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a) Schematic illustrating the experimental design for RNA sequencing analysis of iPSC-derived RPE cells following AKT2 inhibition. Bubble chart plot shows enrichment of b) top ten pathways upregulated pathways, c) top ten downregulated pathways in RNA sequencing analysis of iPSC RPE CFH Y402H treated with AKT2 inhibitor. d) The heatmap shows hierarchical clustering with euclidean distance and Ward’s method of mTOR pathway genes, identifying differentially regulated ( P <0.05, FC>1.5) mTORC1-specific and mTORC2-specific genes in iPSC RPE CFH Y402H cells treated with the AKT2 inhibitor. Upon AKT2 inhibition in iPSC RPE CFH Y402 cells, mTORC2-specific genes are upregulated, while mTORC1-inhibitory genes are DEPTOR and CASTOR2 are upregulated. e) Schematic illustrating the stepwise inhibition strategy utilizing specific inhibitors targeting EGFR, Src, PI3K, mTORC1, mTORC2, and AKT2. f) Western blot analysis displaying the expression <t>of</t> <t>phospho-AKT1,</t> phospho-AKT2, mTOR Ser2448, mTOR Ser2481 and phospho-TERT Ser824 following treatment with inhibitors targeting EGFR (gefitinib; 5μM), Src (PP1; 10μM), PI3K (Wortmannin; 100nM), mTORC1 (Rapamycin; 10nM), mTORC1/2 (Torin1; 50nM) with or without AKT2 inhibition, for 48 hours. A total of 20 µg of protein was loaded in each well, with H3 serving as the loading control. g) Nuclear-cytoplasmic fractionation results demonstrating the expression levels of <t>RPTOR,</t> RICTOR and phosphor-TERT proteins following AKT2 inhibition. A reduction in RPTOR protein levels and an increase in RICTOR protein levels were observed following AKT2 inhibition. A total of 25 µg of protein was loaded in each well. GAPDH was utilized as a control for the cytoplasmic fraction, while Lamin A/C served as a nuclear loading control. h) Correlation analysis indicating an inverse relationship between AKT1 compensation (phospho-AKT1 Ser473) and RPTOR protein levels upon AKT2 inhibition. i) Correlation analysis showing an inverse relationship between AKT1 compensation (phospho-AKT1 Ser473) and RICTOR protein levels upon AKT2 inhibition.
Rptor, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 96/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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a) Schematic illustrating the experimental design for RNA sequencing analysis of iPSC-derived RPE cells following AKT2 inhibition. Bubble chart plot shows enrichment of b) top ten pathways upregulated pathways, c) top ten downregulated pathways in RNA sequencing analysis of iPSC RPE CFH Y402H treated with AKT2 inhibitor. d) The heatmap shows hierarchical clustering with euclidean distance and Ward’s method of mTOR pathway genes, identifying differentially regulated ( P <0.05, FC>1.5) mTORC1-specific and mTORC2-specific genes in iPSC RPE CFH Y402H cells treated with the AKT2 inhibitor. Upon AKT2 inhibition in iPSC RPE CFH Y402 cells, mTORC2-specific genes are upregulated, while mTORC1-inhibitory genes are DEPTOR and CASTOR2 are upregulated. e) Schematic illustrating the stepwise inhibition strategy utilizing specific inhibitors targeting EGFR, Src, PI3K, mTORC1, mTORC2, and AKT2. f) Western blot analysis displaying the expression <t>of</t> <t>phospho-AKT1,</t> phospho-AKT2, mTOR Ser2448, mTOR Ser2481 and phospho-TERT Ser824 following treatment with inhibitors targeting EGFR (gefitinib; 5μM), Src (PP1; 10μM), PI3K (Wortmannin; 100nM), mTORC1 (Rapamycin; 10nM), mTORC1/2 (Torin1; 50nM) with or without AKT2 inhibition, for 48 hours. A total of 20 µg of protein was loaded in each well, with H3 serving as the loading control. g) Nuclear-cytoplasmic fractionation results demonstrating the expression levels of <t>RPTOR,</t> RICTOR and phosphor-TERT proteins following AKT2 inhibition. A reduction in RPTOR protein levels and an increase in RICTOR protein levels were observed following AKT2 inhibition. A total of 25 µg of protein was loaded in each well. GAPDH was utilized as a control for the cytoplasmic fraction, while Lamin A/C served as a nuclear loading control. h) Correlation analysis indicating an inverse relationship between AKT1 compensation (phospho-AKT1 Ser473) and RPTOR protein levels upon AKT2 inhibition. i) Correlation analysis showing an inverse relationship between AKT1 compensation (phospho-AKT1 Ser473) and RICTOR protein levels upon AKT2 inhibition.
Anti Rptor, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 96/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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a) Schematic illustrating the experimental design for RNA sequencing analysis of iPSC-derived RPE cells following AKT2 inhibition. Bubble chart plot shows enrichment of b) top ten pathways upregulated pathways, c) top ten downregulated pathways in RNA sequencing analysis of iPSC RPE CFH Y402H treated with AKT2 inhibitor. d) The heatmap shows hierarchical clustering with euclidean distance and Ward’s method of mTOR pathway genes, identifying differentially regulated ( P <0.05, FC>1.5) mTORC1-specific and mTORC2-specific genes in iPSC RPE CFH Y402H cells treated with the AKT2 inhibitor. Upon AKT2 inhibition in iPSC RPE CFH Y402 cells, mTORC2-specific genes are upregulated, while mTORC1-inhibitory genes are DEPTOR and CASTOR2 are upregulated. e) Schematic illustrating the stepwise inhibition strategy utilizing specific inhibitors targeting EGFR, Src, PI3K, mTORC1, mTORC2, and AKT2. f) Western blot analysis displaying the expression <t>of</t> <t>phospho-AKT1,</t> phospho-AKT2, mTOR Ser2448, mTOR Ser2481 and phospho-TERT Ser824 following treatment with inhibitors targeting EGFR (gefitinib; 5μM), Src (PP1; 10μM), PI3K (Wortmannin; 100nM), mTORC1 (Rapamycin; 10nM), mTORC1/2 (Torin1; 50nM) with or without AKT2 inhibition, for 48 hours. A total of 20 µg of protein was loaded in each well, with H3 serving as the loading control. g) Nuclear-cytoplasmic fractionation results demonstrating the expression levels of <t>RPTOR,</t> RICTOR and phosphor-TERT proteins following AKT2 inhibition. A reduction in RPTOR protein levels and an increase in RICTOR protein levels were observed following AKT2 inhibition. A total of 25 µg of protein was loaded in each well. GAPDH was utilized as a control for the cytoplasmic fraction, while Lamin A/C served as a nuclear loading control. h) Correlation analysis indicating an inverse relationship between AKT1 compensation (phospho-AKT1 Ser473) and RPTOR protein levels upon AKT2 inhibition. i) Correlation analysis showing an inverse relationship between AKT1 compensation (phospho-AKT1 Ser473) and RICTOR protein levels upon AKT2 inhibition.
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a) Schematic illustrating the experimental design for RNA sequencing analysis of iPSC-derived RPE cells following AKT2 inhibition. Bubble chart plot shows enrichment of b) top ten pathways upregulated pathways, c) top ten downregulated pathways in RNA sequencing analysis of iPSC RPE CFH Y402H treated with AKT2 inhibitor. d) The heatmap shows hierarchical clustering with euclidean distance and Ward’s method of mTOR pathway genes, identifying differentially regulated ( P <0.05, FC>1.5) mTORC1-specific and mTORC2-specific genes in iPSC RPE CFH Y402H cells treated with the AKT2 inhibitor. Upon AKT2 inhibition in iPSC RPE CFH Y402 cells, mTORC2-specific genes are upregulated, while mTORC1-inhibitory genes are DEPTOR and CASTOR2 are upregulated. e) Schematic illustrating the stepwise inhibition strategy utilizing specific inhibitors targeting EGFR, Src, PI3K, mTORC1, mTORC2, and AKT2. f) Western blot analysis displaying the expression <t>of</t> <t>phospho-AKT1,</t> phospho-AKT2, mTOR Ser2448, mTOR Ser2481 and phospho-TERT Ser824 following treatment with inhibitors targeting EGFR (gefitinib; 5μM), Src (PP1; 10μM), PI3K (Wortmannin; 100nM), mTORC1 (Rapamycin; 10nM), mTORC1/2 (Torin1; 50nM) with or without AKT2 inhibition, for 48 hours. A total of 20 µg of protein was loaded in each well, with H3 serving as the loading control. g) Nuclear-cytoplasmic fractionation results demonstrating the expression levels of <t>RPTOR,</t> RICTOR and phosphor-TERT proteins following AKT2 inhibition. A reduction in RPTOR protein levels and an increase in RICTOR protein levels were observed following AKT2 inhibition. A total of 25 µg of protein was loaded in each well. GAPDH was utilized as a control for the cytoplasmic fraction, while Lamin A/C served as a nuclear loading control. h) Correlation analysis indicating an inverse relationship between AKT1 compensation (phospho-AKT1 Ser473) and RPTOR protein levels upon AKT2 inhibition. i) Correlation analysis showing an inverse relationship between AKT1 compensation (phospho-AKT1 Ser473) and RICTOR protein levels upon AKT2 inhibition.
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a) Schematic illustrating the experimental design for RNA sequencing analysis of iPSC-derived RPE cells following AKT2 inhibition. Bubble chart plot shows enrichment of b) top ten pathways upregulated pathways, c) top ten downregulated pathways in RNA sequencing analysis of iPSC RPE CFH Y402H treated with AKT2 inhibitor. d) The heatmap shows hierarchical clustering with euclidean distance and Ward’s method of mTOR pathway genes, identifying differentially regulated ( P <0.05, FC>1.5) mTORC1-specific and mTORC2-specific genes in iPSC RPE CFH Y402H cells treated with the AKT2 inhibitor. Upon AKT2 inhibition in iPSC RPE CFH Y402 cells, mTORC2-specific genes are upregulated, while mTORC1-inhibitory genes are DEPTOR and CASTOR2 are upregulated. e) Schematic illustrating the stepwise inhibition strategy utilizing specific inhibitors targeting EGFR, Src, PI3K, mTORC1, mTORC2, and AKT2. f) Western blot analysis displaying the expression <t>of</t> <t>phospho-AKT1,</t> phospho-AKT2, mTOR Ser2448, mTOR Ser2481 and phospho-TERT Ser824 following treatment with inhibitors targeting EGFR (gefitinib; 5μM), Src (PP1; 10μM), PI3K (Wortmannin; 100nM), mTORC1 (Rapamycin; 10nM), mTORC1/2 (Torin1; 50nM) with or without AKT2 inhibition, for 48 hours. A total of 20 µg of protein was loaded in each well, with H3 serving as the loading control. g) Nuclear-cytoplasmic fractionation results demonstrating the expression levels of <t>RPTOR,</t> RICTOR and phosphor-TERT proteins following AKT2 inhibition. A reduction in RPTOR protein levels and an increase in RICTOR protein levels were observed following AKT2 inhibition. A total of 25 µg of protein was loaded in each well. GAPDH was utilized as a control for the cytoplasmic fraction, while Lamin A/C served as a nuclear loading control. h) Correlation analysis indicating an inverse relationship between AKT1 compensation (phospho-AKT1 Ser473) and RPTOR protein levels upon AKT2 inhibition. i) Correlation analysis showing an inverse relationship between AKT1 compensation (phospho-AKT1 Ser473) and RICTOR protein levels upon AKT2 inhibition.
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a) Schematic illustrating the experimental design for RNA sequencing analysis of iPSC-derived RPE cells following AKT2 inhibition. Bubble chart plot shows enrichment of b) top ten pathways upregulated pathways, c) top ten downregulated pathways in RNA sequencing analysis of iPSC RPE CFH Y402H treated with AKT2 inhibitor. d) The heatmap shows hierarchical clustering with euclidean distance and Ward’s method of mTOR pathway genes, identifying differentially regulated ( P <0.05, FC>1.5) mTORC1-specific and mTORC2-specific genes in iPSC RPE CFH Y402H cells treated with the AKT2 inhibitor. Upon AKT2 inhibition in iPSC RPE CFH Y402 cells, mTORC2-specific genes are upregulated, while mTORC1-inhibitory genes are DEPTOR and CASTOR2 are upregulated. e) Schematic illustrating the stepwise inhibition strategy utilizing specific inhibitors targeting EGFR, Src, PI3K, mTORC1, mTORC2, and AKT2. f) Western blot analysis displaying the expression <t>of</t> <t>phospho-AKT1,</t> phospho-AKT2, mTOR Ser2448, mTOR Ser2481 and phospho-TERT Ser824 following treatment with inhibitors targeting EGFR (gefitinib; 5μM), Src (PP1; 10μM), PI3K (Wortmannin; 100nM), mTORC1 (Rapamycin; 10nM), mTORC1/2 (Torin1; 50nM) with or without AKT2 inhibition, for 48 hours. A total of 20 µg of protein was loaded in each well, with H3 serving as the loading control. g) Nuclear-cytoplasmic fractionation results demonstrating the expression levels of <t>RPTOR,</t> RICTOR and phosphor-TERT proteins following AKT2 inhibition. A reduction in RPTOR protein levels and an increase in RICTOR protein levels were observed following AKT2 inhibition. A total of 25 µg of protein was loaded in each well. GAPDH was utilized as a control for the cytoplasmic fraction, while Lamin A/C served as a nuclear loading control. h) Correlation analysis indicating an inverse relationship between AKT1 compensation (phospho-AKT1 Ser473) and RPTOR protein levels upon AKT2 inhibition. i) Correlation analysis showing an inverse relationship between AKT1 compensation (phospho-AKT1 Ser473) and RICTOR protein levels upon AKT2 inhibition.
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a) Schematic illustrating the experimental design for RNA sequencing analysis of iPSC-derived RPE cells following AKT2 inhibition. Bubble chart plot shows enrichment of b) top ten pathways upregulated pathways, c) top ten downregulated pathways in RNA sequencing analysis of iPSC RPE CFH Y402H treated with AKT2 inhibitor. d) The heatmap shows hierarchical clustering with euclidean distance and Ward’s method of mTOR pathway genes, identifying differentially regulated ( P <0.05, FC>1.5) mTORC1-specific and mTORC2-specific genes in iPSC RPE CFH Y402H cells treated with the AKT2 inhibitor. Upon AKT2 inhibition in iPSC RPE CFH Y402 cells, mTORC2-specific genes are upregulated, while mTORC1-inhibitory genes are DEPTOR and CASTOR2 are upregulated. e) Schematic illustrating the stepwise inhibition strategy utilizing specific inhibitors targeting EGFR, Src, PI3K, mTORC1, mTORC2, and AKT2. f) Western blot analysis displaying the expression of phospho-AKT1, phospho-AKT2, mTOR Ser2448, mTOR Ser2481 and phospho-TERT Ser824 following treatment with inhibitors targeting EGFR (gefitinib; 5μM), Src (PP1; 10μM), PI3K (Wortmannin; 100nM), mTORC1 (Rapamycin; 10nM), mTORC1/2 (Torin1; 50nM) with or without AKT2 inhibition, for 48 hours. A total of 20 µg of protein was loaded in each well, with H3 serving as the loading control. g) Nuclear-cytoplasmic fractionation results demonstrating the expression levels of RPTOR, RICTOR and phosphor-TERT proteins following AKT2 inhibition. A reduction in RPTOR protein levels and an increase in RICTOR protein levels were observed following AKT2 inhibition. A total of 25 µg of protein was loaded in each well. GAPDH was utilized as a control for the cytoplasmic fraction, while Lamin A/C served as a nuclear loading control. h) Correlation analysis indicating an inverse relationship between AKT1 compensation (phospho-AKT1 Ser473) and RPTOR protein levels upon AKT2 inhibition. i) Correlation analysis showing an inverse relationship between AKT1 compensation (phospho-AKT1 Ser473) and RICTOR protein levels upon AKT2 inhibition.

Journal: bioRxiv

Article Title: A non-canonical AKT1-TERT pathway coordinates autophagy and ERphagy

doi: 10.1101/2025.11.24.690135

Figure Lengend Snippet: a) Schematic illustrating the experimental design for RNA sequencing analysis of iPSC-derived RPE cells following AKT2 inhibition. Bubble chart plot shows enrichment of b) top ten pathways upregulated pathways, c) top ten downregulated pathways in RNA sequencing analysis of iPSC RPE CFH Y402H treated with AKT2 inhibitor. d) The heatmap shows hierarchical clustering with euclidean distance and Ward’s method of mTOR pathway genes, identifying differentially regulated ( P <0.05, FC>1.5) mTORC1-specific and mTORC2-specific genes in iPSC RPE CFH Y402H cells treated with the AKT2 inhibitor. Upon AKT2 inhibition in iPSC RPE CFH Y402 cells, mTORC2-specific genes are upregulated, while mTORC1-inhibitory genes are DEPTOR and CASTOR2 are upregulated. e) Schematic illustrating the stepwise inhibition strategy utilizing specific inhibitors targeting EGFR, Src, PI3K, mTORC1, mTORC2, and AKT2. f) Western blot analysis displaying the expression of phospho-AKT1, phospho-AKT2, mTOR Ser2448, mTOR Ser2481 and phospho-TERT Ser824 following treatment with inhibitors targeting EGFR (gefitinib; 5μM), Src (PP1; 10μM), PI3K (Wortmannin; 100nM), mTORC1 (Rapamycin; 10nM), mTORC1/2 (Torin1; 50nM) with or without AKT2 inhibition, for 48 hours. A total of 20 µg of protein was loaded in each well, with H3 serving as the loading control. g) Nuclear-cytoplasmic fractionation results demonstrating the expression levels of RPTOR, RICTOR and phosphor-TERT proteins following AKT2 inhibition. A reduction in RPTOR protein levels and an increase in RICTOR protein levels were observed following AKT2 inhibition. A total of 25 µg of protein was loaded in each well. GAPDH was utilized as a control for the cytoplasmic fraction, while Lamin A/C served as a nuclear loading control. h) Correlation analysis indicating an inverse relationship between AKT1 compensation (phospho-AKT1 Ser473) and RPTOR protein levels upon AKT2 inhibition. i) Correlation analysis showing an inverse relationship between AKT1 compensation (phospho-AKT1 Ser473) and RICTOR protein levels upon AKT2 inhibition.

Article Snippet: The primary antibodies Akt1 (2938S), Akt2 (3063S), mTOR (2983S), RPTOR (2280S), RICTOR (2114S), PERK (5683T), p-PERK (3179S), eIF2α (9722S), p-eIF2α (9721S), ATF4 (11815S), BiP (3177T), CHOP (2895T), FOXO3a (12829S), p-FOXO3a (9464S), β-actin (4970S), Vinculin (13901S) and c-MYC (5605T) were purchased from Cell Signaling Technology, Inc. TERT (NB100-317) was purchased from Novus Biologicals.

Techniques: RNA Sequencing, Derivative Assay, Inhibition, Western Blot, Expressing, Control, Fractionation