Journal: Nucleic acids research

Article Title: Distinct functions for the paralogous RBM41 and U11/U12-65K proteins in the minor spliceosome.

doi: 10.1093/nar/gkae070

Figure Lengend Snippet: Figure 2. RBM41 interacts with the U12 and U6atac snRNAs in vitro . ( A ) Consensus RNA motifs bound by RBM41 in vitro and matching sequences in the U12 and U6atac snRNAs. The consensus motif (obtained from ENCODE database ( 81 ), entry ENCSR637HFY) determined by Ray et al. ( 58 ) using the RNAcompete method is shown. ( B ) RNA hairpins used in EMSA experiments and their location in the U12 and U6atac snRNAs. ( C ) EMSA analysis of RBM41 and U11 / U12-65K RRM binding to U12 (top panel) and U6atac snRNA (bottom panel) hairpins. EMSA was carried out using recombinant RBM41 RRM (residues 267–413) or 65K C-terminal RRM (residues 380–517) and 32 P-labeled U12, U6atac or negative control RNA hairpins shown in panel B. ( D ) Binding curves and dissociation constants for the interaction of RBM41 and 65K RRMs with U12 and U6atac hairpins. The inset shows a low protein concentration range (0–10 μM) of the same binding curves.

Article Snippet: For BioID cell line construction, full-length RBM41 (1–413), RBM41 N-terminal fragment (1–258), RBM41 Cterminal fragment (259–413) and full-length 65K were cloned into MAC-tag-N vector (Addgene #108078) using Gateway cloning as described ( 34 ).

Techniques: In Vitro, Binding Assay, Recombinant, Labeling, Negative Control, Protein Concentration

Journal: Nucleic acids research

Article Title: Distinct functions for the paralogous RBM41 and U11/U12-65K proteins in the minor spliceosome.

doi: 10.1093/nar/gkae070

Figure Lengend Snippet: Figure 3. RBM41 specifically associates with minor spliceosomal snRNPs. ( A ) RNA immunoprecipitation with V5-tagged RBM41 and 65K. V5-RBM41 or V5-65K expression vector or empty vector were transfected into HEK293 cells. 24 h later, RNA immunoprecipitation with anti-V5 antibody or control antibody was carried out in native conditions and co-immunoprecipitated RNA analyzed by northern blot using the indicated probes. ( B ) RNA immunoprecipitation with endogenous RBM41. RIP was carried out in native conditions in either HeLa nuclear extract (left) or HEK293 total lysate (right) using an antibody against endogenous RBM41 or control antibody. ( C ) V5-RBM41 constructs used for RNA immunoprecipitation in panel D. ( D ) Effect of truncations and RRM mutations on the snRNP association of RBM41.V5-tagged RBM41 constructs shown in C were transfected into HEK293 cells and RNA immunoprecipitation carried out using anti-V5 or control antibody.

Article Snippet: For BioID cell line construction, full-length RBM41 (1–413), RBM41 N-terminal fragment (1–258), RBM41 Cterminal fragment (259–413) and full-length 65K were cloned into MAC-tag-N vector (Addgene #108078) using Gateway cloning as described ( 34 ).

Techniques: RNA Immunoprecipitation, Expressing, Plasmid Preparation, Transfection, Control, Immunoprecipitation, Northern Blot, Construct

Journal: Nucleic acids research

Article Title: Distinct functions for the paralogous RBM41 and U11/U12-65K proteins in the minor spliceosome.

doi: 10.1093/nar/gkae070

Figure Lengend Snippet: Figure 4. RBM41 and U11 / U12-65K partition into distinct snRNP comple x es. ( A ) Glycerol gradient analysis of RBM41 and U11 / U12-65K in HeLa nuclear extract. Nuclear extract was loaded on top of a 10–30% glycerol gradient. After ultracentrifugation, the gradient was fractionated, protein and RNA isolated and analyzed by western and northern blot using the antibodies and probes indicated on the left. Location of the U11, U12 and U6atac mono-snRNPs, U11 / U12 di-snRNP and U4at ac / U6at ac di-snRNP are inferred based on the snRNA profiles. ( B ) Domain str uct ures of MAC-tagged RBM41 and 65K constructs used for BioID. N-terminal MAC tag is not drawn to scale. ( C ) Spectral count fold changes for U11 / U12 di-snRNP proteins in BioID datasets. ( D ) Immunoprecipitation of U11 and U12 snRNAs by anti-31K, anti-48K, anti-59K, anti-65K and anti-Sm antibodies in HEK293 total lysate f ollo w ed b y Northern blot detection of the U11 and U12 snRNAs.

Article Snippet: For BioID cell line construction, full-length RBM41 (1–413), RBM41 N-terminal fragment (1–258), RBM41 Cterminal fragment (259–413) and full-length 65K were cloned into MAC-tag-N vector (Addgene #108078) using Gateway cloning as described ( 34 ).

Techniques: Isolation, Western Blot, Northern Blot, Construct, Immunoprecipitation

Journal: Nucleic acids research

Article Title: Distinct functions for the paralogous RBM41 and U11/U12-65K proteins in the minor spliceosome.

doi: 10.1093/nar/gkae070

Figure Lengend Snippet: Figure 5. RBM41 interacts with DHX8 and localizes to Cajal bodies. ( A ) Spectral counts for DHX8 in RBM41 and U11 / U12-65K BioID datasets. ( B ) Immunoprecipitation with anti-V5 or control antibody f ollo w ed b y w estern blot in Flp-In™T-REx™293 cell lines e xpressing V5-RBM41 or V5-65K. T he asterisk indicates a non-specific band detected in both control and anti-31K IPs and likely represents cross-reaction of the anti-rabbit secondary antibody with light chain from the IP antibody. ( C ) RNA immunoprecipitation with e x ogenously e xpressed V5-tagged proteins f ollo w ed b y R T-PCR. T he indicated pCI-neo constructs for expressing V5-tagged proteins or empty pCI-neo vectors were transfected into HEK293 cells. 24 h later, RIP was carried out using anti-V5 antibody and RNA extracted from the beads analyzed by RT-PCR. Amplification across the branch junction was used to detect U2- and U12-type intron lariats and lariat intermediates from the f ollo wing introns: SPCS2 introns 3–4 (U12) and 2–3 (U2), SUDS3 introns 7–8 (U12) and 9–10 (U2), WDR11 introns 28–29 (U12) and 27–28 (U2). ( D ) RNA immunoprecipitation with endogenous RBM41 in HEK293 cells f ollo w ed b y R T-PCR. ( E ) Spectral counts for coilin in RBM41 and U11 / U12-65K BioID datasets. ( F ) Anti-RBM41 immunofluorescence in HEK293 cells transfected with a vector f or e xpressing coilin-GFP.

Article Snippet: For BioID cell line construction, full-length RBM41 (1–413), RBM41 N-terminal fragment (1–258), RBM41 Cterminal fragment (259–413) and full-length 65K were cloned into MAC-tag-N vector (Addgene #108078) using Gateway cloning as described ( 34 ).

Techniques: Immunoprecipitation, Control, RNA Immunoprecipitation, Construct, Expressing, Transfection, Reverse Transcription Polymerase Chain Reaction, Amplification, Immunofluorescence, Plasmid Preparation

Journal: Nucleic acids research

Article Title: Distinct functions for the paralogous RBM41 and U11/U12-65K proteins in the minor spliceosome.

doi: 10.1093/nar/gkae070

Figure Lengend Snippet: Figure 6. RBM41 knockout influences the splicing of U12-type introns. ( A ) Western blot analysis of RBM41 knockout and matching control cell lines used in the RNAseq analysis. ( B ) Comparison of the statistically significant (Whippet Probability > 0.9) alternative splicing events in the genes containing only U2-type introns and e v ents either within or near proximity (immediate up- or downstream exons and introns) of the U12-type introns. AA - alternative acceptor, AD – alternative donor, CE – core e x on. ( C ) R epresentativ e sashimi plots showing Intron retention ( NOL11 ), Alternative U12-type 3 ′ ss choice ( THOC2 ) and loss of both exon skipping and alternative U12-type 3 ′ ss usage in RBM41 knockout cells ( TCTN1 ). The percentages refer to the intron retention le v els ( NOL11 ), the alternative 3 ′ splice usage le v els ( TH OC2 ) or e x on skipping le v els ( TCTN1 ) as indicated by the arches in the Sashimi plot. ( D ) Validation of the THOC2 and TCTN1 alternative splicing changes using a set of three independent RBM41 knockout cell lines and their matching controls.

Article Snippet: For BioID cell line construction, full-length RBM41 (1–413), RBM41 N-terminal fragment (1–258), RBM41 Cterminal fragment (259–413) and full-length 65K were cloned into MAC-tag-N vector (Addgene #108078) using Gateway cloning as described ( 34 ).

Techniques: Knock-Out, Western Blot, Control, Comparison, Alternative Splicing, Biomarker Discovery

Journal: International Journal of Molecular Sciences

Article Title: Synaptic Alterations in a Transgenic Model of Tuberous Sclerosis Complex: Relevance to Autism Spectrum Disorders

doi: 10.3390/ijms221810058

Figure Lengend Snippet: The effect of Tsc2 haploinsufficiency on body weight in mice. ( a ) The protein level of Tsc2 in the brain cortex of Tsc2 +/+ and Tsc2 +/− two-month-old male mice was analysed by Western blotting. Typical blots are presented. ( b ) Densitometric analysis of immunoreactivity of Tsc2 in the brain cortex. Data were normalised to GAPDH. n = 3. ( c , d ) The effect of Tsc2 insufficiency on the body weight and brain weight. ( e ) Example photographs of the brains of Tsc2 +/+ and Tsc2 +/− two-month-old male mice. n = 30. Data represent the mean values ± SEMs. ** p < 0.01 vs. wild-type animals, as determined by Student’s t -test.

Article Snippet: Rabbit monoclonal anti-Tsc2 antibody was purchased from Novus Biologicals (Centennial, CO, USA).

Techniques: Western Blot

Journal: International Journal of Molecular Sciences

Article Title: Synaptic Alterations in a Transgenic Model of Tuberous Sclerosis Complex: Relevance to Autism Spectrum Disorders

doi: 10.3390/ijms221810058

Figure Lengend Snippet: The effect of Tsc2 haploinsufficiency on the mRNA levels for synaptic proteins.

Article Snippet: Rabbit monoclonal anti-Tsc2 antibody was purchased from Novus Biologicals (Centennial, CO, USA).

Techniques:

Journal: International Journal of Molecular Sciences

Article Title: Synaptic Alterations in a Transgenic Model of Tuberous Sclerosis Complex: Relevance to Autism Spectrum Disorders

doi: 10.3390/ijms221810058

Figure Lengend Snippet: The effect of Tsc2 haploinsufficiency on levels of synaptic proteins. The immunoreactivity of synaptic proteins in the hippocampus (hipp.), cortex (ctx.), and cerebellum (cer.) was determined by using Western blotting. Representative pictures are presented. GAPDH was used as a loading control. n.d.—not detected; densitometric analysis is presented in .

Article Snippet: Rabbit monoclonal anti-Tsc2 antibody was purchased from Novus Biologicals (Centennial, CO, USA).

Techniques: Western Blot, Control

Journal: International Journal of Molecular Sciences

Article Title: Synaptic Alterations in a Transgenic Model of Tuberous Sclerosis Complex: Relevance to Autism Spectrum Disorders

doi: 10.3390/ijms221810058

Figure Lengend Snippet: The effect of Tsc2 haploinsufficiency on levels of synaptic proteins—densitometric analysis.

Article Snippet: Rabbit monoclonal anti-Tsc2 antibody was purchased from Novus Biologicals (Centennial, CO, USA).

Techniques:

Journal: International Journal of Molecular Sciences

Article Title: Synaptic Alterations in a Transgenic Model of Tuberous Sclerosis Complex: Relevance to Autism Spectrum Disorders

doi: 10.3390/ijms221810058

Figure Lengend Snippet: Pathological alterations of neuronal ultrastructure found in the brains of Tsc2 +/− mice.

Article Snippet: Rabbit monoclonal anti-Tsc2 antibody was purchased from Novus Biologicals (Centennial, CO, USA).

Techniques:

Journal: International Journal of Molecular Sciences

Article Title: Synaptic Alterations in a Transgenic Model of Tuberous Sclerosis Complex: Relevance to Autism Spectrum Disorders

doi: 10.3390/ijms221810058

Figure Lengend Snippet: The effect of Tsc2 haploinsufficiency on the ultrastructure of neuronal cells in the CA1/CA2 ( a , b ). and CA2/CA3 ( c , d ) regions of the hippocampus, brain cortex ( e , f ), and cerebellum ( g , h ). ( a , c , e , g )—control group Tsc2 +/+ ; ( b , d , f , h )—Tsc2 +/− group. Typical electronograms at low magnification are presented.

Article Snippet: Rabbit monoclonal anti-Tsc2 antibody was purchased from Novus Biologicals (Centennial, CO, USA).

Techniques: Control

Journal: International Journal of Molecular Sciences

Article Title: Synaptic Alterations in a Transgenic Model of Tuberous Sclerosis Complex: Relevance to Autism Spectrum Disorders

doi: 10.3390/ijms221810058

Figure Lengend Snippet: The effect of Tsc2 haploinsufficiency on the number of synaptic vesicles in ( a ) cerebral cortex, ( b ) cerebellum, ( c ) CA1/CA2 region of the hippocampus, and ( d ) CA2/CA3 region of the hippocampus. The quantitative analysis of microphotographs was performed for 5 animals in each group; 10 random synapses from each animal were taken for analysis ( n = 50). Data were not normally distributed and are presented as medians with interquartile ranges, minimums, and maximums. * p ˂ 0.05 vs. wild-type animals, as determined using the Mann–Whitney test.

Article Snippet: Rabbit monoclonal anti-Tsc2 antibody was purchased from Novus Biologicals (Centennial, CO, USA).

Techniques: MANN-WHITNEY

Journal: International Journal of Molecular Sciences

Article Title: Synaptic Alterations in a Transgenic Model of Tuberous Sclerosis Complex: Relevance to Autism Spectrum Disorders

doi: 10.3390/ijms221810058

Figure Lengend Snippet: The effect of Tsc2 haploinsufficiency in mice on behaviour (exploratory activity). The exploratory activity of male mice was analysed in an open-field test. ( a ) The total distance travelled by animals, ( b ) the number of entries to the central zone, ( c ) the time spent in the central zone, ( d ) the number of defecation events, ( e ) the number of rearing events, ( f ) the number of climbing events, ( g ) the number of grooming events, and ( h ) the total time spent on self-grooming. Data ( a – c , e , f ) represent the mean values ± SEMs from n = 30 independent experiments. Data ( d , g , h ) not normally distributed are presented as medians with interquartile ranges, minimums, and maximums ( n = 30). Each data point is from a separate animal.

Article Snippet: Rabbit monoclonal anti-Tsc2 antibody was purchased from Novus Biologicals (Centennial, CO, USA).

Techniques: Activity Assay

Journal: International Journal of Molecular Sciences

Article Title: Synaptic Alterations in a Transgenic Model of Tuberous Sclerosis Complex: Relevance to Autism Spectrum Disorders

doi: 10.3390/ijms221810058

Figure Lengend Snippet: The effect of Tsc2 haploinsufficiency in mice on behaviour (sociability). ( a ) The time spent by the tested animal in the chamber; ( b ) the time spent by the tested animal on the exploration of cages. Data represent the mean values ± SEMs from n = 20 independent experiments. Each data point is from a separate animal.

Article Snippet: Rabbit monoclonal anti-Tsc2 antibody was purchased from Novus Biologicals (Centennial, CO, USA).

Techniques:

Journal: International Journal of Molecular Sciences

Article Title: Synaptic Alterations in a Transgenic Model of Tuberous Sclerosis Complex: Relevance to Autism Spectrum Disorders

doi: 10.3390/ijms221810058

Figure Lengend Snippet: The effect of Tsc2 haploinsufficiency in mice on behaviour (cognitive function). The memory of male mice was analysed in a novel object recognition test. Index of discrimination (ID) was calculated, as described in the Methods section. Presented results are means ± SEMs from n = 10 animals in each group. * p < 0.05, compared to control, as determined using Student’s t -test. Each data point is from a separate animal.

Article Snippet: Rabbit monoclonal anti-Tsc2 antibody was purchased from Novus Biologicals (Centennial, CO, USA).

Techniques: Control

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

Journal: iScience

Article Title: Growth factor-independent mTORC1 signaling promotes primary cilia length via suppression of autophagy

doi: 10.1016/j.isci.2025.114204

Figure Lengend Snippet: Genetic perturbation of mTORC1 signaling demonstrates that it promotes the elongation of primary cilia (A and B) Immunoblot (A) and primary cilia length (B) in doxycycline-inducible shScrambled and shMTOR RPE1 cells treated with doxycycline (0.5 μg/mL) for 7 days. (C) Immunoblot of isogenic clonal RPE1 cell lines with sgRNAs targeting TSC1, TSC2, or TBC1D7. Cells were serum-starved overnight. TSC1 and TSC2 are indicated by black arrowheads. (D) Primary cilia length in RPE1 lines used in (C). (E) Immunoblot of parental, sgTSC2, and human TSC2-rescued sgTSC2 RPE1 cells treated with Torin1 for 1 h. (F) Primary cilia length in RPE1 cell lines used in (E) following Torin1 treatment. (G) Immunoblot of Tsc2 +/+ , Tsc2 −/− , and human TSC2-rescued Tsc2 −/− MEFs after overnight serum starvation. (H) Primary cilia length in MEF cell lines used in (G). (I) Immunoblot of parental and sgNPRL2 RPE1 cells treated with overnight amino acid starvation followed by 1 h of amino acid stimulation. (J) Primary cilia length in parental and sgNPRL2 RPE1 cells treated with DMSO, Torin1, leucine deprivation, or complete amino acid deprivation. All treatments for primary cilia assessment were applied during the final 24 h in serum-free conditions (48 h total): DMSO (0.1%), rapamycin (20 nM), and Torin1 (250 nM). Data are represented as mean ± standard deviation (SD). Statistical analysis was performed using two-way ANOVA followed by Šídák’s multiple comparisons test. The significance threshold (α) was set at 0.05, and all p values are reported.

Article Snippet: Flag-tagged human TSC2 (Flag-TSC2) cDNA from pcDNA3 Flag TSC2 (Addgene; 14129) was PCR-amplified using Phusion High-Fidelity DNA Polymerase (Thermo Scientific; F531S) with primers containing sequences homologous to the insertion site in pMXS-IRES-Blast, a retroviral vector (Cell Biolabs, Inc; RTV-106): 5’-CTAGCTAGTTAATTAAatggactacaaagacgatgacgataaagc-3’ 5’-CGCCGGCCCTCGAGtcacacaaactcggtgaagtcctcc-3’ (Uppercase: homologous to pMXS-IRES-Blast; lowercase: homologous to Flag-TSC2).

Techniques: Western Blot, Standard Deviation

Journal: iScience

Article Title: Growth factor-independent mTORC1 signaling promotes primary cilia length via suppression of autophagy

doi: 10.1016/j.isci.2025.114204

Figure Lengend Snippet: mTORC1 signaling promotes elongation of primary cilia via autophagy suppression (A) Schematic diagram showing the generation of the GFP-LC3B-RFP autophagy reporter RPE1 cell line and flow cytometry analysis used to assess autophagic flux. (B and C) Flow cytometric analysis (B) and quantification of autophagic flux (C) in RPE1 cells treated with FBS (10%), bafilomycin A1, rapamycin, Torin1, or leucine deprivation for the indicated times. (D and E) Immunoblot (D) and primary cilia length (E) in RPE1 cells treated with DMSO, rapamycin, chloroquine, or bafilomycin A1. (F and G) Immunoblot (F) and primary cilia length (G) in RPE1 cells treated with DMSO, chloroquine, or bafilomycin A1, alone or in combination with rapamycin. (H) Primary cilia length in RPE1 cells treated with DMSO, ULK1/2 inhibitor (SBP-7455), or vacuoar protein sorting 34, class III PI 3-kinase (VPS34) inhibitor (VPS34-IN1). (I) Immunoblot of parental and sgATG7 RPE1 cells treated with bafilomycin A1 for the final 3 h under overnight serum starvation. p62 is indicated by a black arrowhead. (J) Primary cilia length in RPE1 cells used in (I) following treatment with DMSO, Torin1, or bafilomycin A1. (K) Immunoblot of parental and sgTFEB RPE1 cells (left) and primary cilia length in parental or sgTFEB RPE1 cells treated with DMSO, Torin1, or bafilomycin A1 (right). (L) Immunoblot of parental, sgTSC2, and human TSC2-rescued sgTSC2 RPE1 cells treated with bafilomycin A1 for the final 3 h under overnight serum starvation. (M) Primary cilia length in RPE1 cells used in (L) following treatment with DMSO or bafilomycin A1. All treatments were applied during the final 24 h in serum-free conditions (48 h total), unless otherwise specified: DMSO (0.1%), rapamycin (20 nM), Torin1 (250 nM), chloroquine (50 μM), bafilomycin A1 (100 nM), SBP-7455 (10 μM), and VPS34-IN1 (10 μM). Data are represented as mean ± standard deviation (SD). Statistical analysis was performed using two-way ANOVA followed by Šídák’s multiple comparisons test. The significance threshold (α) was set at 0.05, and all p values are reported.

Article Snippet: Flag-tagged human TSC2 (Flag-TSC2) cDNA from pcDNA3 Flag TSC2 (Addgene; 14129) was PCR-amplified using Phusion High-Fidelity DNA Polymerase (Thermo Scientific; F531S) with primers containing sequences homologous to the insertion site in pMXS-IRES-Blast, a retroviral vector (Cell Biolabs, Inc; RTV-106): 5’-CTAGCTAGTTAATTAAatggactacaaagacgatgacgataaagc-3’ 5’-CGCCGGCCCTCGAGtcacacaaactcggtgaagtcctcc-3’ (Uppercase: homologous to pMXS-IRES-Blast; lowercase: homologous to Flag-TSC2).

Techniques: Flow Cytometry, Western Blot, Standard Deviation