pcdna3 1 sars cov 2 s rbd 8xhis tag mammalian expression plasmid  (Addgene inc)

Journal: Cell Death & Disease

Article Title: Uncomplexed-TSC1 deploys novel mTORC1-independent pathway to exacerbate the liver glycogen storage in TSC

doi: 10.1038/s41419-025-08161-3

Figure Lengend Snippet: a Confirmation of Tsc1 mutant mice using Sanger sequencing. b Confirmation of Tsc2 mutant mice using Sanger sequencing. c MRI revealed imaging characteristics of the kidneys in 12 to 13 months mice (WT, Tsc1 c.2500-2503delAACA , Tsc2 c.1113delA ). d Macroscopic appearance of 12 to 13 months mouse tissues (WT, Tsc1 c.2500-2503delAACA , Tsc2 c.1113delA ). e Representative images of HE staining, SMA IHC staining, CD31 IHC staining, HMB45 IHC staining and PAS staining in liver tissues isolated from mice aged 12 to 13 months (WT, Tsc1 c.2500-2503delAACA , Tsc2 c.1113delA − ). n = 6 for each genotype; each group contains mice from three different litters. Bar, 100 μm in ( e ). f Representative images of HE staining and PAS staining in kidney tissues isolated from mice aged 12 to 13 months (WT, Tsc1 c.2500-2503delAACA , Tsc2 c.1113delA ). n = 6 for each genotype; each group contains mice from three different litters. Bar, 100 μm in ( f ). Data are shown as the mean ± SD, n = 6, two-tailed unpaired Student’s t -test.

Article Snippet: The pcDNA3.1-myc- TSC1 and pcDNA3.1-Flag- TSC2 plasmids were preserved by our lab; pcDNA3.1- METTL3 -WT-3*HA was purchased from PPL (Jiangsu, China); pcDNA3.1- METTL3 -mut-3*HA were constructed in our Lab; pcDNA3.1- KDM5A -Flag purchased from Cyagen (Suzhou, China) for overexpression experiments.

Techniques: Mutagenesis, Sequencing, Imaging, Staining, Immunohistochemistry, Isolation, Two Tailed Test

Journal: Cell Death & Disease

Article Title: Uncomplexed-TSC1 deploys novel mTORC1-independent pathway to exacerbate the liver glycogen storage in TSC

doi: 10.1038/s41419-025-08161-3

Figure Lengend Snippet: a Western blot (WB) analysis of mTORC1 activity in MEFs (WT, Tsc1 −/− , Tsc2 −/− ) and HepG2 (si-NC, si- TSC1 , si- TSC2 ). b , c Analysis of glycogen levels in MEFs (WT, Tsc1 −/− , Tsc2 −/− ) and HepG2 (si-NC, si- TSC1 , si- TSC2 ) using Periodic Acid-Schiff (PAS) staining and a glycogen assay kit. Bar, 20 μm in ( b ). d Detection of mTORC1 activity and p-GSK3β(s9) levels in MEFs (WT, Tsc1 −/− , Tsc2 −/− ) using WB at 24 h post treatment of 300 nM rapamycin. e , f Analysis of glycogen levels in MEFs (WT, Tsc1 −/− , Tsc2 −/− ) at 24 h post treatment of 300 nM rapamycin or control (DMSO) using PAS staining and glycogen assay kit. Bar, 20 μm in ( e ). Data are shown as the mean ± SD, n = 3, two-tailed unpaired Student’s t -test.

Article Snippet: The pcDNA3.1-myc- TSC1 and pcDNA3.1-Flag- TSC2 plasmids were preserved by our lab; pcDNA3.1- METTL3 -WT-3*HA was purchased from PPL (Jiangsu, China); pcDNA3.1- METTL3 -mut-3*HA were constructed in our Lab; pcDNA3.1- KDM5A -Flag purchased from Cyagen (Suzhou, China) for overexpression experiments.

Techniques: Western Blot, Activity Assay, Staining, Control, Two Tailed Test

Journal: Cell Death & Disease

Article Title: Uncomplexed-TSC1 deploys novel mTORC1-independent pathway to exacerbate the liver glycogen storage in TSC

doi: 10.1038/s41419-025-08161-3

Figure Lengend Snippet: a , b Detection of m 6 A levels in MEFs (WT, Tsc1 −/− , Tsc2 −/− ) and HepG2 (si-NC, si- TSC1 , si- TSC2 ) using an m 6 A quantification assay and m 6 A dot blot. c The main enzymes involved in m 6 A modification, created with Figdraw. d WB analysis of METTL3, METTL14, WTAP, FTO and ALKBH5 expression in MEFs (WT, Tsc1 −/− , Tsc2 −/− ) and HepG2 (si-NC, si- TSC1 , si- TSC2 ). e WB analysis of the expression of WTAP, METTL3 and p-P70S6 (Thr389) in MEFs (WT, Tsc1 −/− , Tsc2 −/− ) at 24 h post-treatment with 300 nM rapamycin or DMSO. f , g Detection of m 6 A levels in Tsc2 −/− MEFs (si-NC, si- Wtap- 1, si- Wtap- 2) using m 6 A quantification assay and m 6 A dot blot. h , i Detection of m 6 A levels in Tsc2 −/− MEFs (si-NC, si- Mettl3- 1, si- Mettl3- 2) using m 6 A quantification assay and m 6 A dot blot. Data are shown as the mean ± SD, n = 3, two-tailed unpaired Student’s t -test.

Article Snippet: The pcDNA3.1-myc- TSC1 and pcDNA3.1-Flag- TSC2 plasmids were preserved by our lab; pcDNA3.1- METTL3 -WT-3*HA was purchased from PPL (Jiangsu, China); pcDNA3.1- METTL3 -mut-3*HA were constructed in our Lab; pcDNA3.1- KDM5A -Flag purchased from Cyagen (Suzhou, China) for overexpression experiments.

Techniques: Dot Blot, Modification, Expressing, Two Tailed Test

Journal: Cell Death & Disease

Article Title: Uncomplexed-TSC1 deploys novel mTORC1-independent pathway to exacerbate the liver glycogen storage in TSC

doi: 10.1038/s41419-025-08161-3

Figure Lengend Snippet: a WB analysis of mTORC1 activity, METTL3 and WTAP protein levels in MEFs (WT, Tsc1 −/− , Tsc2 −/− , Tsc1/2 −/− ) and HepG2 (si-NC, si- TSC1 , si- TSC2 , si- TSC1 + TSC2 ). b Detection of METTL3 expression in WT MEFs and HepG2 with transient overexpression of TSC1 or TSC2 . c Schematic representation of the presence of TSC1, TSC2, and TSC complex forms in four types of MEFs, created using Figdraw. TSC1 knockout ( Tsc1 –/– MEFs) causes loss of TSC1 and increased expression of uncomplexed-TSC2, due to loss of TSC1 leads to reductions of TSC2 at the same time, only increased a part of expression of uncomplexed-TSC2. TSC2 knockout ( Tsc2 –/– MEFs) causes the loss of TSC2 and increased uncomplexed-TSC1 level, double knockout ( Tsc1/2 –/– MEFs) caused loss of TSC1 and TSC2. d , e Analysis of TSC1, TSC2 and TSC complex in samples prepared from WT MEFs and HepG2 post s ucrose density-gradient centrifugation using WB (fractions 1 to 9 were arranged from top to bottom). f Analysis of mTORC1 activity in HepG2 with transient overexpression of TSC1 or TSC2 . g , h Analysis of glycogen levels in MEFs and HepG2 using PAS staining and the glycogen assay kit at 48 h post transient overexpression of TSC1 or TSC2 . Bar, 20 μm in ( g ). i , j Quantification of glycogen levels in MEFs (WT, Tsc1 −/− , Tsc2 −/− , Tsc1/2 −/− ) and HepG2 (si-NC, si- TSC1 , si- TSC2 , si- TSC1 + TSC2 ). Bar, 20 μm in ( i ). Data are shown as the mean ± SD, n = 3, two-tailed unpaired Student’s t -test.

Article Snippet: The pcDNA3.1-myc- TSC1 and pcDNA3.1-Flag- TSC2 plasmids were preserved by our lab; pcDNA3.1- METTL3 -WT-3*HA was purchased from PPL (Jiangsu, China); pcDNA3.1- METTL3 -mut-3*HA were constructed in our Lab; pcDNA3.1- KDM5A -Flag purchased from Cyagen (Suzhou, China) for overexpression experiments.

Techniques: Activity Assay, Expressing, Over Expression, Knock-Out, Double Knockout, Gradient Centrifugation, Staining, Two Tailed Test

Journal: Cell Death & Disease

Article Title: Uncomplexed-TSC1 deploys novel mTORC1-independent pathway to exacerbate the liver glycogen storage in TSC

doi: 10.1038/s41419-025-08161-3

Figure Lengend Snippet: a Analysis of Mettl3 mRNA expression in all MEFs using qRT-PCR. b Analysis of METTL3 mRNA expression in oe-NC, oe- TSC1 and oe- TSC2 HepG2 using qRT-PCR. c Venn diagram showing overlaps between differential genes of three RNA-seq with GO molecular function including chromatin remodeling. d WB analysis of KDM5A in HepG2 (si-NC, si- TSC1 , si- TSC2 , si- TSC1 + TSC2 ). e Analysis of KDM5A expression in MEFs (WT, Tsc1 −/− , Tsc 2 −/− , Tsc1/2 −/− ) at 24 h post treatment of 300 nM rapamycin or DMSO. f Analysis of KDM5A expression in oe-NC, oe- TSC1 and oe- TSC2 HepG2 using WB. g Detection of KDM5A and METTL3 in HepG2 at 48 h post transient overexpression or knockdown of KDM5A by transfection with overexpressing vector (oe- KDM5A ) or KDM5A -siRNA (si- KDM5A -1, si- KDM5A -2). h ChIP-PCR analysis of H3K4me3 modification within the METTL3 promoter in oe-NC and oe- KDM5A HepG2. i ChIP-PCR analysis of H3K4me3 modification within the METTL3 promoter in oe-NC, oe- TSC1 and oe- TSC2 HepG2. j ChIP-PCR analysis of KDM5A binding with METTL3 promoter in oe-NC, oe- TSC1 and oe- TSC2 HepG2. k ChIP-PCR analysis of TFs TBP, ETS1 and NRF1 binding with METTL3 promoter in oe-NC, oe- TSC1 and oe- TSC2 HepG2. l , m Detection of glycogen levels in HepG2 using PAS staining and the glycogen assay kit at 48 h post transfection with TSC1 overexpressing vector, TSC1 overexpressing vector + KDM5A overexpressing vector, or TSC1 overexpressing vector + KDM5A overexpressing vector + METTL3 overexpressing vector. Bar, 20 μm in ( l ). Data are shown as the mean ± SD, n = 3, two-tailed unpaired Student’s t -test.

Article Snippet: The pcDNA3.1-myc- TSC1 and pcDNA3.1-Flag- TSC2 plasmids were preserved by our lab; pcDNA3.1- METTL3 -WT-3*HA was purchased from PPL (Jiangsu, China); pcDNA3.1- METTL3 -mut-3*HA were constructed in our Lab; pcDNA3.1- KDM5A -Flag purchased from Cyagen (Suzhou, China) for overexpression experiments.

Techniques: Expressing, Quantitative RT-PCR, RNA Sequencing, Over Expression, Knockdown, Transfection, Plasmid Preparation, Modification, Binding Assay, Staining, Two Tailed Test

Journal: Cell Death & Disease

Article Title: Uncomplexed-TSC1 deploys novel mTORC1-independent pathway to exacerbate the liver glycogen storage in TSC

doi: 10.1038/s41419-025-08161-3

Figure Lengend Snippet: a WB analysis of METTL3 and GYS2 in Tsc2 −/− MEFs (si- M3 -1, si- M3 -2 ) . b WB analysis of METTL3 and GYS2 in oe-NC and oe- M3 HepG2. c WB analysis of GYS2 in HepG2 (si-NC, si- TSC1 , si- TSC2 , si- TSC1 + TSC2 ). d Analysis of GYS2 in MEFs (WT, Tsc1 −/− , Tsc 2 −/− , Tsc1/2 −/− ) at 24 h post-treatment with 300 nM rapamycin or DMSO using WB. e , f Detection of METTL3 and GYS2 in HepG2 at 48 h post the transfection of si- TSC2 or oe- TSC1 , and si- METTL3 . g M 6 A methylation detection of GYS2 in oe-NC and oe- TSC1 HepG2. h M 6 A methylation detection of GYS2 in oe-NC and oe- M3 HepG2. i Detection of the mRNA half-life of GYS2 in oe-NC and oe- TSC1 HepG2. j Detection of the half-life of mRNA GYS2 in oe-NC and oe- M3 HepG2. k Detection of IGF2BP2 and GYS2 in HepG2 at 48 h post the transfection of si- TSC2 and si- IGF2BP2 . l Detection of the mRNA half-life of GYS2 in si-NC and si- IGF2BP2 . m WB quantification of knockdown efficacy of GYS2 in HepG2 (si- GYS2 -1, si- GYS2 -2 targeting human GYS2 ). n , o Analysis of glycogen levels in HepG2 at 48 h post transfection of oe- M3 and si- GYS2 ; Bar, 20 μm in ( n ). Data are shown as the mean ± SD, n = 3, two-tailed unpaired Student’s t -test.

Article Snippet: The pcDNA3.1-myc- TSC1 and pcDNA3.1-Flag- TSC2 plasmids were preserved by our lab; pcDNA3.1- METTL3 -WT-3*HA was purchased from PPL (Jiangsu, China); pcDNA3.1- METTL3 -mut-3*HA were constructed in our Lab; pcDNA3.1- KDM5A -Flag purchased from Cyagen (Suzhou, China) for overexpression experiments.

Techniques: Transfection, Methylation, Knockdown, Two Tailed Test

Journal: Cell Death & Disease

Article Title: Uncomplexed-TSC1 deploys novel mTORC1-independent pathway to exacerbate the liver glycogen storage in TSC

doi: 10.1038/s41419-025-08161-3

Figure Lengend Snippet: a Representative images of PAS staining in liver tissues isolated from mice aged 12 to 13 months (WT, Tsc1 +/− , Tsc2 +/− and Tsc1 +/− / Tsc2 +/− ). n = 6 for each genotype; each group contains mice from three different litters. Bar, 100 μm in ( a ). b Representative images of PAS staining from tumor of nude mice (WT, Tsc1 −/− , Tsc2 −/− ). n = 5 for each genotype. Bar, 50 μm in ( b ). c Representative images of METTL3 IHC staining in liver tissue isolated from mice aged 13 to 14 months and METTL3 IHC scores of all groups. n = 6 for each genotype; each group contains mice from three different litters. Bar, 50 μm in ( c ). d Representative images of GYS2 IHC staining in liver tissue isolated from mice aged 13 to 14 months and GYS2 IHC scores of all groups. n = 6 for each genotype; each group contains mice from three different litters. Bar, 50 μm in ( d ). e In vivo fluorescence imaging of all mice injected rAAV8, n = 5 for each group; each group contains mice from three different litters. f Representative images of HE staining, SMA IHC staining, CD31 IHC staining and PAS staining in liver tissues isolated from mice injected rAAV8 after 6 weeks. n = 5 for each group; each group contains mice from three different litters. Bar, 100 μm in ( f ). g Tumor sizes in each group at 20 days post drug treatment, n = 5 for each group. h The tumor volume in each group were monitored every 5 days starting from 1 day post drug treatment. n = 5 for each group. i The tumor weight in each group 20 days after drug treatment. j Representative images of PAS staining from tumor 20 days post drug treatment of nude mice. n = 5 for each group. Bar, 50 μm in j . k Schematic of the proposed mechanism, created by Figdraw. Data are shown as the mean ± SD, n = 5 ~ 6, two-way RM ANOVA and Tukey’s multiple comparisons test for ( h ), two-tailed unpaired Student’s t -test for others.

Article Snippet: The pcDNA3.1-myc- TSC1 and pcDNA3.1-Flag- TSC2 plasmids were preserved by our lab; pcDNA3.1- METTL3 -WT-3*HA was purchased from PPL (Jiangsu, China); pcDNA3.1- METTL3 -mut-3*HA were constructed in our Lab; pcDNA3.1- KDM5A -Flag purchased from Cyagen (Suzhou, China) for overexpression experiments.

Techniques: Staining, Isolation, Immunohistochemistry, In Vivo, Fluorescence, Imaging, Injection, Two Tailed Test