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Sangon Biotech tsc2 shrna sequences
Tsc2 Shrna Sequences, supplied by Sangon Biotech, used in various techniques. Bioz Stars score: 90/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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tsc2 shrna sequences - by Bioz Stars, 2026-10

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Article Title: Salidroside attenuates cerebral ischemia/reperfusion injury by regulating TSC2-induced autophagy.
Article Snippet: Salidroside (SAL), an antioxidant derived from Rhodiola rosea, exerts neuroprotective effects in cerebral ischemia/reperfusion (I/R) injury; however, the mechanisms have not been fully elucidated.. The present study established a rat model of middle cerebral artery occlusion/reperfusion (MCAO/R) and a cellular model of oxygen–glucose deprivation/reoxygenation (OGD/R) to explore the roles and mechanisms of SAL in cerebral I/R injury.. The rat model of MCAO/R was established and rats were treated with different doses of SAL.

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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).

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

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.

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.

Techniques: Staining, Luciferase

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.

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.

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

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.

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.

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

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.

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.

Techniques: Inhibition, Generated, Gene Expression

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