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ATCC gd3 synthases
Gd3 Synthases, supplied by ATCC, used in various techniques. Bioz Stars score: 97/100, based on 390 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
https://www.bioz.com/product/gd3+synthases/Phoenix-ECO/pmc12997301-319-19-40
Average 97 stars, based on 390 article reviews

gd3 synthases - by Bioz Stars, 2026-09

97/100 stars

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Article Title: Tumors located in the brain impair the frequency and phenotype of dendritic cells in blood and tumor
Article Snippet: Cell lines were generated from parental lines CT2A (Merck), GL261 (Division of Cancer Treatment and Diagnosis Tumor Repository, NCI) and SB28-luc/GFP (DSMZ; Leibniz-InstitutDSMZ Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH). .. The cell lines were engineered to express the GD2 antigen by transduction with ecotropic retrovirus encoding murine GD2 and GD3 synthases (MP9956:SFG.GD3synthase-2A-GD2synthase was a gift from Martin Pule Addgene plasmid # 75013; http://n2t.net/addgene:75013 ; RRID:Addgene_75013; retrovirus generated from the Phoenix-Eco (ATCC Phoenix-ECO CRL-3214) packaging line), with two-rounds of FACS sorting to achieve a >99% GD2+ line. ..

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Effects of knockdown of ganglioside GM3 synthase or GD3 synthase siRNA on malignant melanoma cells. ( A ) Knockdown of ganglioside GM3 or GD3 synthase on malignant melanoma SK-MEL-28 and G-361 cells. siRNA against ganglioside GM3 or GD3 synthase was transfected with Lipofectamin ® 3000 reagent, and protein levels of ganglioside GM3 and GD3 synthase were examined after four days of transfection using immunoblotting (left panel). Blue and red squares represent ganglioside GM3 and GD3 synthase protein levels, respectively (right panel). ACTB was used as loading control. ACTB: β-actin. All data are presented as mean percentage levels ± SD ( n = 3, * p < 0.05). ( B ) RT-PCR analysis of melanoma SK-MEL-28 and G-361 cells treated with ganglioside GM3 and GD3 synthase siRNA (left panel). Blue and red squares represent ganglioside GM3 and GD3 synthase protein levels, respectively (right panel). ACTB was used as loading control. ACTB: β-actin. All data are presented as mean percentage levels ± SD ( n = 3, * p < 0.05).

Journal: International Journal of Molecular Sciences

Article Title: Quercetin Induces Mitochondrial Apoptosis and Downregulates Ganglioside GD3 Expression in Melanoma Cells

doi: 10.3390/ijms25105146

Figure Lengend Snippet: Effects of knockdown of ganglioside GM3 synthase or GD3 synthase siRNA on malignant melanoma cells. ( A ) Knockdown of ganglioside GM3 or GD3 synthase on malignant melanoma SK-MEL-28 and G-361 cells. siRNA against ganglioside GM3 or GD3 synthase was transfected with Lipofectamin ® 3000 reagent, and protein levels of ganglioside GM3 and GD3 synthase were examined after four days of transfection using immunoblotting (left panel). Blue and red squares represent ganglioside GM3 and GD3 synthase protein levels, respectively (right panel). ACTB was used as loading control. ACTB: β-actin. All data are presented as mean percentage levels ± SD ( n = 3, * p < 0.05). ( B ) RT-PCR analysis of melanoma SK-MEL-28 and G-361 cells treated with ganglioside GM3 and GD3 synthase siRNA (left panel). Blue and red squares represent ganglioside GM3 and GD3 synthase protein levels, respectively (right panel). ACTB was used as loading control. ACTB: β-actin. All data are presented as mean percentage levels ± SD ( n = 3, * p < 0.05).

Article Snippet: Ganglioside GM3 and GD3 synthase-specific siRNA, as well as control siRNA, were custom-synthesized by Thermo Fisher Scientific (Frederick, MD, USA).

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

Ganglioside siRNA inhibits relative protein levels of FAK, paxillin, and Akt in malignant melanoma cells. ( A ) Knockdown of ganglioside GM3 or GD3 synthase on malignant melanoma SK-MEL-28 (left panel) and G-361 (right panel) cells. Effects of knockdown of ganglioside GM3 or GD3 synthase on phosphorylation levels of paxillin and Akt in malignant melanoma SK-MEL-28 and G-361 cells. Reduction in tyrosine phosphorylation was observed, and its total protein level was not affected. ACTB was used as loading control. ACTB: β-actin. All data shown were representative of at least three independent experiments. ( B ) Knockdown of ganglioside GM3 or GD3 synthase treated with or without quercetin on malignant melanoma SK-MEL-28 and G-361 cells. Protein levels of FAK, paxillin, and Akt and phosphorylation levels of Akt and paxillin were examined after 24 h in untreated and quercetin-treated (250 µM) panel cells using immunoblotting analysis. ACTB was used as loading control. ACTB: β-actin. All data shown were representative of at least three independent experiments.

Journal: International Journal of Molecular Sciences

Article Title: Quercetin Induces Mitochondrial Apoptosis and Downregulates Ganglioside GD3 Expression in Melanoma Cells

doi: 10.3390/ijms25105146

Figure Lengend Snippet: Ganglioside siRNA inhibits relative protein levels of FAK, paxillin, and Akt in malignant melanoma cells. ( A ) Knockdown of ganglioside GM3 or GD3 synthase on malignant melanoma SK-MEL-28 (left panel) and G-361 (right panel) cells. Effects of knockdown of ganglioside GM3 or GD3 synthase on phosphorylation levels of paxillin and Akt in malignant melanoma SK-MEL-28 and G-361 cells. Reduction in tyrosine phosphorylation was observed, and its total protein level was not affected. ACTB was used as loading control. ACTB: β-actin. All data shown were representative of at least three independent experiments. ( B ) Knockdown of ganglioside GM3 or GD3 synthase treated with or without quercetin on malignant melanoma SK-MEL-28 and G-361 cells. Protein levels of FAK, paxillin, and Akt and phosphorylation levels of Akt and paxillin were examined after 24 h in untreated and quercetin-treated (250 µM) panel cells using immunoblotting analysis. ACTB was used as loading control. ACTB: β-actin. All data shown were representative of at least three independent experiments.

Article Snippet: Ganglioside GM3 and GD3 synthase-specific siRNA, as well as control siRNA, were custom-synthesized by Thermo Fisher Scientific (Frederick, MD, USA).

Techniques: Knockdown, Phospho-proteomics, Control, Western Blot

( 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 .

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

( 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 .

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 .

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

(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 .

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 .

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

( 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 .

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 .

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

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