human tsc2 flag tsc2 cdna  (Addgene inc)

Journal: bioRxiv

Article Title: Growth Factor-Independent mTORC1 Signaling Promotes Primary Cilia Length via Suppression of Autophagy

doi: 10.1101/2025.05.07.652626

Figure Lengend Snippet: (A–B) (A) Immunoblot and (B) primary cilia length in doxycycline-inducible shMTOR RPE1 cells treated with doxycycline (0.5 µg/mL) for 7 days. (C) Immunoblot of clonal RPE1 cell lines with sgRNAs targeting TSC1, TSC2, or TBC1D7. Cells were serum-starved overnight. (D) Primary cilia length in RPE1 lines used in (C). TSC1 and TSC2 are indicated by black arrowheads. (E) Immunoblot of sgTSC2 RPE1 cells, human TSC2-rescued sgTSC2 cells, and parental controls treated with Torin1 for 1 hour in growth media with serum. (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 hour 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 hours in serum-free conditions (48 h total): DMSO (0.1%), rapamycin (20 nM), Torin1 (250 nM). 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

Journal: bioRxiv

Article Title: Growth Factor-Independent mTORC1 Signaling Promotes Primary Cilia Length via Suppression of Autophagy

doi: 10.1101/2025.05.07.652626

Figure Lengend Snippet: (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–C) (B) Flow cytometric analysis and (C) quantification of autophagic flux in RPE1 cells treated with FBS (10%), bafilomycin A1, rapamycin, Torin1, or leucine deprivation for the indicated times. (D) Immunoblot and (E) primary cilia length in RPE1 cells treated with DMSO, rapamycin, chloroquine, or bafilomycin A1. (F–G) (F) Immunoblot and (G) primary cilia length 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 VPS34 inhibitor (VPS34-IN1). (I) Immunoblot of parental and sgATG7 RPE1 cells treated with bafilomycin A1 for the final 3 hours under overnight serum starvation. (J) Primary cilia length in RPE1 cells used in (I) following treatment with DMSO, Torin1, or bafilomycin A1. p62 is indicated by a black arrowhead. (K) Primary cilia length in parental, sgULK1, or sgBECN1 RPE1 cells. (L) Immunoblot of parental, sgTSC2, and human TSC2-rescued sgTSC2 RPE1 cells treated with bafilomycin A1 for the final 3 hours 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 hours 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), VPS34-IN1 (10 µM). 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

Journal: Scientific Reports

Article Title: TSC complex decrease the expression of mTOR by regulated miR-199b-3p

doi: 10.1038/s41598-025-85706-8

Figure Lengend Snippet: The information of the patients involved in this article.

Article Snippet: The antibodies involved in this study were as belows: TSC1 monoclonal antibody (Stanta Cruz, Cat.No.sc-377386), TSC2 monoclonal mouse antibody (Stanta Cruz, Cat. No. sc-271314), TSC2 monoclonal rabbit antibody (Cell Signaling.

Techniques: Mutagenesis

Journal: Scientific Reports

Article Title: TSC complex decrease the expression of mTOR by regulated miR-199b-3p

doi: 10.1038/s41598-025-85706-8

Figure Lengend Snippet: Expression of TSC2 and phenotypes of the fibroblasts with or without TSC2-Q371fs, the WT was present as the average of all healthy volunteers. ( A ) Linear schematic of the protein structure of TSC2-WT, TSC2-Q371fs and the location of the primes and the qRT-PCR results, relative expression levels were normalized with GAPDH . ( B ) Immunofluorescence analysis using TSC2 (red) antibody and DAPI (blue) in Bar 15 μm. ( C ) The western blot of the endogenous TSC2 and the statistics, the originals gels can be seen in the supplementary figure . ( D ) Proliferation rate of fibroblasts. ( E ) Apoptosis assay of fibroblasts and the statistics.

Article Snippet: The antibodies involved in this study were as belows: TSC1 monoclonal antibody (Stanta Cruz, Cat.No.sc-377386), TSC2 monoclonal mouse antibody (Stanta Cruz, Cat. No. sc-271314), TSC2 monoclonal rabbit antibody (Cell Signaling.

Techniques: Expressing, Quantitative RT-PCR, Immunofluorescence, Western Blot, Apoptosis Assay

Journal: Scientific Reports

Article Title: TSC complex decrease the expression of mTOR by regulated miR-199b-3p

doi: 10.1038/s41598-025-85706-8

Figure Lengend Snippet: Functional analysis of the variations ( A ) Quantification of mRNA level using qRT-PCR, the WT represents the mixture of the equal amount total RNA from the three healthy fibroblasts. Relative expression levels were normalized with GAPDH . ( B ) Evaluation of the rescue experiment by western blot (the WT group was the WT2). the originals gels can be seen in the supplementary figure S2. ( C ) The co-ip experiment of TSC1-WT and TSC1-N837fs. The originals gels can be seen in the supplementary figure S3. ( D ) The measurement of TSC complex using non-denaturing PAGE. The originals gels can be seen in the supplementary figure S3 (E) The co-ip experiment of TSC2-WT and TSC2-Q371fs. The originals gels can be seen in the supplementary figure S4 ( F ) The measurement of TSC complex using non-denaturing PAGE. The originals gels can be seen in the supplementary figure S4.

Article Snippet: The antibodies involved in this study were as belows: TSC1 monoclonal antibody (Stanta Cruz, Cat.No.sc-377386), TSC2 monoclonal mouse antibody (Stanta Cruz, Cat. No. sc-271314), TSC2 monoclonal rabbit antibody (Cell Signaling.

Techniques: Functional Assay, Quantitative RT-PCR, Expressing, Western Blot, Co-Immunoprecipitation Assay

Journal: Scientific Reports

Article Title: TSC complex decrease the expression of mTOR by regulated miR-199b-3p

doi: 10.1038/s41598-025-85706-8

Figure Lengend Snippet: TSC complex regulates the endogenous content of miR199b-3p, * p < 0.05, ** p < 0.01,*** p < 0.001 comparison with WT. ( A ) The efficiency of knockdown TSC1 in shTSC1-HEK293T cell line. The originals gels can be seen in the supplementary figure S6. ( B ) The expression of miR-199b-3p in shTSC1-HEK293T cells and overexpression TSC1-WT or TSC1-N837fs in HEK293T cell line. ( C ) Quantitative analysis of mTOR in shTSC1-HEK293T cell line and overexpression TSC1-WT or TSC1 N837fs in HEK293T cell line using western blot (low panel) and the statistics (up panel). The originals gels can be seen in the supplementary figure S6. (D) The efficiency of knockdown TSC2 in shTSC2-HEK293T cell line. The originals gels can be seen in the supplementary figure S6. ( E ) The expression of miR-199b-3p in shTSC2-HEK293T cell line and overexpression TSC2-WT or TSC2- Q371fs in HEK293T cell line. ( F ) Quantitative analysis of mTOR in shTSC2-HEK293T cell line (low panel) and overexpression TSC2-WT or TSC2- Q371fs in HEK293T cell line using Western blot (low panel) and the statistics analysis (up panel). The originals gels can be seen in the supplementary figure S6.

Article Snippet: The antibodies involved in this study were as belows: TSC1 monoclonal antibody (Stanta Cruz, Cat.No.sc-377386), TSC2 monoclonal mouse antibody (Stanta Cruz, Cat. No. sc-271314), TSC2 monoclonal rabbit antibody (Cell Signaling.

Techniques: Comparison, Knockdown, Expressing, Over Expression, Western Blot

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: Thoracic Cancer

Article Title: Tumor‐derived exosomal miR ‐148b‐3p mediates M2 macrophage polarization via TSC2 / mTORC1 to promote breast cancer migration and invasion

doi: 10.1111/1759-7714.14891

Figure Lengend Snippet: miR‐148b‐3p is upregulated in breast cancer cell‐derived exosomes. (a) Heatmap of differential miRNA expression between MDA‐MB‐231‐exo and MCF‐10A‐exo based on GEO Dataset (GSE50429, FDR corrected p ‐value <0.05). (b) Venn diagram of upregulated miRNAs from GSE50429 and the target miRNAs of TSC2 predicted by Targetscan 7.1 software, which was calculated using Venny 2.1 software. (c) Expression levels of four potential upregulated miRNAs in exosomes derived from MDA‐MB‐231 were evaluated by qRT‐PCR. (d, e) Correlations between miR‐148b‐3p expression levels and lymph node metastasis and tumor stage in primary BRCA samples based on TCGA dataset from UALCAN. (f) The Kaplan–Meier survival curves of BC patients are shown according to the expression levels of serum exosomal miR‐148b‐3p ( p = 0.0017 by log‐rank analysis). (g) qRT‐PCR was utilized to evaluate the miR‐148b‐3p expression in the conditioned medium of MDA‐MB‐231 cells pretreated with control medium or RNase A (2 mg/mL) alone or in combination with Triton X‐100 (0.1%) for 0.5 h. (h, i) qRT‐PCR was utilized to detect miR‐148b‐3p expression in the conditioned medium of MDA‐MB‐231 cells pretreated with GW4869 or depleted exosomes by ultracentrifugation. The Student's t ‐test was adopted to analyze the statistical significance of the difference between two groups, and one‐way ANOVA test was utilized to analyze multiple groups (* p < 0.05, ** p < 0.01, *** p < 0.001).

Article Snippet: Transfection siRNA against human ACO1, SFRS13A and TSC2 were purchased from ThermoFisher.

Techniques: Derivative Assay, Expressing, Software, Quantitative RT-PCR, Control

Journal: Thoracic Cancer

Article Title: Tumor‐derived exosomal miR ‐148b‐3p mediates M2 macrophage polarization via TSC2 / mTORC1 to promote breast cancer migration and invasion

doi: 10.1111/1759-7714.14891

Figure Lengend Snippet: TSC2 is the direct target of miR‐148b‐3p in macrophages. (a, b) qRT‐PCR and WB assays were carried out to evaluate TSC2 expression in macrophages after incubated with ectopic miR‐148b‐3p mimic or inhibitor. (c, d) qRT‐PCR and western blot assays were done to determine TSC2 expression in macrophages after incubated with TSC2‐overexpression plasmids. “TSC2‐OE” refers to “macrophages transfected with TSC2‐overexpression plasmids”. (e) Sequencing of the wild‐type and mutant target sites in 3′UTR of TSC2 mRNA. (f) Luciferase reporter activity was measured to verify the target association between miR‐148b‐3p and TSC2. The student's t ‐test was adopted to analyze the statistical significance of the difference between two groups, and one‐way ANOVA test was utilized to analyze multiple groups (* p < 0.05, ** p < 0.01, *** p < 0.001).

Article Snippet: Transfection siRNA against human ACO1, SFRS13A and TSC2 were purchased from ThermoFisher.

Techniques: Quantitative RT-PCR, Expressing, Incubation, Western Blot, Over Expression, Transfection, Sequencing, Mutagenesis, Luciferase, Activity Assay

Journal: Thoracic Cancer

Article Title: Tumor‐derived exosomal miR ‐148b‐3p mediates M2 macrophage polarization via TSC2 / mTORC1 to promote breast cancer migration and invasion

doi: 10.1111/1759-7714.14891

Figure Lengend Snippet: Exosomal miR‐148b‐3p regulates macrophage polarization by inhibiting TSC2, thus promoting breast cancer migration and invasion. (a) qRT‐PCR was used to evaluate M2 markers (CD163, IL‐10 and arginase‐1) expression in macrophages transfected with small interfering RNAs (si‐TSC2) for downregulating TSC2 expression. “M + si‐TSC2” refers to “macrophages transfected with si‐TSC2”. (b) The identical method used in (a) was applied to macrophages. The different conditioned macrophage supernatants were harvested to detect the release of IL‐10 and TNF‐α by ELISA. (c, d) The identical method used in (a) was applied to macrophages, and an EdU assay was performed to evaluate the proliferation of MDA‐MB‐231 cells. MDA‐MB‐231 cells were cocultured with the conditioned medium from macrophages for 48 h, and the positive cell ratio is shown. Scale bar, 50 μm. Original magnification ×200. (e, f) The identical method used in A was applied to macrophages, which were subsequently cultured with MDA‐MB‐231 cells in the indirect coculture system in vitro. After incubation for 24 h, MDA‐MB‐231 cells were detached from the upper chamber, and the migration and invasion ability of MDA‐MB‐231 cells was detected by transwell aasay. Representative pictures of migratory or invasive cells and quantifications are shown. Scale bar, 100 μm. Original magnification ×100. The Student's t ‐test was adopted to analyze the statistical significance of the difference between two groups, and one‐way ANOVA test was utilized to analyze multiple groups (* p < 0.05, ** p < 0.01).

Article Snippet: Transfection siRNA against human ACO1, SFRS13A and TSC2 were purchased from ThermoFisher.

Techniques: Migration, Quantitative RT-PCR, Expressing, Transfection, Enzyme-linked Immunosorbent Assay, EdU Assay, Cell Culture, In Vitro, Incubation

Journal: Thoracic Cancer

Article Title: Tumor‐derived exosomal miR ‐148b‐3p mediates M2 macrophage polarization via TSC2 / mTORC1 to promote breast cancer migration and invasion

doi: 10.1111/1759-7714.14891

Figure Lengend Snippet: Exosomal miR‐148b‐3p regulates macrophage polarization by inhibiting TSC2 and activating the mTORC1 signaling pathway. (a) Western blot (WB) analysis of mTORC1 activity markers (p‐S6, S6) and M2‐related gene CD206 expression in M0 macrophages treated with PBS, MDA‐MB‐231‐CM, MDA‐MB‐231‐exo or (b) miR‐NC, miR‐148b‐3p mimic, miR‐148b‐3p inhibitor. (c) WB assay of mTORC1 activity markers (p‐S6, S6), TSC2 and M2‐related gene CD206 expression in M0 macrophages treated with si‐NC as control, si‐TSC2#1 and si‐TSC2#2 for downregulating TSC2 expression. (d) WB analysis of mTORC1 activity markers (p‐S6, S6) and M2‐related gene CD206 expression in M0 macrophages treated with the indicated treatment. Each experiment was conducted at least three times. (e) Schematic diagram of exosomal miR‐148b‐3p promoted M2 macrophage polarization via the TSC2/mTORC1 signaling pathway was drawn using Figdraw.

Article Snippet: Transfection siRNA against human ACO1, SFRS13A and TSC2 were purchased from ThermoFisher.

Techniques: Western Blot, Activity Assay, Expressing, Control

Journal: Cell Death & Disease

Article Title: Targeting SPHK1/S1PR3-regulated S-1-P metabolic disorder triggers autophagic cell death in pulmonary lymphangiomyomatosis (LAM)

doi: 10.1038/s41419-022-05511-3

Figure Lengend Snippet: A Immunohistochemistry staining of ASAH1, SPHK1 and phospho-S6(Ser235/236) was performed on rat-derived ELT3-V3 (TSC2-deficient, V3) and ELT3-T3 (TSC2-expression, T3) cell xenograft tumors of SCID mice. Representative images of five mice in each group. B Immunoblot analysis of ASAH1, SPHK1, S1PR3, p-ERK, pS6(235/236) protein levels in 621-101 cells treated with vehicle control or 10 nM estrogen for 24 h. β-actin was used as the loading control. C – F were treated with vehicle control or 20 nM rapamycin for 24 h. The mRNA levels of TSC2, ASAH1 , and SPHK1 were detected by RT-qPCR in ( C ) 621-101 and 621-103 cells, ( E ) Mouse embryo fibrosis cells (TSC2− MEFs and TSC2 + MEFs). Data showed the mean of three sets of independent samples. Protein expressions of tuberin, SPHK1, pS6(ser235/236) and ASAH1 were determined by immunoblot analysis in ( D ) 621-101 and 621-103 cells, ( F ) Mouse embryo fibrosis cells (TSC2 - MEFs and TSC2 + MEFs). β-actin was used as loading control. G , H TSC2-addback cells (621-103) were transfected with human TSC2 siRNAs or control siRNA for 24 h. Gene expressions of TSC2 , SPHK1 , and ASAH1 were determined by RT-qPCR, and protein levels of Tuberin, SPHK1, pS6(ser235/236) and ASAH1 were detected by immunoblotting. β-actin was used as loading control. Student’s t test, * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001.

Article Snippet: Human S1PR3 siRNAs (50 nmol/L, Genepharma, #1, #2, #3) and human TSC2 siRNAs (designed by Invitrogen) were transfected into cells using lipofectamine 2000 RNAIMAX (Invitrogen) according to the manufacturer’s protocols.

Techniques: Immunohistochemistry, Staining, Derivative Assay, Expressing, Western Blot, Quantitative RT-PCR, Transfection

Journal: Cell Death & Disease

Article Title: Targeting SPHK1/S1PR3-regulated S-1-P metabolic disorder triggers autophagic cell death in pulmonary lymphangiomyomatosis (LAM)

doi: 10.1038/s41419-022-05511-3

Figure Lengend Snippet: A LAM patient-derived TSC2-deficient (621-101) cells were transfected with shRNA- SPHK1 (#1 and #2) or a negative control (shNC). The mRNA and protein levels of SPHK1 were determined by RT-qPCR and immunoblotting, respectively. B Viability of 621-101shNC and 621- 101 shSPHK1 cells cultured in serum-free medium for 24 h was measured by MTT assay (upper panel). The cell death rate was measured by PI/CV assay (lower panel). The results are representative of eight independent samples per group. C 621-101shNC and 621- 101 shSPHK1 cells were seeded in 6-well plates for 24 h and then stained with an Annexin V:FITC apoptosis detection kit. Cell death was analyzed by flow cytometry. D The capacities for 621-101shNC and 621- 101 shSPHK1 cell migration (upper panel) and invasion (lower panel) were determined in a Transwell chamber without or with Matrigel. Cells were cultured in serum-free medium for 48 h and stained with crystal violet. Representative images of three repeats in each group. E 621-101shNC and 621- 101 shSPHK1 cells transfected with firefly luciferase were injected intravenously into SCID mice ( n = 5). Lung colonization was determined by bioluminescence imaging 0 h, 2 h, 6 h, and 12 h post injection. Luminescence color scale: 0 h, 2 h, 6 h, and 12 h (min = 1 × 10 6 , max = 1 × 10 7 ). The statistical analysis (lower panel) indicated the relative photon flux change. The results are representative of five mice per group. Student’s t test, ** P < 0.01, *** P < 0.001, and **** P < 0.0001.

Article Snippet: Human S1PR3 siRNAs (50 nmol/L, Genepharma, #1, #2, #3) and human TSC2 siRNAs (designed by Invitrogen) were transfected into cells using lipofectamine 2000 RNAIMAX (Invitrogen) according to the manufacturer’s protocols.

Techniques: Derivative Assay, Transfection, shRNA, Negative Control, Quantitative RT-PCR, Western Blot, Cell Culture, MTT Assay, Staining, Flow Cytometry, Migration, Luciferase, Injection, Imaging

Journal: Cell Death & Disease

Article Title: Targeting SPHK1/S1PR3-regulated S-1-P metabolic disorder triggers autophagic cell death in pulmonary lymphangiomyomatosis (LAM)

doi: 10.1038/s41419-022-05511-3

Figure Lengend Snippet: A Cell viability of 621-101 and 621-103 cells treated with the specific SPHK1 inhibitor PF543 at the indicated concentrations for 24 h (left panel) and 48 h (middle panel) and ELT3-V3 and ELT3-T3 treated with the indicated concentrations of PF543 for 24 h (right panel) was measured by MTT assay in serum-free condition. The results are representative of eight independent samples per group. B The death of 621-101 and 621-103 cells treated with vehicle control or PF543 (2.5 μM) in serum-free conditions for 24 h was evaluated by PI/CV assay. The results are representative of eight independent samples per group. C 621-101 cells treated with vehicle control or PF543 (2.5 μM) for 24 h were stained with the reagent in an Annexin V: FITC apoptosis detection kit. Cell death was analyzed by flow cytometry ( n = 3). D The mRNA and protein levels of SPHK1 in TSC2-deficient 621-10 cells and TSC2-addback 621-103 cells treated with vehicle control or PF543 were determined by RT-qPCR (upper panel) and immunoblot assay (lower panel). β-actin was used as the loading control. E Migration (left panel) and invasion (right panel) of 621-101 cells treated with vehicle control or PF543 (2.5 μM) under serum-free conditions for 48 h were measured in Transwell chambers without or with Matrigel. The cells were stained with crystal violet. Representative images of three repeats in each group. Student’s t test, * P < 0.05, ** P < 0.01, *** P < 0.001, and **** P < 0.0001.

Article Snippet: Human S1PR3 siRNAs (50 nmol/L, Genepharma, #1, #2, #3) and human TSC2 siRNAs (designed by Invitrogen) were transfected into cells using lipofectamine 2000 RNAIMAX (Invitrogen) according to the manufacturer’s protocols.

Techniques: MTT Assay, Staining, Flow Cytometry, Quantitative RT-PCR, Western Blot, Migration

Journal: Cell Death & Disease

Article Title: Targeting SPHK1/S1PR3-regulated S-1-P metabolic disorder triggers autophagic cell death in pulmonary lymphangiomyomatosis (LAM)

doi: 10.1038/s41419-022-05511-3

Figure Lengend Snippet: A S1P in conditioned medium of cultured 621-101 and 621-103 cells (upper panel) or 621-103 cells (middle panel) and HEK293T cells (lower panel) transfected with siRNA- TSC2 or siControl was quantified by ELISA. The Data were normalized to the control group. The results are representative of six sets of independent samples per group. B Viability of 621-101 and 621-103 cells (left panel) or ELT3-V3 and ELT3-T3 cells (right panel) treated with S1P for 24 h at the indicated concentrations was measured by MTT assay under serum-free conditions. C The death rate of 621-101 cells treated with 2 μM S1P for 24 h was determined by PI/CV assay under serum-free conditions. B , C Results are representative of eight independent samples per group. D RT-qPCR analyzed the mRNA level of SGPL1 and SPHK1 in 621-101 and 621-103 cells treated with 2 μM S1P or vehicle control for 24 h. The data show the mean of three sets of independent samples. E The mRNA levels of S1PR1 - S1PR5 in 621-101 and 621-103 cells were detected by RT-qPCR assay. F RT-qPCR analyzed the mRNA levels of TSC2 , S1PR1 , S1PR2 , S1PR3 , S1PR4 , and S1PR5 in siRNA- TSC2 -transfected 621-103 cells or siRNA-control cells. G 621-101 and 621-103 cells were treated with 2 μM S1P or vehicle control for 24 h. Tuberin, SPHK1, S1PR1, S1PR3, and phospho-S6 (S235/236) were analyzed by immunoblotting assay. β-actin was used as the loading control. The results are representative of three different experiments. H , I 621-101 and 621-103 cells were treated with 20 nM rapamycin or vehicle control for 24 h. The S1PR1 and S1PR3 levels were performed by RT-qPCR ( H ). Protein levels of tuberin, S1PR1, S1PR3, and phospho-S6 (S235/236) were determined by immunoblotting ( I ). β-actin was used as the loading control. The results are representative of three different experiments. Student’s t test, * P < 0.05, ** P < 0.01, *** P < 0.001, and **** P < 0.0001.

Article Snippet: Human S1PR3 siRNAs (50 nmol/L, Genepharma, #1, #2, #3) and human TSC2 siRNAs (designed by Invitrogen) were transfected into cells using lipofectamine 2000 RNAIMAX (Invitrogen) according to the manufacturer’s protocols.

Techniques: Cell Culture, Transfection, Enzyme-linked Immunosorbent Assay, MTT Assay, Quantitative RT-PCR, Western Blot

Journal: Cell Death & Disease

Article Title: Targeting SPHK1/S1PR3-regulated S-1-P metabolic disorder triggers autophagic cell death in pulmonary lymphangiomyomatosis (LAM)

doi: 10.1038/s41419-022-05511-3

Figure Lengend Snippet: A Gene expression of ATG5 in 621-101 cells treated with vehicle control or specific SPHK1 inhibitor PF543 (2.5 μM) was detected by RT-qPCR. 621-101 cells. B 621-101 cells were transfected with shRNA- SPHK1 or negative control (shN.C). Immunoblotting was performed to determine the levels of phospho-S6 (235/236), LC3I and LC3II in 621-101 shRNA- SPHK1 cells and control cells treated with vehicle control, rapamycin (Rapa, 10 nM) or CQ (5 µM). C 621-101 cells were treated with 20 nM rapamycin and 5 µM CQ, with or without 2.5 µM PF543 for 24 h. Levels of tuberin, p62, phospho-S6 (235/236), LC3I/LC3II, cl-caspase 3 and SPHK1 were assessed by immunoblotting. 621-103 cells were added as TSC2-addback control. D , E 621-101 cells were transfected with S1PR3 -siRNA or negative control (siControl). Gene expression of ATG5 and SQSTM1 was analyzed by RT-qPCR ( D ), and protein levels of p62, LC3I/LC3II were determined by immunoblotting ( E ). F 621-101 cells were treated with vehicle control or selective S1PR3 inhibitor TY52156 (2 μM), and the gene expression of ATG5 , ATG7 , ATG12 , BECN1 and SQSTM1 was detected by RT-qPCR. G 621-101 cells were treated with 20 nM Rapa and 5 µM CQ with or without 2 µM TY52156 for 24 h. Protein levels of tuberin, p62, phospho-S6 (235/236), LC3I/LC3II and S1PR3 were assessed by immunoblotting. 621-103 cells were added as TSC2-addback control. Student’s t test, * P < 0.05, ** P < 0.01, *** P < 0.001.

Article Snippet: Human S1PR3 siRNAs (50 nmol/L, Genepharma, #1, #2, #3) and human TSC2 siRNAs (designed by Invitrogen) were transfected into cells using lipofectamine 2000 RNAIMAX (Invitrogen) according to the manufacturer’s protocols.

Techniques: Expressing, Quantitative RT-PCR, Transfection, shRNA, Negative Control, Western Blot

Journal: Cell Death & Disease

Article Title: Targeting SPHK1/S1PR3-regulated S-1-P metabolic disorder triggers autophagic cell death in pulmonary lymphangiomyomatosis (LAM)

doi: 10.1038/s41419-022-05511-3

Figure Lengend Snippet: Simplified graph representation of abnormal sphingosine metabolism signaling that promotes tumorigenesis of TSC2-deficient cells.

Article Snippet: Human S1PR3 siRNAs (50 nmol/L, Genepharma, #1, #2, #3) and human TSC2 siRNAs (designed by Invitrogen) were transfected into cells using lipofectamine 2000 RNAIMAX (Invitrogen) according to the manufacturer’s protocols.

Techniques: