plko 1 shrna plasmid  (Addgene inc)

Journal: bioRxiv

Article Title: Driving proteomic imbalance to combat Neurofibromatosis type I (NF1)-associated malignancy

doi: 10.1101/2024.05.24.595838

Figure Lengend Snippet: (A) Immunoblotting detection of JNK activation, mTORC1 activity, and protein polyubiquitination in S462 cells following stable TSC2 knockdown and/or HSF1 inhibition by 10 μM DTHIB for 3 days. (B) Quantitation of soluble AOs by flow cytometry using A11 antibody staining in S462 cells with and without TSC2 knockdown and 10 μM DTHIB treatment (mean ± SD, n=3 independent experiments, Two-way ANOVA). (C) Quantitation of cytotoxicity by flow cytometry using Live-or-Dye stains in S462 cells with and without stable TSC2 knockdown and DTHIB (10 μM) or combined DTHIB and LY2584702 (20 μM) treatment (mean ± SD, n=3 independent experiments, Two-way ANOVA). Cells were pre-treated with LY2584702 for 1 day followed by DTHIB treatment for another 3 days. (D) Quantitation of cytotoxicity by flow cytometry using Live-or-Dye stains in S462 cells with and without stable TSC2 knockdown and DTHIB (10 μM) or combined DTHIB and CR (30 μM) treatment (mean ± SD, n=3 independent experiments, Two-way ANOVA). Cells were pre-treated with CR for 1 day followed by DTHIB treatment for another 3 days. (E) Immunoblotting detection of mTORC1 stimulation by 500 μM NV-5138 in S462 cells with and without 10 μM DTHIB treatment. (F) Quantitation of soluble AOs by flow cytometry using A11 antibody staining in S462 cells with and without 500 μM NV-5138 stimulation and 10 μM DTHIB treatment (mean ± SD, n=3 independent experiments, One-way ANOVA). (G) Quantitation of cytotoxicity by flow cytometry using Live-or-Dye stains in S462 cells with and without 500 μM NV-5138 stimulation and 10 μM DTHIB or combined DTHIB and CR treatment (mean ± SD, n=5 independent experiments, One-way ANOVA). (H) Quantitation of cytotoxicity by flow cytometry using Live-or-Dye stains in immortalized human Schwann cells with and without 500 μM NV-5138 stimulation and 10 μM DTHIB treatment (mean ± SD, n=3 independent experiments, One-way ANOVA). (I) Immunoblotting detection of mTORC1 stimulation by 500 μM NV-5138 in immortalized human Schwann cells with and without 10 μM DTHIB treatment.

Article Snippet: The pLKO.1 shRNA plasmid targeting human TSC2 was a gift from Do-Hyung Kim (Cat#15478, Addgene).

Techniques: Western Blot, Activation Assay, Activity Assay, Knockdown, Inhibition, Quantitation Assay, Flow Cytometry, Staining

Journal: bioRxiv

Article Title: Driving proteomic imbalance to combat Neurofibromatosis type I (NF1)-associated malignancy

doi: 10.1101/2024.05.24.595838

Figure Lengend Snippet: (A) and (B) Quantitation of cytotoxicity by flow cytometry using Live-or-Dye stains combined with cleaved caspase 3 (Asp175) antibody staining. S462 cells with and without stable TSC2 knockdown were treated with DMSO, 10 μM DTHIB, or combined DTHIB and 30 μM Q-VD-OPh (mean ± SD, n=3 independent experiments, Two-way ANOVA). (C) Quantitation of cytotoxicity by flow cytometry using Live-or-Dye stains in S462 cells with and without stable TSC2 knockdown. Cells were treated with 10 μM DTHIB alone or co-treated with 10 μM DTHIB and 30 μM Necrostatin-1 or 20 μM Liproxstatin-1 for 3 days (mean ± SD, n=3 independent experiments, Two-way ANOVA). (D) Immunoblotting detection of ferroptosis markers in S462 cells with and without stable TSC2 knockdown treated with 10 μM DTHIB alone or co-treated with 10 μM DTHIB and 20 μM liproxstatin-1 for 3 days. Erastin was included as a positive control to induce canonical ferroptosis. (E) Immunoblotting detection of autophagy markers in S462 cells with stable TSC2 knockdown treated with 10 μM DTHIB alone or co-treated with 10 μM DTHIB and 3 μM wortmannin for 3 days. Rapamycin was included as a positive control to induce autophagy. (F) Quantitation of cytotoxicity by flow cytometry using Live-or-Dye stains in S462 cells with and without stable TSC2 knockdown. Cells were treated with 10 μM DTHIB alone or co-treated with 10 μM DTHIB and 3 μM wortmannin for 3 days (mean ± SD, n=3 independent experiments, Two-way ANOVA). (G) Schematic depiction of instigation of cell death by severe proteomic imbalance, owing to simultaneous mTORC1 stimulation ( ) and HSF1 inhibition ( ). In cancer cells, constitutive HSF1 activation provides extra chaperoning capacity to cope with elevated protein misfolding, partly due to enhanced protein synthesis and widespread genetic mutations. Nevertheless, amyloids still emerge, although at low levels. Importantly, HSF1 can neutralize highly toxic amyloid oligomers, averting lethal consequences. By contrast, HSF1 inhibition diminishes chaperoning capacity, insufficient to counterbalance the robust protein translation. mTORC1 stimulation further aggravates this proteomic imbalance, which, in turn, strongly promotes amyloidogenesis. In consequence, the amounts of amyloid oligomers exceed the neutralizing capacity of HSF1, leading to cell death; nonetheless, it remains unclear how this non-apoptotic, non-autophagic death occurs. (H) Schematic depiction of the concept of driving proteomic imbalance to combat malignancy. On the one hand, in cancer cells, mTORC1 is inevitably activated to stimulate protein translation, markedly augmenting protein quantity. On the other hand, the extra chaperoning capacity governed by HSF1, albeit dispensable for normal life, becomes necessary to ensure sufficient protein quality in cancer cells, thereby counterbalancing augmented protein quantity and suppressing proteomic instability. Thus, proteomic balance promotes malignant growth. By contrast, disrupting proteomic balance, through HSF1 inhibition, is sufficient to provoke proteomic instability and elicit tumor suppression. However, simultaneous mTORC1 stimulation can remarkably drive proteomic imbalance, causing severe proteomic instability and profound tumor suppression.

Article Snippet: The pLKO.1 shRNA plasmid targeting human TSC2 was a gift from Do-Hyung Kim (Cat#15478, Addgene).

Techniques: Quantitation Assay, Flow Cytometry, Staining, Knockdown, Western Blot, Positive Control, Inhibition, Activation Assay

Journal: Brain : a journal of neurology

Article Title: Mammalian target of rapamycin complex 1 activation sensitizes human glioma cells to hypoxia-induced cell death.

doi: 10.1093/brain/awx196

Figure Lengend Snippet: Figure 1 TSC2 gene suppression activates mTORC1 signalling during nutrient deprivation and hypoxia. (A) LNT-229 and LN-308 TSC2sh and control cells (non-targeting sequence, NTsh) were analysed by qPCR. TSC2 gene suppression was confirmed. Values are normalized to 18S as well as SDHA housekeeping gene expression (n = 3, mean SD). (B) LNT-229 and LN-308 cells were incubated for 4 h, as indicated. Cellular lysates were analysed by immunoblot with antibodies for TSC2, P-Akt (Ser 473), Akt, P-S6K1 (Thr 389), S6K1, P-S6RP (Ser 240/244 and Ser 235/235), S6RP, P-4E-BP1 (Ser 65), 4E-BP1 or actin. The same incubation conditions were used for immunoprecipitation of GTP-RHEB. The precipitate was then analysed by immunoblot with an antibody for RHEB as well as an immunoblot with an antibody for actin as a loading control of the employed lysate (lower two lanes). (C) G55 (left) and LN-428 (right) NTsh and TSC2sh cells were analysed by qPCR. TSC2 gene suppression was confirmed, values are normalized to 18S as well as SDHA housekeeping gene expression (n = 3, mean SD). (D) G55 (left) and LN-428 (right) cells were incubated for 4 h, as indicated. Cellular lysates were analysed by immunoblot with antibodies for TSC2, P-S6RP (Ser 240/244 and Ser 235/235), S6RP, P-4E-BP1 (Ser 65), 4E-BP1 or actin. (E) LNT-229 and LN-308 cells were incubated in serum-free and serum containing (10% FCS) culture conditions without glucose restriction (25 mM glucose) for 5 days. Cell density was measured by crystal violet staining at the beginning of cultivation and after 5 days (n = 4, mean SD).

Article Snippet: Generation of TSC2 gene suppressed cells The pLKO.1 plasmids targeting TSC2 (TSC2sh) and the pLKO.1 plasmid with a non-targeting shRNA sequence (NTsh) were ordered from Addgene (Addgene #15478, #1864).

Techniques: Control, Sequencing, Gene Expression, Incubation, Western Blot, Immunoprecipitation, Staining

Journal: Brain : a journal of neurology

Article Title: Mammalian target of rapamycin complex 1 activation sensitizes human glioma cells to hypoxia-induced cell death.

doi: 10.1093/brain/awx196

Figure Lengend Snippet: Figure 2 TSC2 gene suppression sensitizes human malignant glioma cells to hypoxia-induced cell death. Cells were exposed to glucose restricted (2 mM glucose) serum-free DMEM under normoxic conditions or 0.1% oxygen until cell death (24 h), ATP depletion (12 h) or an increase in ROS (8 h) was observed. (A) Cell death was quantified by LDH release and by propidium iodide staining (n = 4, mean SD, *P 5 0.05, **P 5 0.01). (B) G55 and LN-428 NTsh and TSC2sh cells were exposed to glucose restricted (2 mM glucose) serum-free DMEM under normoxic conditions or 0.1% oxygen until cell death (24 h) was observed. Cell death was quantified by LDH release (n = 4, mean SD, *P 5 0.05, **P 5 0.01). (C) ATP was quantified by a luciferase-based assay. The ratio of ATP concentrations in hypoxia to normoxia is depicted (n = 5, mean SD, **P 5 0.01). (D) ROS levels were measured by H2DCFDA-FACS (n = 3, mean SD, *P 5 0.05, **P 5 0.01). n.s. = not significant; ROS = reactive oxygen species.

Article Snippet: Generation of TSC2 gene suppressed cells The pLKO.1 plasmids targeting TSC2 (TSC2sh) and the pLKO.1 plasmid with a non-targeting shRNA sequence (NTsh) were ordered from Addgene (Addgene #15478, #1864).

Techniques: Staining, Luciferase

Journal: Brain : a journal of neurology

Article Title: Mammalian target of rapamycin complex 1 activation sensitizes human glioma cells to hypoxia-induced cell death.

doi: 10.1093/brain/awx196

Figure Lengend Snippet: Figure 3 TSC2sh gene suppression has no effect on glucose consumption and lactate production, but alters metabolic pathways. (A) Cells were exposed to glucose restricted (2 mM glucose) serum-free DMEM under normoxic conditions or 0.1% oxygen for 8 h. Glucose consumption and lactate production were determined in the supernatant (n = 3, mean SD). (B) Cells were exposed to glucose restricted (2 mM glucose) serum-free DMEM under normoxic conditions or 0.1% oxygen for 8 h. Intracellular metabolites were analysed either by LC-MS-MS analysis or GC-MS analysis (n = 3, mean SD, *P 5 0.05, **P 5 0.01). (C) LNT-229 and LN-308 NTsh and TSC2sh cells were exposed to glucose restricted (2 mM glucose) serum-free DMEM under normoxic conditions or 0.1% oxygen for 4 h, cDNA and cellular lysates of cells were generated. Gene expression of G6PD was quantified by qPCR, values are normalized to 18S as well as SDHA housekeeping gene expression (n = 3, mean SD, *P 5 0.05) (left). Cellular lysates were analysed by immunoblot with antibodies for G6PD or actin. Quantification of G6PD protein content of three independent experiments is provided (n = 3, mean SD, *P 5 0.05, **P 5 0.01) and one representative immunoblot is shown (middle). LNT-229 and LN-308 wild-type cells were exposed to medium containing 10% FCS with 25 mM glucose with 200 nM rapamycin, 200 nM torin2 or vehicle for 72 h. Cellular lysates were analysed by immunoblot with antibodies for G6PD, P-S6RP (Ser 240/244 and Ser 235/235), S6RP, P-4E-BP1 (Ser 65), 4E-BP1 or actin. n.s. = not significant.

Article Snippet: Generation of TSC2 gene suppressed cells The pLKO.1 plasmids targeting TSC2 (TSC2sh) and the pLKO.1 plasmid with a non-targeting shRNA sequence (NTsh) were ordered from Addgene (Addgene #15478, #1864).

Techniques: Liquid Chromatography with Mass Spectroscopy, Gas Chromatography-Mass Spectrometry, Generated, Gene Expression, Western Blot

Journal: Brain : a journal of neurology

Article Title: Mammalian target of rapamycin complex 1 activation sensitizes human glioma cells to hypoxia-induced cell death.

doi: 10.1093/brain/awx196

Figure Lengend Snippet: Figure 4 TSC2 gene suppression induces oxygen consumption and expression of genes of mitochondrial oxidative function. (A) LNT-229 NTsh and TSC2sh cells were incubated in medium containing 10% FCS with 25 mM glucose. Oxygen consumption was measured by a fluorescence-based assay (left, n = 3, mean, **P 5 0.01). Using an Oxygraph-2k system steady state oxygen consumption was also monitored (right, n = 5 mean + SD, **P 5 0.01). (B) cDNA of LNT-229 and LN-308 NTsh and TSC2sh cells cultured in standard conditions was generated. Gene expression of PPARGC1A/PGC-1a and PPARGC1B/PGC-1b, ERRSA/ERR-a, NRF1, MT-CYB, MT-CO1, MT-CO2, SCO2, ATP5G1, COX5A and IDH3A was quantified by qPCR, values are normalized to 18S as well as SDHA housekeeping gene expression (n = 3, mean SD, *P 5 0.05, **P 5 0.01). n.s. = not significant.

Article Snippet: Generation of TSC2 gene suppressed cells The pLKO.1 plasmids targeting TSC2 (TSC2sh) and the pLKO.1 plasmid with a non-targeting shRNA sequence (NTsh) were ordered from Addgene (Addgene #15478, #1864).

Techniques: Expressing, Incubation, Cell Culture, Generated, Gene Expression

Journal: Brain : a journal of neurology

Article Title: Mammalian target of rapamycin complex 1 activation sensitizes human glioma cells to hypoxia-induced cell death.

doi: 10.1093/brain/awx196

Figure Lengend Snippet: Figure 6 Inhibition of mTORC1 with rapamycin reverts the phenotype of TSC2sh cells. (A) LNT-229 NTsh and TSC2sh cells were exposed to medium containing 10% FCS with 25 mM glucose with 200 nM rapamycin or vehicle. Oxygen consumption was measured with a fluorescence-based assay (n = 3, mean, *P 5 0.05, **P 5 0.01). (B) cDNA of LNT-229 NTsh and TSC2sh cells treated with 200 nM rapamycin or vehicle for 24 h in DMEM medium containing 10% FCS and 25 mM glucose was generated. Gene expression of PPARGC1A/PGC-1a, PPARGC1B/ PGC-1b and ERRSA/ERR-a was measured by qPCR, values are normalized to 18S as well as SDHA housekeeping gene expression (n = 3, mean SD, **P 5 0.01). n.s. = not significant.

Article Snippet: Generation of TSC2 gene suppressed cells The pLKO.1 plasmids targeting TSC2 (TSC2sh) and the pLKO.1 plasmid with a non-targeting shRNA sequence (NTsh) were ordered from Addgene (Addgene #15478, #1864).

Techniques: Inhibition, Generated, Gene Expression

Journal: Brain : a journal of neurology

Article Title: Mammalian target of rapamycin complex 1 activation sensitizes human glioma cells to hypoxia-induced cell death.

doi: 10.1093/brain/awx196

Figure Lengend Snippet: Figure 1 TSC2 gene suppression activates mTORC1 signalling during nutrient deprivation and hypoxia. (A) LNT-229 and LN-308 TSC2sh and control cells (non-targeting sequence, NTsh) were analysed by qPCR. TSC2 gene suppression was confirmed. Values are normalized to 18S as well as SDHA housekeeping gene expression (n = 3, mean SD). (B) LNT-229 and LN-308 cells were incubated for 4 h, as indicated. Cellular lysates were analysed by immunoblot with antibodies for TSC2, P-Akt (Ser 473), Akt, P-S6K1 (Thr 389), S6K1, P-S6RP (Ser 240/244 and Ser 235/235), S6RP, P-4E-BP1 (Ser 65), 4E-BP1 or actin. The same incubation conditions were used for immunoprecipitation of GTP-RHEB. The precipitate was then analysed by immunoblot with an antibody for RHEB as well as an immunoblot with an antibody for actin as a loading control of the employed lysate (lower two lanes). (C) G55 (left) and LN-428 (right) NTsh and TSC2sh cells were analysed by qPCR. TSC2 gene suppression was confirmed, values are normalized to 18S as well as SDHA housekeeping gene expression (n = 3, mean SD). (D) G55 (left) and LN-428 (right) cells were incubated for 4 h, as indicated. Cellular lysates were analysed by immunoblot with antibodies for TSC2, P-S6RP (Ser 240/244 and Ser 235/235), S6RP, P-4E-BP1 (Ser 65), 4E-BP1 or actin. (E) LNT-229 and LN-308 cells were incubated in serum-free and serum containing (10% FCS) culture conditions without glucose restriction (25 mM glucose) for 5 days. Cell density was measured by crystal violet staining at the beginning of cultivation and after 5 days (n = 4, mean SD).

Article Snippet: Generation of TSC2 gene suppressed cells The pLKO.1 plasmids targeting TSC2 (TSC2sh) and the pLKO.1 plasmid with a non-targeting shRNA sequence (NTsh) were ordered from Addgene (Addgene #15478, #1864).

Techniques: Control, Sequencing, Gene Expression, Incubation, Western Blot, Immunoprecipitation, Staining

Journal: Brain : a journal of neurology

Article Title: Mammalian target of rapamycin complex 1 activation sensitizes human glioma cells to hypoxia-induced cell death.

doi: 10.1093/brain/awx196

Figure Lengend Snippet: Figure 2 TSC2 gene suppression sensitizes human malignant glioma cells to hypoxia-induced cell death. Cells were exposed to glucose restricted (2 mM glucose) serum-free DMEM under normoxic conditions or 0.1% oxygen until cell death (24 h), ATP depletion (12 h) or an increase in ROS (8 h) was observed. (A) Cell death was quantified by LDH release and by propidium iodide staining (n = 4, mean SD, *P 5 0.05, **P 5 0.01). (B) G55 and LN-428 NTsh and TSC2sh cells were exposed to glucose restricted (2 mM glucose) serum-free DMEM under normoxic conditions or 0.1% oxygen until cell death (24 h) was observed. Cell death was quantified by LDH release (n = 4, mean SD, *P 5 0.05, **P 5 0.01). (C) ATP was quantified by a luciferase-based assay. The ratio of ATP concentrations in hypoxia to normoxia is depicted (n = 5, mean SD, **P 5 0.01). (D) ROS levels were measured by H2DCFDA-FACS (n = 3, mean SD, *P 5 0.05, **P 5 0.01). n.s. = not significant; ROS = reactive oxygen species.

Article Snippet: Generation of TSC2 gene suppressed cells The pLKO.1 plasmids targeting TSC2 (TSC2sh) and the pLKO.1 plasmid with a non-targeting shRNA sequence (NTsh) were ordered from Addgene (Addgene #15478, #1864).

Techniques: Staining, Luciferase

Journal: Brain : a journal of neurology

Article Title: Mammalian target of rapamycin complex 1 activation sensitizes human glioma cells to hypoxia-induced cell death.

doi: 10.1093/brain/awx196

Figure Lengend Snippet: Figure 3 TSC2sh gene suppression has no effect on glucose consumption and lactate production, but alters metabolic pathways. (A) Cells were exposed to glucose restricted (2 mM glucose) serum-free DMEM under normoxic conditions or 0.1% oxygen for 8 h. Glucose consumption and lactate production were determined in the supernatant (n = 3, mean SD). (B) Cells were exposed to glucose restricted (2 mM glucose) serum-free DMEM under normoxic conditions or 0.1% oxygen for 8 h. Intracellular metabolites were analysed either by LC-MS-MS analysis or GC-MS analysis (n = 3, mean SD, *P 5 0.05, **P 5 0.01). (C) LNT-229 and LN-308 NTsh and TSC2sh cells were exposed to glucose restricted (2 mM glucose) serum-free DMEM under normoxic conditions or 0.1% oxygen for 4 h, cDNA and cellular lysates of cells were generated. Gene expression of G6PD was quantified by qPCR, values are normalized to 18S as well as SDHA housekeeping gene expression (n = 3, mean SD, *P 5 0.05) (left). Cellular lysates were analysed by immunoblot with antibodies for G6PD or actin. Quantification of G6PD protein content of three independent experiments is provided (n = 3, mean SD, *P 5 0.05, **P 5 0.01) and one representative immunoblot is shown (middle). LNT-229 and LN-308 wild-type cells were exposed to medium containing 10% FCS with 25 mM glucose with 200 nM rapamycin, 200 nM torin2 or vehicle for 72 h. Cellular lysates were analysed by immunoblot with antibodies for G6PD, P-S6RP (Ser 240/244 and Ser 235/235), S6RP, P-4E-BP1 (Ser 65), 4E-BP1 or actin. n.s. = not significant.

Article Snippet: Generation of TSC2 gene suppressed cells The pLKO.1 plasmids targeting TSC2 (TSC2sh) and the pLKO.1 plasmid with a non-targeting shRNA sequence (NTsh) were ordered from Addgene (Addgene #15478, #1864).

Techniques: Liquid Chromatography with Mass Spectroscopy, Gas Chromatography-Mass Spectrometry, Generated, Gene Expression, Western Blot

Journal: Brain : a journal of neurology

Article Title: Mammalian target of rapamycin complex 1 activation sensitizes human glioma cells to hypoxia-induced cell death.

doi: 10.1093/brain/awx196

Figure Lengend Snippet: Figure 4 TSC2 gene suppression induces oxygen consumption and expression of genes of mitochondrial oxidative function. (A) LNT-229 NTsh and TSC2sh cells were incubated in medium containing 10% FCS with 25 mM glucose. Oxygen consumption was measured by a fluorescence-based assay (left, n = 3, mean, **P 5 0.01). Using an Oxygraph-2k system steady state oxygen consumption was also monitored (right, n = 5 mean + SD, **P 5 0.01). (B) cDNA of LNT-229 and LN-308 NTsh and TSC2sh cells cultured in standard conditions was generated. Gene expression of PPARGC1A/PGC-1a and PPARGC1B/PGC-1b, ERRSA/ERR-a, NRF1, MT-CYB, MT-CO1, MT-CO2, SCO2, ATP5G1, COX5A and IDH3A was quantified by qPCR, values are normalized to 18S as well as SDHA housekeeping gene expression (n = 3, mean SD, *P 5 0.05, **P 5 0.01). n.s. = not significant.

Article Snippet: Generation of TSC2 gene suppressed cells The pLKO.1 plasmids targeting TSC2 (TSC2sh) and the pLKO.1 plasmid with a non-targeting shRNA sequence (NTsh) were ordered from Addgene (Addgene #15478, #1864).

Techniques: Expressing, Incubation, Cell Culture, Generated, Gene Expression

Journal: Brain : a journal of neurology

Article Title: Mammalian target of rapamycin complex 1 activation sensitizes human glioma cells to hypoxia-induced cell death.

doi: 10.1093/brain/awx196

Figure Lengend Snippet: Figure 6 Inhibition of mTORC1 with rapamycin reverts the phenotype of TSC2sh cells. (A) LNT-229 NTsh and TSC2sh cells were exposed to medium containing 10% FCS with 25 mM glucose with 200 nM rapamycin or vehicle. Oxygen consumption was measured with a fluorescence-based assay (n = 3, mean, *P 5 0.05, **P 5 0.01). (B) cDNA of LNT-229 NTsh and TSC2sh cells treated with 200 nM rapamycin or vehicle for 24 h in DMEM medium containing 10% FCS and 25 mM glucose was generated. Gene expression of PPARGC1A/PGC-1a, PPARGC1B/ PGC-1b and ERRSA/ERR-a was measured by qPCR, values are normalized to 18S as well as SDHA housekeeping gene expression (n = 3, mean SD, **P 5 0.01). n.s. = not significant.

Article Snippet: Generation of TSC2 gene suppressed cells The pLKO.1 plasmids targeting TSC2 (TSC2sh) and the pLKO.1 plasmid with a non-targeting shRNA sequence (NTsh) were ordered from Addgene (Addgene #15478, #1864).

Techniques: Inhibition, Generated, Gene Expression

Journal: Brain : a journal of neurology

Article Title: Mammalian target of rapamycin complex 1 activation sensitizes human glioma cells to hypoxia-induced cell death.

doi: 10.1093/brain/awx196

Figure Lengend Snippet: Figure 7 Model of deregulated mTORC1 signalling and vulnerability to starvation conditions in glioblastoma. Oxygen concentration as well as nutrient availability decline with increasing distance from blood vessels. When the oxygen concen- tration falls below a threshold, HIF-1a is induced. Depending on the quality of their sensor, cells inhibit mTORC1 signalling via TSC1/2 activation for adaptation. When cellular sensors are deregulated, e.g. in TSC2sh cells, starvation- and hypoxia-induced cell death is more widespread. Other indirect mTORC1 activating mutations like mutated EGFR (e.g. EGFR vIII mutation) and loss of PTEN might add to a deregulated mTORC1 sensor in glioblastomas.

Article Snippet: Generation of TSC2 gene suppressed cells The pLKO.1 plasmids targeting TSC2 (TSC2sh) and the pLKO.1 plasmid with a non-targeting shRNA sequence (NTsh) were ordered from Addgene (Addgene #15478, #1864).

Techniques: Concentration Assay, Activation Assay, Mutagenesis