 to inhibit antitumor T cells and drive tumor immune evasion

doi: 10.1038/s41467-023-36881-7

Figure Lengend Snippet: YY2 protein expression in Tsc2−/− 105K cells ( a ) and Tsc2 KO MEFs ( b ) treated with 20 nM rapamycin (Rapa) or vehicle for 24 hr ( n = 3). YY2 protein expression in Tsc2−/− 105K cells ( c ) and Tsc2 KO MEFs ( d ) treated with 10 μM PF-4708671 (S6K1 inhibitor) or vehicle for 24 hr ( n = 3). e Immunoblot analysis of HeLa cells transfected with eGFP-YY2, wild-type S6K (HA-WT-S6K), or kinase-dead S6K (HA-KD-S6K) for 48 hr ( n = 3). f Immunoblot analysis of in vitro kinase assays of recombinant active S6K in the presence or absence of myc-YY2 as a substrate. eIF4B was used as a positive control ( n = 3). g Immunoblot analysis of immunoprecipitated (IP) GFP-YY2 and the whole-cell-lysate (WCL) from HeLa cells expressing GFP-YY2 ( n = 3). h Immunoblot analysis of immunoprecipitated GFP-YY2 and the WCL from HeLa cells expressing GFP-YY2 ( n = 3). i The evolutionarily conserved putative S6K phosphorylation site in YY2. j Immunoblot analysis of immunoprecipitated GFP-YY2 and the WCL from HeLa cells stably expressing wild-type (WT) GFP-YY2 or mutant (T336A) GFP-YY2, treated with 40 μM PF-4708671 for 1 hr ( n = 3). k Immunoblot analysis of immunoprecipitated GFP-YY2 and the WCL from HeLa cells expressing WT GFP-YY2 or T336A GFP-YY2 ( n = 3). l , m Network view of predicted E3 ubiquitin ligase-YY2 interactions by UbiBrowser 2.0. Confidence level of ( m ). n Decreased EGFP-YY2 expression in HeLa cells transfected with Smurf1-FLAG ( n = 3). o Decreased Smurf1 ubiquitination in HeLa cells transfected with S6K. Immunoblot analysis of GFP-immunoprecipitates from EGFP-YY2 HeLa cells transfected with Smurf1-FLAG or HA-WT-S6K for 24 hr ( n = 3). p Immunoblot analysis of GFP-and IgG control immunoprecipitates from EGFP-YY2 HeLa cells transfected with Smurf1-FLAG ( n = 3). q Immunoblot analysis of B7-H3 expression in Tsc2−/− 105K cells, transfected with control or myc-YY2 for 48 hr and treated with 20 nM rapamycin or vehicle for the last 24 hr ( n = 3). Source data is provided in the Source data file.

Article Snippet: Plasmids encoding wild-type YY2 (Myc-DDK-YY2, Cat#MR218884) was purchased from Origene, USA, wild-type S6K (pRK7-HA-S6K1-WT, Cat# 8984), kinase-dead S6K (pRK7-HA-S6K1-KR, Cat# 8985), wild-type Smurf1 (pCMV5B-Flag-Smurf1 wt, Cat# 11752), wild-type mTOR (pcDNA3-FLAG-MTOR, Cat#26603), L1460P mutant mTOR (pcDNA3-FLAG-MTOR-L1460P, Cat#69006), C1483F mutant mTOR (pcDNA3-FLAG-MTOR-C1483F, Cat#69008), S2215Y mutant mTOR (pcDNA3-FLAG-MTOR-S2215Y, Cat#69013), and R2505P mutant mTOR (pcDNA3-FLAG-MTOR-R2505P, Cat#69015) were purchased from Addgene, USA.

Techniques: Expressing, Western Blot, Transfection, In Vitro, Recombinant, Positive Control, Immunoprecipitation, Phospho-proteomics, Stable Transfection, Mutagenesis, Ubiquitin Proteomics, Control

Journal: Cellular reprogramming

Article Title: mTOR-mediated activation of p70 S6K induces differentiation of pluripotent human embryonic stem cells.

doi: 10.1089/cell.2010.0011

Figure Lengend Snippet: FIG. 2. H7 hESCs show reduced levels of p70 S6K signaling compared to their differentiated progeny. (A) Representative Western blots from four separate experiments indicating decreased p70 S6K activation in hESCs compared to differentiated, fibroblast- like cells. (B) Representative images showing reduced S6 phosphorylation in H7 hESCs compared to H7 Diffs. S6 phosphorylation appears to be increased along the outer edges of hESC colonies. Scale bar, 40 mm.

Article Snippet: The p70 S6K constructs were generously donated to Addgene by Dr. John Blenis, Addgene catalog number 8989 (p70 S6K CON) and 8984 (p70 S6K WT).

Techniques: Western Blot, Activation Assay, Phospho-proteomics

Journal: Cellular reprogramming

Article Title: mTOR-mediated activation of p70 S6K induces differentiation of pluripotent human embryonic stem cells.

doi: 10.1089/cell.2010.0011

Figure Lengend Snippet: FIG. 3. Suppression of mTORC1-mediated translation does not induce cell death or disrupt pluripotency in H7 hESCs. (A) Rapamycin treatment causes cell death in differentiated, but not undifferentiated cells. Representative images of untreated and treated H7 hESCs and H7 Diffs are shown. Scale bar, 100 mm. (B) Neither treatment with rapamycin nor siRNA against p70 S6K impairs colony formation or affects expression of pluripotency markers in H7 hESCs. Representative images for hESCs treated for 3 days with vehicle control or 20 nM rapamycin are shown. Also shown are representative images from H7 hESCs nucleofected with either control, nonspecific siRNA, or siRNA directed against p70 S6K. Typically, hESC colonies are smaller post nucleofection due to the requirement of single-cell suspensions. Staining for Oct4 and Nanog pluripotency markers are shown. Scale bar, 100 mM. Western blot insert confirms p70 S6K knockdown in hESCs with Actin serving as a loading control.

Article Snippet: The p70 S6K constructs were generously donated to Addgene by Dr. John Blenis, Addgene catalog number 8989 (p70 S6K CON) and 8984 (p70 S6K WT).

Techniques: Expressing, Control, Staining, Western Blot, Knockdown

Journal: Cellular reprogramming

Article Title: mTOR-mediated activation of p70 S6K induces differentiation of pluripotent human embryonic stem cells.

doi: 10.1089/cell.2010.0011

Figure Lengend Snippet: FIG. 5. Expression of constitutively active p70 S6K alters cell morphology and causes a loss of Oct4 expression in H7 hESCs. Constitutively active p70 S6K induces differentia- tion of H7 hESCs. (A) Representative fluorescent imaging of H7 hESC nucleofected with GFP, GFP þ p70 S6K WT, or GFP þ p70 S6K CON and stained for Oct4. Arrows indicate nucleofected cells. Scale bar, 50 mm. (B) Graphical analysis of the percentage of GFP þ cells expressing Oct4. For each condition, approximately 300 cells were analyzed. Statistical significance, p < 0.01.

Article Snippet: The p70 S6K constructs were generously donated to Addgene by Dr. John Blenis, Addgene catalog number 8989 (p70 S6K CON) and 8984 (p70 S6K WT).

Techniques: Expressing, Imaging, Staining

Journal: Cellular reprogramming

Article Title: mTOR-mediated activation of p70 S6K induces differentiation of pluripotent human embryonic stem cells.

doi: 10.1089/cell.2010.0011

Figure Lengend Snippet: FIG. 6. Knockdown of Rictor and TSC2 expression elevates p70 S6K activation and alters hESC morphology. Activation of p70 S6K by Rictor and TSC2 siRNA-mediated knockdown leads to changes in cell mor- phology to a more differentiated pheno- type. (A) Representative Western blots from multiple experiments showing ex- pression of proteins associated with mTORC1 (mammalian target of rapamycin complex 1) and mTORC2 signaling in H7 hESCs and H7 Diffs. (B) Western blot of immunoprecipitations of mTOR or TSC1 probed for Raptor, Rictor, or TSC2, re- spectively, in H7 hESCs and H7 Diffs. (C) Representative phase contrast images of H7 hESCs nucleofected with either control siRNA or Rictor=TSC2 siRNA mixture. Scale bar, 250 mm. (D) Western blot analysis confirming knockdown of TSC2 and Rictor as well as expression of p70 S6K, activated p70 S6K (p-T389), Oct4, and Actin.

Article Snippet: The p70 S6K constructs were generously donated to Addgene by Dr. John Blenis, Addgene catalog number 8989 (p70 S6K CON) and 8984 (p70 S6K WT).

Techniques: Knockdown, Expressing, Activation Assay, Western Blot, Control

Journal: Cellular reprogramming

Article Title: mTOR-mediated activation of p70 S6K induces differentiation of pluripotent human embryonic stem cells.

doi: 10.1089/cell.2010.0011

Figure Lengend Snippet: FIG. 7. Diagram depicting mTORC signal- ing in hESCs and differentiated cells. In pluripotent hESCs (left panel), mTOR protein is mostly associated in mTORC2 (mTOR- Rictor), whereas mTORC1 (mTOR-Raptor) activity is suppressed by the heterodimer TSC1=TSC2. In differentiated cells (right panel), mTOR is highly associated with Raptor over Rictor leading to elevated p70 S6K activity. Increased protein translation, in turn, induces differentiation.

Article Snippet: The p70 S6K constructs were generously donated to Addgene by Dr. John Blenis, Addgene catalog number 8989 (p70 S6K CON) and 8984 (p70 S6K WT).

Techniques: Activity Assay

Journal: Cellular reprogramming

Article Title: mTOR-mediated activation of p70 S6K induces differentiation of pluripotent human embryonic stem cells.

doi: 10.1089/cell.2010.0011

Figure Lengend Snippet: FIG. 2. H7 hESCs show reduced levels of p70 S6K signaling compared to their differentiated progeny. (A) Representative Western blots from four separate experiments indicating decreased p70 S6K activation in hESCs compared to differentiated, fibroblast- like cells. (B) Representative images showing reduced S6 phosphorylation in H7 hESCs compared to H7 Diffs. S6 phosphorylation appears to be increased along the outer edges of hESC colonies. Scale bar, 40 mm.

Article Snippet: The p70 S6K constructs were generously donated to Addgene by Dr. John Blenis, Addgene catalog number 8989 (p70 S6K CON) and 8984 (p70 S6K WT).

Techniques: Western Blot, Activation Assay, Phospho-proteomics

Journal: Cellular reprogramming

Article Title: mTOR-mediated activation of p70 S6K induces differentiation of pluripotent human embryonic stem cells.

doi: 10.1089/cell.2010.0011

Figure Lengend Snippet: FIG. 3. Suppression of mTORC1-mediated translation does not induce cell death or disrupt pluripotency in H7 hESCs. (A) Rapamycin treatment causes cell death in differentiated, but not undifferentiated cells. Representative images of untreated and treated H7 hESCs and H7 Diffs are shown. Scale bar, 100 mm. (B) Neither treatment with rapamycin nor siRNA against p70 S6K impairs colony formation or affects expression of pluripotency markers in H7 hESCs. Representative images for hESCs treated for 3 days with vehicle control or 20 nM rapamycin are shown. Also shown are representative images from H7 hESCs nucleofected with either control, nonspecific siRNA, or siRNA directed against p70 S6K. Typically, hESC colonies are smaller post nucleofection due to the requirement of single-cell suspensions. Staining for Oct4 and Nanog pluripotency markers are shown. Scale bar, 100 mM. Western blot insert confirms p70 S6K knockdown in hESCs with Actin serving as a loading control.

Article Snippet: The p70 S6K constructs were generously donated to Addgene by Dr. John Blenis, Addgene catalog number 8989 (p70 S6K CON) and 8984 (p70 S6K WT).

Techniques: Expressing, Control, Staining, Western Blot, Knockdown

Journal: Cellular reprogramming

Article Title: mTOR-mediated activation of p70 S6K induces differentiation of pluripotent human embryonic stem cells.

doi: 10.1089/cell.2010.0011

Figure Lengend Snippet: FIG. 5. Expression of constitutively active p70 S6K alters cell morphology and causes a loss of Oct4 expression in H7 hESCs. Constitutively active p70 S6K induces differentia- tion of H7 hESCs. (A) Representative fluorescent imaging of H7 hESC nucleofected with GFP, GFP þ p70 S6K WT, or GFP þ p70 S6K CON and stained for Oct4. Arrows indicate nucleofected cells. Scale bar, 50 mm. (B) Graphical analysis of the percentage of GFP þ cells expressing Oct4. For each condition, approximately 300 cells were analyzed. Statistical significance, p < 0.01.

Article Snippet: The p70 S6K constructs were generously donated to Addgene by Dr. John Blenis, Addgene catalog number 8989 (p70 S6K CON) and 8984 (p70 S6K WT).

Techniques: Expressing, Imaging, Staining

Journal: Cellular reprogramming

Article Title: mTOR-mediated activation of p70 S6K induces differentiation of pluripotent human embryonic stem cells.

doi: 10.1089/cell.2010.0011

Figure Lengend Snippet: FIG. 6. Knockdown of Rictor and TSC2 expression elevates p70 S6K activation and alters hESC morphology. Activation of p70 S6K by Rictor and TSC2 siRNA-mediated knockdown leads to changes in cell mor- phology to a more differentiated pheno- type. (A) Representative Western blots from multiple experiments showing ex- pression of proteins associated with mTORC1 (mammalian target of rapamycin complex 1) and mTORC2 signaling in H7 hESCs and H7 Diffs. (B) Western blot of immunoprecipitations of mTOR or TSC1 probed for Raptor, Rictor, or TSC2, re- spectively, in H7 hESCs and H7 Diffs. (C) Representative phase contrast images of H7 hESCs nucleofected with either control siRNA or Rictor=TSC2 siRNA mixture. Scale bar, 250 mm. (D) Western blot analysis confirming knockdown of TSC2 and Rictor as well as expression of p70 S6K, activated p70 S6K (p-T389), Oct4, and Actin.

Article Snippet: The p70 S6K constructs were generously donated to Addgene by Dr. John Blenis, Addgene catalog number 8989 (p70 S6K CON) and 8984 (p70 S6K WT).

Techniques: Knockdown, Expressing, Activation Assay, Western Blot, Control

Journal: Cellular reprogramming

Article Title: mTOR-mediated activation of p70 S6K induces differentiation of pluripotent human embryonic stem cells.

doi: 10.1089/cell.2010.0011

Figure Lengend Snippet: FIG. 7. Diagram depicting mTORC signal- ing in hESCs and differentiated cells. In pluripotent hESCs (left panel), mTOR protein is mostly associated in mTORC2 (mTOR- Rictor), whereas mTORC1 (mTOR-Raptor) activity is suppressed by the heterodimer TSC1=TSC2. In differentiated cells (right panel), mTOR is highly associated with Raptor over Rictor leading to elevated p70 S6K activity. Increased protein translation, in turn, induces differentiation.

Article Snippet: The p70 S6K constructs were generously donated to Addgene by Dr. John Blenis, Addgene catalog number 8989 (p70 S6K CON) and 8984 (p70 S6K WT).

Techniques: Activity Assay