Journal: BMC Cancer

Article Title: Ganglioside GD2 in reception and transduction of cell death signal in tumor cells

doi: 10.1186/1471-2407-14-295

Figure Lengend Snippet: Cross-reactivity of anti-GD2 antibodies with gangliosides GM1, GM2, GD1b and GD3. (A, B) Interaction of anti-GD2 antibodies with gangliosides GD2 vs. GM1, GM2, GD1b and GD3 was assessed by ELISA as described in Methods . Plates were coated with gangliosides GD2, GM1, GM2, GD1b, and GD3 (0.25 μg/well) and incubated with two types of anti-GD2 mAbs (0.156 - 10 μg/ml) ME361 (A) and 14G2a (B) . In (A), the level of cross-reactivity is presented as the ratio for GM2, GD1b and GD3 binding to that of GD2. Mean ± S.E. of three separate experiments are shown, statistical analysis was performed using two-way analysis of variance method. There was a statistically significant difference between groups (P ≤ 0.001), ***P <0.001 as determined by multiple comparisons versus zero cross-reactivity by Dunnett's post-hoc analysis (A) . In (B), multiple comparisons were not performed, since the values of the cross-reactivity were less than 2%.

Article Snippet: Other anti-GD2 14G2a mAbs were purchased from Millipore Inc. Anti-GM2/GD2 synthase and anti-ALCAM antibodies, siRNA and primers for GM2/GD2 and GD3 synthases were purchased from Santa Cruz Biotechnology Inc.

Techniques: Enzyme-linked Immunosorbent Assay, Incubation, Binding Assay

Journal: International Journal of Biological Sciences

Article Title: Disialyl GD2 ganglioside suppresses ICAM-1-mediated invasiveness in human breast cancer MDA-MB231 cells

doi: 10.7150/ijbs.16903

Figure Lengend Snippet: RT-PCR primer sequences

Article Snippet: Glyceraldehydes-3-phosphodehydrogenase (GAPDH) (MAB-8145) (Chemicon, USA), SARK/JNK(#9252), p-SARK/JNK(#9255) (Cell signaling, USA) and GD3 synthase (xw-8145) (ProSci incorporated, USA) were purchased.

Techniques:

Journal: International Journal of Biological Sciences

Article Title: Disialyl GD2 ganglioside suppresses ICAM-1-mediated invasiveness in human breast cancer MDA-MB231 cells

doi: 10.7150/ijbs.16903

Figure Lengend Snippet: Real-time PCR primer sequences

Article Snippet: Glyceraldehydes-3-phosphodehydrogenase (GAPDH) (MAB-8145) (Chemicon, USA), SARK/JNK(#9252), p-SARK/JNK(#9255) (Cell signaling, USA) and GD3 synthase (xw-8145) (ProSci incorporated, USA) were purchased.

Techniques: Real-time Polymerase Chain Reaction

Journal: International Journal of Biological Sciences

Article Title: Disialyl GD2 ganglioside suppresses ICAM-1-mediated invasiveness in human breast cancer MDA-MB231 cells

doi: 10.7150/ijbs.16903

Figure Lengend Snippet: A) GD3 synthase gene expression patterns in human breast cancer cells. Total mRNA was isolated from breast cancer and SK-Mel II melanoma cells. The bar graphs represent the relative band intensity obtained from RT-PCR results by densitometry. For the expression levels of GD3 synthase mRNA, RT-PCR (B) and real-time PCR (C) analyses were performed. The protein expression was analyzed by Western blot (D). The pc3-GD3s cells were cultured with 1 mg/ml G-418. All experiments have been performed at least three times and we examined mean differences between groups by using the error bar graph procedure.

Article Snippet: Glyceraldehydes-3-phosphodehydrogenase (GAPDH) (MAB-8145) (Chemicon, USA), SARK/JNK(#9252), p-SARK/JNK(#9255) (Cell signaling, USA) and GD3 synthase (xw-8145) (ProSci incorporated, USA) were purchased.

Techniques: Gene Expression, Isolation, Reverse Transcription Polymerase Chain Reaction, Expressing, Real-time Polymerase Chain Reaction, Western Blot, Cell Culture

Journal: International Journal of Biological Sciences

Article Title: Disialyl GD2 ganglioside suppresses ICAM-1-mediated invasiveness in human breast cancer MDA-MB231 cells

doi: 10.7150/ijbs.16903

Figure Lengend Snippet: The pc3-GD3s cells show changes in morphology but not in cell growth Cell were cultured in DMEM supplemented with 10% or 5% FBS. The pc3 (◆ ) and pc3-GD3s cells (■) were plated in triplicates. Cell growth in different serum conditions was assessed by the XTT assay after 24 hr (A). The cell morphology was observed by phase contrast microscopy (B). Inhibition of invasiveness in GD3 synthase-transfected MDA-MB231 cells (C). Representative microphotographs of Hematoxylin and Eosin stained cells. Cells that invaded through the gelatin and were located on the underside of the filter were counted. All experiments performed at least three times and we examined mean differences between groups by using the error bar graph procedure.

Article Snippet: Glyceraldehydes-3-phosphodehydrogenase (GAPDH) (MAB-8145) (Chemicon, USA), SARK/JNK(#9252), p-SARK/JNK(#9255) (Cell signaling, USA) and GD3 synthase (xw-8145) (ProSci incorporated, USA) were purchased.

Techniques: Cell Culture, XTT Assay, Microscopy, Inhibition, Transfection, Staining

Journal: International Journal of Biological Sciences

Article Title: Disialyl GD2 ganglioside suppresses ICAM-1-mediated invasiveness in human breast cancer MDA-MB231 cells

doi: 10.7150/ijbs.16903

Figure Lengend Snippet: Differential regulation of ICAM-1 expression in pc3-GD3s-trasnfected SK-BR3 (ER-) and MCF-7 (ER+) cells. Expression levels of GM3 synthase, GD3 synthase and ICAM-1 mRNAs in the pc3-GD3s-transfected SK-BR3 (ER-) were checked by RT-PCR (A) and relative band intensities were compared with each β-actin control (B). β-Actin was included as an internal control. For MCF-7 (ER+) cells, ICAM-1 mRNA level in the pc3-GD3s-transfected MCF-7 cells was examined by Western blot analysis (C) and relative band intensity was also compared with GAPDH control (D). All experiments performed at least three times. We examined mean differences between groups by using the error bar graph procedure.

Article Snippet: Glyceraldehydes-3-phosphodehydrogenase (GAPDH) (MAB-8145) (Chemicon, USA), SARK/JNK(#9252), p-SARK/JNK(#9255) (Cell signaling, USA) and GD3 synthase (xw-8145) (ProSci incorporated, USA) were purchased.

Techniques: Expressing, Transfection, Reverse Transcription Polymerase Chain Reaction, Control, Western Blot

Journal: PLoS ONE

Article Title: GD3 Synthase Overexpression Sensitizes Hepatocarcinoma Cells to Hypoxia and Reduces Tumor Growth by Suppressing the cSrc/NF-κB Survival Pathway

doi: 10.1371/journal.pone.0008059

Figure Lengend Snippet: A. Representative image of lipids extracts resolved by HPTLC from Hep3B cells transfected with and empty vector (3B) or overexpressing GD3 Synthase (3B-GD3) isotopically labeled with 3 H-galactose, and quantification of three independent experiments. Commercial non-labeled standards were run in parallel, being TLC plates stained with 5% orcinol solution. *p<0.05 vs. Hep3B control cells. B. GD3 synthase activity was measured in control Hep3B (3B) and Hep3B GD3-expressing cells (3B-GD3). GD3 Synthase protein levels were analyzed by western blot and compared with β-actin levels, used as a control. C. Confocal immunofluorescence was visualized in empty-vector containing and GD3 Synthase overexpressing Hep3B cells using antibodies anti-GD3 and anti-Mn-SOD. The graphs on the right panel represent the fluorescence intensity profile plotted from a to b direction in the merge images for the different cell lines (Mn-SOD in green and GD3 in red). D. Cell growth under normoxic conditions (n = 3). *p<0.05 vs. Hep3B control cells.

Article Snippet: GD3 synthase RNA silencing was achieved in Hep3B cells with target- specific siRNA duplexes (Santa Cruz) using LipofectamineTM 2000 as transfecting agent and its related scramble siRNA as a control.

Techniques: High Performance Thin Layer Chromatography, Transfection, Plasmid Preparation, Labeling, Staining, Control, Activity Assay, Expressing, Western Blot, Immunofluorescence, Fluorescence

Journal: PLoS ONE

Article Title: GD3 Synthase Overexpression Sensitizes Hepatocarcinoma Cells to Hypoxia and Reduces Tumor Growth by Suppressing the cSrc/NF-κB Survival Pathway

doi: 10.1371/journal.pone.0008059

Figure Lengend Snippet: A. Cell death in Hep3B cells growing under normoxia or hypoxia for 48 h, and treated with increasing doses of GD3. Cell viability measured 24 h later (n = 3). B. Time-dependent viability of Hep3B and Hep3B-GD3 cells exposed to 2% O 2 (n = 3). C, D. ROS generation evaluated with fluorescent probes HE (5 µM) and DCF (1 µM) during 30 min at 37°C (n = 4). *p<0.05 vs. Hep3B cells. #p<0.05 vs. hypoxic Hep3B cells.

Article Snippet: GD3 synthase RNA silencing was achieved in Hep3B cells with target- specific siRNA duplexes (Santa Cruz) using LipofectamineTM 2000 as transfecting agent and its related scramble siRNA as a control.

Techniques:

Journal: PLoS ONE

Article Title: GD3 Synthase Overexpression Sensitizes Hepatocarcinoma Cells to Hypoxia and Reduces Tumor Growth by Suppressing the cSrc/NF-κB Survival Pathway

doi: 10.1371/journal.pone.0008059

Figure Lengend Snippet: A. Representative western blots of p65 and HIF-1α from nuclear extracts of control (3B) and GD3-overexpressing (3B-GD3) cells using c-jun and β-actin as representative nuclear protein and cytosolic marker, respectively (n = 3). B , Luciferase activity in 3B and 3B-GD3 cells transfected with NF-κB luciferase reporter construct (n = 4). C. Representative immunoblots with c-Src, phospho-Tyr416Src and Mn-SOD antibodies and normalized by β-actin levels. D, Mn-SOD mRNA levels were analyzed by real-time PCR after 72 hours (n = 3). E, ROS production, and F, survival of Hep3B-GD3 in the presence or absence of Mn-SOD mimetic, MnTBAP (50 µM) after 72 h hypoxia (n = 4). *p<0.05 vs. Hep3B cells. #p<0.05 vs. hypoxic control.

Article Snippet: GD3 synthase RNA silencing was achieved in Hep3B cells with target- specific siRNA duplexes (Santa Cruz) using LipofectamineTM 2000 as transfecting agent and its related scramble siRNA as a control.

Techniques: Western Blot, Control, Marker, Luciferase, Activity Assay, Transfection, Construct, Real-time Polymerase Chain Reaction

Journal: PLoS ONE

Article Title: GD3 Synthase Overexpression Sensitizes Hepatocarcinoma Cells to Hypoxia and Reduces Tumor Growth by Suppressing the cSrc/NF-κB Survival Pathway

doi: 10.1371/journal.pone.0008059

Figure Lengend Snippet: mRNA levels of PDK1 ( A ) and VEGF ( B ) in Hep3B cells from subcutaneous tumors in nude mice or from cells growing under normal culture conditions with 21% O 2 (n = 3–4). *p<0.05 vs. normoxic Hep3B cells. C , Tumor volume was measured in nude mice injected with Hep3B (empty vector) and Hep3B-GD3 cells (n = 5–6 animals per group). D, E. Representative TUNEL-staining in tumor samples from injected mice, and quantification of TUNEL-positive cells in each group as indicated in (n = 3-4). *p<0.05 vs. Hep3B-injected mice. F. mRNA levels of Mn-SOD from Hep3B and Hep3B-GD3 subcutaneous tumors (n = 4). *p<0.05 vs. Hep3B-injected mice.

Article Snippet: GD3 synthase RNA silencing was achieved in Hep3B cells with target- specific siRNA duplexes (Santa Cruz) using LipofectamineTM 2000 as transfecting agent and its related scramble siRNA as a control.

Techniques: Injection, Plasmid Preparation, TUNEL Assay, Staining

Journal: PLoS ONE

Article Title: GD3 Synthase Overexpression Sensitizes Hepatocarcinoma Cells to Hypoxia and Reduces Tumor Growth by Suppressing the cSrc/NF-κB Survival Pathway

doi: 10.1371/journal.pone.0008059

Figure Lengend Snippet: In Hep3B cells, NF-κB activation via cSrc plays a key role in the resistance to hypoxia, in part, by inducing Mn-SOD expression. However, in Hep3B-GD3 cells the overgeneration of GD3 blocks the cSrc/NF-κB pathway potentiating the mitochondrial ROS production during hypoxia and contributing to hypoxia-mediated cell death.

Article Snippet: GD3 synthase RNA silencing was achieved in Hep3B cells with target- specific siRNA duplexes (Santa Cruz) using LipofectamineTM 2000 as transfecting agent and its related scramble siRNA as a control.

Techniques: Activation Assay, Expressing

Journal: PLOS Biology

Article Title: The mTORC2 subunit RICTOR drives breast cancer progression by promoting ganglioside biosynthesis through transcriptional and epigenetic mechanisms

doi: 10.1371/journal.pbio.3003362

Figure Lengend Snippet: ( A ) A schematic presentation showing the ceramide-glucosylceramide rheostat connecting the sphingolipid and ganglioside metabolic pathways. ( B ) The glucosylceramide to ceramide ratio (mean ± SEM, n = 27) for luminal tumor tissues (labeled as T) and adjacent normal tissues (labeled as N) indicates that the balance is shifted towards glucosylceramides. ( C ) Heat map showing the levels of different ganglioside species in luminal tumor tissues and adjacent normal tissues. ( D – H ) Absolute quantification (mean ± SEM, n = 5) of GM3 (D), GD3 (E), GD2 (F), GM2 (G), and GM1 (H) gangliosides in luminal tumor tissues and adjacent normal tissues shows an increase in GM3 and GD3 gangliosides and a decrease in GM1 gangliosides in luminal tumor tissues. ( I ) Immunoblot confirming an increase in UGCG expression in MCF-7_UGCG OE cells. ( J ) Absolute quantification of glucosylceramides (mean ± SEM, n = 5) confirms an increase in MCF-7_UGCG OE cells over MCF-7 cells. ( K – O ) Absolute quantification (mean ± SEM, n = 5) of GM3 (K), GD3 (L), GD2 (M), GM2 (N), and GM1 (O) ganglioside species shows an increase of GM3, GD3, and GM2 gangliosides and a decrease of GM1 gangliosides in MCF-7_UGCG OE cells compared to MCF-7 cells. ( P ) Cell proliferation (mean ± SEM, n = 5) assay demonstrates an increase in the proliferation of MCF-7_UGCG OE cells over MCF-7_VECT OE cells. ( Q ) Tumor growth kinetics reveal enhanced growth of MCF-7_UGCG OE tumors compared to MCF-7_VECT OE tumors (mean ± SEM, n = 5–6). ( R ) Immunoblots show the expression of RICTOR, RAPTOR, AKT, pAKT Ser473 , SGK1, pSGK1 Ser78 , 4EBP1, p4EBP1 Thr37 , and p70S6K in MCF-7_UGCG OE cells in comparison to MCF-7_UGCG DEAD cells. ( S ) Immunoblots for RICTOR, pAKT Ser473 , and UGCG in tumor tissues from luminal cancer patients show higher expression than adjacent normal tissues. ( T ) A schematic diagram showing the questions to be answered to understand the mTORC2-mediated regulation of the sphingolipid metabolic pathway and its role in tumor progression. Data among groups were analyzed using a paired Student t test (for patient data), One-way ANOVA among multiple groups or Two-way ANOVA in time-dependent studies. P -value: * p < 0.05, ** p < 0.01, *** p < 0.0005, **** p < 0.0001. Numerical data can be found in .

Article Snippet: MCF-7, BT-474, MDA-MB-453, HCT-116, HEK-293 cells (ATCC, USA), DMEM media (Cat# D5648) Sigma, USA, MEBM media (Cat# CC-3151) Lonza, Switzerland, MEM media (Cat# AL081) HiMedia, USA, DPBS (Cat# D5652) Sigma, USA, FBS (Cat# 10270) Gibco, USA, lipid-free FBS (Cat# S148L) Biowest, USA, Penicillin-Streptomycin (Cat# 113-98-43810-74-0) HyClone, USA, Lipofectamine 2000, (Cat# 11668019) Invitrogen, USA, Lipofectamine 3000 (Cat# L3000015) Invitrogen, USA, Trypsin (Cat# TCL007) HiMedia, USA, Puromycin (Cat# P7255) Sigma, USA, G418 (Cat# A1720) Sigma, USA, Haemocytometer (Cat# Z359629) Bright-Line, USA, shRNA Control (Cat# SHC202V) Sigma, USA, shRNA RICTOR Virus Particles (Cat# SHCLNV, TRCN0000289691, TRCN0000307119, TRCN0000296313, TRCN0000307122) Sigma, USA, shRNA RICTOR Glycerol stocks (SHCLNG, TRCN0000296313, TRCN0000307122,), Sigma, USA, shRNA UGCG Glycerol stocks (SHCLNG, TRCN0000036128, TRCN0000036126, TRCN0000300623) Sigma, USA, ZFX Glycerol stocks (Cat# SHCLNG TRCN0000017308, TRCN0000017309, TRCN0000017310), Sigma, USA, shRNA ST8SIA1 (Cat# SHCLNG TRCN0000417447, TRCN0000036044, TRCN0000036046), Sigma, USA, UGCG siRNA (Cat# AM51331) Ambion, USA, ZFX siRNA (Cat# L-006572-00-0005) Dharmacon, USA, Scrambled siRNA (Cat# D-001810-10-05) Dharmacon, USA, GD3 Synthase (ST8SIA1) siRNA (Cat# EHU025731-20UG) Merck, USA, 5-Aza-2′-deoxycytidine (DAC) (Cat#A3656-10MG) Sigma, USA, KDOAM25 Hydrochloride hydrate (Cat# SML2774-5MG) Sigma, USA, MK-2206 dihydrochloride (Cat# HY-10358) MedChem Express, USA, Eliglustat (Cat# HY-14885) MedChem Express, USA, Hygromycin (Cat# PCT1503), HiMedia, USA, Puromycin (Cat# P8833), Sigma, USA.

Techniques: Labeling, Quantitative Proteomics, Western Blot, Expressing, Comparison

Journal: PLOS Biology

Article Title: The mTORC2 subunit RICTOR drives breast cancer progression by promoting ganglioside biosynthesis through transcriptional and epigenetic mechanisms

doi: 10.1371/journal.pbio.3003362

Figure Lengend Snippet: ( A ) Immunoblots confirm knockdown of RICTOR expression in MCF-7_RICTOR SH cells. ( B ) Immunoblots show changes in expression of RICTOR, RAPTOR, and their downstream effectors in MCF-7_RICTOR SH cells compared to MCF-7_SCRAM SH cells. ( C ) Cell proliferation studies show a decrease in the proliferation of MCF-7_RICTOR SH cells (mean ± SEM, n = 4) compared to MCF-7_SCRAM SH cells. ( D ) Tumor growth kinetics show significantly slower growth of MCF-7_RICTOR SH (mean ± SEM, n = 5) tumors compared to the MCF-7_SCRAM SH tumors. ( E ) Heat map representing normalized absolute quantitation of ceramides and glucosylceramides in MCF-7_RICTOR SH and MCF-7 cells. ( F ) Fold change (mean ± SEM, n = 5) in different sphingolipid species reveals an increase in ceramides and a decrease in glucosylceramides in MCF-7_RICTOR SH cells compared to MCF-7 cells. ( G , H ) qRT-PCR (mean ± SEM, n = 4) ( G ) and immunoblots and their quantification (mean ± SEM, n = 3) (H) demonstrate downregulation of UGCG without any change in GBA1 expression in MCF-7_RICTOR SH cells compared to MCF-7_SCRAM SH cells. ( I – M ) Absolute quantification (mean ± SEM, n = 3) of GM3 (I), GD3 (J), GD2 (K), GM2 (L), and GM1 (M) ganglioside species shows a decrease in GM3, GD3, GM2, and GM1 gangliosides in MCF-7_RICTOR SH cells compared to MCF-7 cells. ( N ) Immunoblot confirming UGCG overexpression in MCF-7_RICTOR SH cells. ( O ) The absolute quantification of glucosylceramides (mean ± SEM, n = 4) in MCF-7_RICTOR SH _UGCG OE cells compared to MCF-7_RICTOR SH cells confirms an increase in glucosylceramides. ( P ) Cell proliferation assay demonstrates an increase in cell proliferation (mean ± SEM, n = 4) of MCF-7_RICTOR SH cells on UGCG overexpression. ( Q ) A schematic diagram showing the role of putative factors modulating the RICTOR/pAKT-mediated UGCG expression that can lead to altered glucosylceramides, thereby controlling tumor progression. Data among groups were analyzed using an unpaired Student t test or One-way ANOVA among multiple groups or by Two-way ANOVA in time-dependent studies. p -value: * p < 0.05, ** p < 0.01, *** p < 0.0005, **** p < 0.0001. Numerical data can be found in .

Article Snippet: MCF-7, BT-474, MDA-MB-453, HCT-116, HEK-293 cells (ATCC, USA), DMEM media (Cat# D5648) Sigma, USA, MEBM media (Cat# CC-3151) Lonza, Switzerland, MEM media (Cat# AL081) HiMedia, USA, DPBS (Cat# D5652) Sigma, USA, FBS (Cat# 10270) Gibco, USA, lipid-free FBS (Cat# S148L) Biowest, USA, Penicillin-Streptomycin (Cat# 113-98-43810-74-0) HyClone, USA, Lipofectamine 2000, (Cat# 11668019) Invitrogen, USA, Lipofectamine 3000 (Cat# L3000015) Invitrogen, USA, Trypsin (Cat# TCL007) HiMedia, USA, Puromycin (Cat# P7255) Sigma, USA, G418 (Cat# A1720) Sigma, USA, Haemocytometer (Cat# Z359629) Bright-Line, USA, shRNA Control (Cat# SHC202V) Sigma, USA, shRNA RICTOR Virus Particles (Cat# SHCLNV, TRCN0000289691, TRCN0000307119, TRCN0000296313, TRCN0000307122) Sigma, USA, shRNA RICTOR Glycerol stocks (SHCLNG, TRCN0000296313, TRCN0000307122,), Sigma, USA, shRNA UGCG Glycerol stocks (SHCLNG, TRCN0000036128, TRCN0000036126, TRCN0000300623) Sigma, USA, ZFX Glycerol stocks (Cat# SHCLNG TRCN0000017308, TRCN0000017309, TRCN0000017310), Sigma, USA, shRNA ST8SIA1 (Cat# SHCLNG TRCN0000417447, TRCN0000036044, TRCN0000036046), Sigma, USA, UGCG siRNA (Cat# AM51331) Ambion, USA, ZFX siRNA (Cat# L-006572-00-0005) Dharmacon, USA, Scrambled siRNA (Cat# D-001810-10-05) Dharmacon, USA, GD3 Synthase (ST8SIA1) siRNA (Cat# EHU025731-20UG) Merck, USA, 5-Aza-2′-deoxycytidine (DAC) (Cat#A3656-10MG) Sigma, USA, KDOAM25 Hydrochloride hydrate (Cat# SML2774-5MG) Sigma, USA, MK-2206 dihydrochloride (Cat# HY-10358) MedChem Express, USA, Eliglustat (Cat# HY-14885) MedChem Express, USA, Hygromycin (Cat# PCT1503), HiMedia, USA, Puromycin (Cat# P8833), Sigma, USA.

Techniques: Western Blot, Knockdown, Expressing, Quantitation Assay, Quantitative RT-PCR, Quantitative Proteomics, Over Expression, Proliferation Assay

Journal: PLOS Biology

Article Title: The mTORC2 subunit RICTOR drives breast cancer progression by promoting ganglioside biosynthesis through transcriptional and epigenetic mechanisms

doi: 10.1371/journal.pbio.3003362

Figure Lengend Snippet: (A) A schematic diagram showing the workflow to identify RICTOR-regulated transcription factors that bind to the UGCG promoter. (B) Results from qRT-PCR (mean ± SEM, n = 3) confirm reduced expression of RICTOR-regulated ELF1 , ZFX , and CTCF transcription factors in MCF-7_RICTOR SH cells. (C) ChIP-qPCR (mean ± SEM, n = 3) results show reduced binding of ZFX to UGCG promoter in MCF-7_RICTOR SH cells. (D) EMSA shows the binding of ZFX to UGCG promoter (lanes 2 and 3) in MCF-7_ZFX OE cells, shift-ablation assay in MCF-7_ZFX OE cells (lane 4), competition assay with specific (lane 5) and unrelated oligo as a control (lane 6). “*” denotes nonspecific complexes. (E) EMSA comparing endogenous ZFX-DNA binding activity in MCF-7_SCRAM SH and MCF-7_RICTOR SH cells. (F, G) Immunoblots (F) and their quantification (mean ± SEM, n = 3) (G) confirm downregulation of ZFX in MCF-7_RICTOR SH cells. ( H , I ) Immunoblots (H) and their quantification (mean ± SEM, n = 3) (I) confirm overexpression and silencing of ZFX in MCF-7_ZFX OE and MCF-7_ZFX SL cells. (J, K) Immunoblots (J) and their quantification (mean ± SEM, n = 3) (K) show upregulation and downregulation of UGCG upon overexpression and silencing of ZFX in MCF-7_ZFX OE and MCF-7_ZFX SL cells. (L, M) Fold change (mean ± SEM, n = 5) in ceramides (L) and glucosylceramides (M) confirms a decrease in ceramides and an increase in glucosylceramides in MCF-7_ZFX OE cells. In contrast, MCF-7_ZFX SL cells show higher ceramides and reduced glucosylceramides. (N–R) Absolute quantification (mean ± SEM, n = 3-5) of GM3 (N), GD3 (O), GD2 (P), GM2 (Q), and GM1 (R) gangliosides shows an increase in GM3, GD3, and GM2 gangliosides and attenuated GM1 gangliosides on ZFX overexpression in MCF-7 cells. (S) Cell proliferation (mean ± SEM, n = 4) demonstrates increased proliferation of MCF-7_ZFX OE cells, whereas MCF-7_ZFX SL cells show reduced cell proliferation. (T) Tumor growth kinetics recorded a significantly higher growth of MCF-7_ZFX OE (mean ± SEM, n = 4-6) than MCF-7_VECT OE tumors. ( U , V ) Cell proliferation demonstrates a decrease in proliferation of MCF-7_ZFX OE cells on UGCG silencing (U) (mean ± SEM, n = 4), whereas MCF-7_ZFX SH cells show enhanced cell proliferation on UGCG overexpression (mean ± SEM, n = 3) (V). ( W ) siRNA-mediated silencing of UGCG leads to reduced tumor growth kinetics in MCF-7_ZFX OE tumors. Data among two groups were analyzed using an unpaired Student t test, among multiple groups using One-way ANOVA, and by Two-way ANOVA in time-dependent studies. p -value: * p < 0.05, ** p < 0.01, *** p < 0.0005, **** p < 0.0001. Numerical data can be found in .

Article Snippet: MCF-7, BT-474, MDA-MB-453, HCT-116, HEK-293 cells (ATCC, USA), DMEM media (Cat# D5648) Sigma, USA, MEBM media (Cat# CC-3151) Lonza, Switzerland, MEM media (Cat# AL081) HiMedia, USA, DPBS (Cat# D5652) Sigma, USA, FBS (Cat# 10270) Gibco, USA, lipid-free FBS (Cat# S148L) Biowest, USA, Penicillin-Streptomycin (Cat# 113-98-43810-74-0) HyClone, USA, Lipofectamine 2000, (Cat# 11668019) Invitrogen, USA, Lipofectamine 3000 (Cat# L3000015) Invitrogen, USA, Trypsin (Cat# TCL007) HiMedia, USA, Puromycin (Cat# P7255) Sigma, USA, G418 (Cat# A1720) Sigma, USA, Haemocytometer (Cat# Z359629) Bright-Line, USA, shRNA Control (Cat# SHC202V) Sigma, USA, shRNA RICTOR Virus Particles (Cat# SHCLNV, TRCN0000289691, TRCN0000307119, TRCN0000296313, TRCN0000307122) Sigma, USA, shRNA RICTOR Glycerol stocks (SHCLNG, TRCN0000296313, TRCN0000307122,), Sigma, USA, shRNA UGCG Glycerol stocks (SHCLNG, TRCN0000036128, TRCN0000036126, TRCN0000300623) Sigma, USA, ZFX Glycerol stocks (Cat# SHCLNG TRCN0000017308, TRCN0000017309, TRCN0000017310), Sigma, USA, shRNA ST8SIA1 (Cat# SHCLNG TRCN0000417447, TRCN0000036044, TRCN0000036046), Sigma, USA, UGCG siRNA (Cat# AM51331) Ambion, USA, ZFX siRNA (Cat# L-006572-00-0005) Dharmacon, USA, Scrambled siRNA (Cat# D-001810-10-05) Dharmacon, USA, GD3 Synthase (ST8SIA1) siRNA (Cat# EHU025731-20UG) Merck, USA, 5-Aza-2′-deoxycytidine (DAC) (Cat#A3656-10MG) Sigma, USA, KDOAM25 Hydrochloride hydrate (Cat# SML2774-5MG) Sigma, USA, MK-2206 dihydrochloride (Cat# HY-10358) MedChem Express, USA, Eliglustat (Cat# HY-14885) MedChem Express, USA, Hygromycin (Cat# PCT1503), HiMedia, USA, Puromycin (Cat# P8833), Sigma, USA.

Techniques: Quantitative RT-PCR, Expressing, ChIP-qPCR, Binding Assay, Competitive Binding Assay, Control, Activity Assay, Western Blot, Over Expression, Quantitative Proteomics

Journal: PLOS Biology

Article Title: The mTORC2 subunit RICTOR drives breast cancer progression by promoting ganglioside biosynthesis through transcriptional and epigenetic mechanisms

doi: 10.1371/journal.pbio.3003362

Figure Lengend Snippet: ( A ) Immunoblots reveal an increase in pEGFR Y1068 , pEGFR Y1173 , pAKT S473 , and pERK1/2 (Y202, Y204) in MCF-7_UGCG OE and MCF-7_ZFX OE cells compared to MCF-7 cells. ( B ) Immunoblots show attenuated EGFR activation on shRNA-mediated silencing of GD3 synthase (ST8SIA1) in MCF-7 cells. ( C ) Cell proliferation assay demonstrates a decrease in cell proliferation (mean ± SEM, n = 3) of MCF-7_ST8SIA1 SH cells compared to MCF-7 cells. ( D ) Immunoblots show enhanced EGFR activation on overexpression of ST8SIA1 in MCF-7 cells. ( E ) Cell proliferation assay demonstrates increased proliferation (mean ± SEM, n = 3) of MCF-7_ST8SIA1 OE cells compared to MCF-7 cells. ( F ) Absolute quantification (mean ± SEM, n = 3–4) of GD3 gangliosides validates the silencing and overexpression of ST8SIA1 in MCF-7 cells. ( G ) Immunoblots confirm overexpression of B3GALT4 in MCF-7 cells. ( H ) Cell proliferation assay demonstrates decreased cell proliferation (mean ± SEM, n = 4) of MCF-7_B3GALT4 OE cells compared to MCF-7 cells. ( I ) Absolute quantification (mean ± SEM, n = 5) of gangliosides validates the overexpression of B3GALT4. ( J ) Cell proliferation assay (mean ± SEM, n = 3) showing an increase in proliferation of MCF-7_RICTOR SH cells upon supplementing GD3 gangliosides and a decrease in cell proliferation upon feeding with GM1 gangliosides. ( K ) Immunoblots show attenuated EGFR activation on siRNA-mediated silencing of ST8SIA1 in MCF-7_ZFX OE cells. ( L ) Cell proliferation assay demonstrates a decrease in cell proliferation (mean ± SEM, n = 4) of MCF-7_ZFX OE cells on siRNA-mediated inhibition of ST8SIA1. ( M ) Tumor growth kinetics using xenograft studies show a decrease in growth kinetics (mean ± SEM, n = 4–6) of MCF-7_ZFX OE tumors on siRNA-mediated inhibition of ST8SIA1. Data among groups were analyzed using an unpaired Student t test, among multiple groups using One-way ANOVA, and by Two-way ANOVA in time-dependent studies. p -value: * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001. Numerical data can be found in .

Article Snippet: MCF-7, BT-474, MDA-MB-453, HCT-116, HEK-293 cells (ATCC, USA), DMEM media (Cat# D5648) Sigma, USA, MEBM media (Cat# CC-3151) Lonza, Switzerland, MEM media (Cat# AL081) HiMedia, USA, DPBS (Cat# D5652) Sigma, USA, FBS (Cat# 10270) Gibco, USA, lipid-free FBS (Cat# S148L) Biowest, USA, Penicillin-Streptomycin (Cat# 113-98-43810-74-0) HyClone, USA, Lipofectamine 2000, (Cat# 11668019) Invitrogen, USA, Lipofectamine 3000 (Cat# L3000015) Invitrogen, USA, Trypsin (Cat# TCL007) HiMedia, USA, Puromycin (Cat# P7255) Sigma, USA, G418 (Cat# A1720) Sigma, USA, Haemocytometer (Cat# Z359629) Bright-Line, USA, shRNA Control (Cat# SHC202V) Sigma, USA, shRNA RICTOR Virus Particles (Cat# SHCLNV, TRCN0000289691, TRCN0000307119, TRCN0000296313, TRCN0000307122) Sigma, USA, shRNA RICTOR Glycerol stocks (SHCLNG, TRCN0000296313, TRCN0000307122,), Sigma, USA, shRNA UGCG Glycerol stocks (SHCLNG, TRCN0000036128, TRCN0000036126, TRCN0000300623) Sigma, USA, ZFX Glycerol stocks (Cat# SHCLNG TRCN0000017308, TRCN0000017309, TRCN0000017310), Sigma, USA, shRNA ST8SIA1 (Cat# SHCLNG TRCN0000417447, TRCN0000036044, TRCN0000036046), Sigma, USA, UGCG siRNA (Cat# AM51331) Ambion, USA, ZFX siRNA (Cat# L-006572-00-0005) Dharmacon, USA, Scrambled siRNA (Cat# D-001810-10-05) Dharmacon, USA, GD3 Synthase (ST8SIA1) siRNA (Cat# EHU025731-20UG) Merck, USA, 5-Aza-2′-deoxycytidine (DAC) (Cat#A3656-10MG) Sigma, USA, KDOAM25 Hydrochloride hydrate (Cat# SML2774-5MG) Sigma, USA, MK-2206 dihydrochloride (Cat# HY-10358) MedChem Express, USA, Eliglustat (Cat# HY-14885) MedChem Express, USA, Hygromycin (Cat# PCT1503), HiMedia, USA, Puromycin (Cat# P8833), Sigma, USA.

Techniques: Western Blot, Activation Assay, shRNA, Proliferation Assay, Over Expression, Quantitative Proteomics, Inhibition