Structured Review

DSMZ patu 8902
CAF-derived pyruvate confers resistance to complex I inhibitors in vitro. ( A ) Absolute quantitation of aspartate (Asp) (left) and α-ketoglutarate concentrations (µM) in human cancer-associated fibroblasts (hCAF) or mouse CAF (mCAF) conditioned media (CM), relative to Dulbecco’s modified Eagle medium (DMEM) control. ( B ) Relative proliferation of MIAPaCa-2 ishGOT2.1 −Dox (n=3) or +Dox (n=3) cultured in normal DMEM, 250 µM pyruvate, or a combination of 50 µM Asp and 500 µM dimethyl-α-ketoglutarate (dmαKG). ( C ) Relative cell number of <t>PaTu-8902</t> treated with the indicated concentrations of rotenone (left), phenformin (middle), or IACS-010759 (right), and cultured in normal DMEM, hCAF CM, or 1 mM pyruvate. For all panels, data represent mean ± SD. *p
Patu 8902, supplied by DSMZ, used in various techniques. Bioz Stars score: 92/100, based on 10 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
https://www.bioz.com/result/patu 8902/product/DSMZ
Average 92 stars, based on 10 article reviews
Price from $9.99 to $1999.99
patu 8902 - by Bioz Stars, 2022-11
92/100 stars

Images

1) Product Images from "Metabolic requirement for GOT2 in pancreatic cancer depends on environmental context"

Article Title: Metabolic requirement for GOT2 in pancreatic cancer depends on environmental context

Journal: eLife

doi: 10.7554/eLife.73245

CAF-derived pyruvate confers resistance to complex I inhibitors in vitro. ( A ) Absolute quantitation of aspartate (Asp) (left) and α-ketoglutarate concentrations (µM) in human cancer-associated fibroblasts (hCAF) or mouse CAF (mCAF) conditioned media (CM), relative to Dulbecco’s modified Eagle medium (DMEM) control. ( B ) Relative proliferation of MIAPaCa-2 ishGOT2.1 −Dox (n=3) or +Dox (n=3) cultured in normal DMEM, 250 µM pyruvate, or a combination of 50 µM Asp and 500 µM dimethyl-α-ketoglutarate (dmαKG). ( C ) Relative cell number of PaTu-8902 treated with the indicated concentrations of rotenone (left), phenformin (middle), or IACS-010759 (right), and cultured in normal DMEM, hCAF CM, or 1 mM pyruvate. For all panels, data represent mean ± SD. *p
Figure Legend Snippet: CAF-derived pyruvate confers resistance to complex I inhibitors in vitro. ( A ) Absolute quantitation of aspartate (Asp) (left) and α-ketoglutarate concentrations (µM) in human cancer-associated fibroblasts (hCAF) or mouse CAF (mCAF) conditioned media (CM), relative to Dulbecco’s modified Eagle medium (DMEM) control. ( B ) Relative proliferation of MIAPaCa-2 ishGOT2.1 −Dox (n=3) or +Dox (n=3) cultured in normal DMEM, 250 µM pyruvate, or a combination of 50 µM Asp and 500 µM dimethyl-α-ketoglutarate (dmαKG). ( C ) Relative cell number of PaTu-8902 treated with the indicated concentrations of rotenone (left), phenformin (middle), or IACS-010759 (right), and cultured in normal DMEM, hCAF CM, or 1 mM pyruvate. For all panels, data represent mean ± SD. *p

Techniques Used: Derivative Assay, In Vitro, Quantitation Assay, Modification, Cell Culture

LDHA inhibition does not sensitize tumors to GOT2 KD in vivo. ( A,B ) Tumor volume (left) and weight (right) of PaTu-8902 ( A ) and MIAPaCa-2 ( B ) ishGOT2.1 flank xenografts in NOD scid gamma (NSG) mice fed normal chow (−Dox, n=6) or doxycycline chow (+Dox, n=6) and treated with PBS control or 2 mg/kg FX11. ( C ) Relative proliferation of PaTu-8902 (top) and MIAPaCa-2 (bottom) ishRNA −Dox (n=3) or +Dox (n=3) cultured in normal Dulbecco’s modified Eagle medium (DMEM), 0.25 mM pyruvate, or 2% BSA, normalized to Day 0 for each condition.
Figure Legend Snippet: LDHA inhibition does not sensitize tumors to GOT2 KD in vivo. ( A,B ) Tumor volume (left) and weight (right) of PaTu-8902 ( A ) and MIAPaCa-2 ( B ) ishGOT2.1 flank xenografts in NOD scid gamma (NSG) mice fed normal chow (−Dox, n=6) or doxycycline chow (+Dox, n=6) and treated with PBS control or 2 mg/kg FX11. ( C ) Relative proliferation of PaTu-8902 (top) and MIAPaCa-2 (bottom) ishRNA −Dox (n=3) or +Dox (n=3) cultured in normal Dulbecco’s modified Eagle medium (DMEM), 0.25 mM pyruvate, or 2% BSA, normalized to Day 0 for each condition.

Techniques Used: Inhibition, In Vivo, Mouse Assay, Cell Culture, Modification

GOT2 KD can be rescued by cytosolic, but not mitochondrial, expression of LbNOX. ( A ) Immunoblots of glutamate-oxaloacetate transaminase 2 (GOT2), FLAG, and VINCULIN loading controls from PaTu-8902 (left) or MIAPaCa-2 (right) ishNT or GOT2.1 cells expressing doxycycline-inducible expression of empty vector (EV) or FLAG-tagged cytosolic Lactobacillus NADH oxidase (LbNOX) or mitochondrial LbNOX (mLbNOX). ( B ) Relative colony formation of PaTu-8902 (left) and MIAPaCa-2 (right) ishGOT2.1 −Dox (n=3) or +Dox (n=3) expressing EV, LbNOX, or mLbNOX, normalized to −Dox for each condition. ( C ) Relative proliferation of PaTu-8902 (top) and MIAPaCa-2 (bottom) ishNT −Dox (n=3) or +Dox (n=3) expressing EV, LbNOX, or mLbNOX and treated with dimethyl sulfoxide (DMSO) vehicle control (left) or 1 µM piericidin (right), normalized to Day 0 for each condition. ( D ) Ion abundances of NAD+ (left) and NADH (right) in PaTu-8902 (top) and MIAPaCa-2 (bottom) ishGOT2.1 −Dox (n=3) and +Dox (n=3). ( E ) Relative extracellular pyruvate/lactate ratios in PaTu-8902 (left) and MIAPaCa-2 (right) ishGOT2.1 −Dox (n=3) and +Dox (n=3). ( F ) Ion abundances of extracellular pyruvate (left) and lactate (right) in PaTu-8902 (top) and MIAPaCa-2 (bottom) ishGOT2.1 −Dox (n=3) and +Dox (n=3). ( G ) Heatmap of log2 fold changes in metabolite abundances between PaTu-8902 (left) and MIAPaCa-2 (right) ishGOT2.1 −Dox (n=3) and +Dox (n=3) expressing EV, LbNOX, or mLbNOX. G6 P : glucose-6-phosphate; F6 P : fructose-6-phosphate; FBP: fructose-1,6-bisphosphate; DHAP: dihydroxyacetone phosphate; 2PG: 2-phosphoglycerate; PEP: phosphoenol pyruvate; X5 P : xylulose-5-phosphate; R5 P : ribose-5-phosphate; S7 P : seduoheptulose-7-phosphate; αKG: α-ketoglutarate; and P : pentose phosphate pathway. For all panels, data represent mean ± SD. *p
Figure Legend Snippet: GOT2 KD can be rescued by cytosolic, but not mitochondrial, expression of LbNOX. ( A ) Immunoblots of glutamate-oxaloacetate transaminase 2 (GOT2), FLAG, and VINCULIN loading controls from PaTu-8902 (left) or MIAPaCa-2 (right) ishNT or GOT2.1 cells expressing doxycycline-inducible expression of empty vector (EV) or FLAG-tagged cytosolic Lactobacillus NADH oxidase (LbNOX) or mitochondrial LbNOX (mLbNOX). ( B ) Relative colony formation of PaTu-8902 (left) and MIAPaCa-2 (right) ishGOT2.1 −Dox (n=3) or +Dox (n=3) expressing EV, LbNOX, or mLbNOX, normalized to −Dox for each condition. ( C ) Relative proliferation of PaTu-8902 (top) and MIAPaCa-2 (bottom) ishNT −Dox (n=3) or +Dox (n=3) expressing EV, LbNOX, or mLbNOX and treated with dimethyl sulfoxide (DMSO) vehicle control (left) or 1 µM piericidin (right), normalized to Day 0 for each condition. ( D ) Ion abundances of NAD+ (left) and NADH (right) in PaTu-8902 (top) and MIAPaCa-2 (bottom) ishGOT2.1 −Dox (n=3) and +Dox (n=3). ( E ) Relative extracellular pyruvate/lactate ratios in PaTu-8902 (left) and MIAPaCa-2 (right) ishGOT2.1 −Dox (n=3) and +Dox (n=3). ( F ) Ion abundances of extracellular pyruvate (left) and lactate (right) in PaTu-8902 (top) and MIAPaCa-2 (bottom) ishGOT2.1 −Dox (n=3) and +Dox (n=3). ( G ) Heatmap of log2 fold changes in metabolite abundances between PaTu-8902 (left) and MIAPaCa-2 (right) ishGOT2.1 −Dox (n=3) and +Dox (n=3) expressing EV, LbNOX, or mLbNOX. G6 P : glucose-6-phosphate; F6 P : fructose-6-phosphate; FBP: fructose-1,6-bisphosphate; DHAP: dihydroxyacetone phosphate; 2PG: 2-phosphoglycerate; PEP: phosphoenol pyruvate; X5 P : xylulose-5-phosphate; R5 P : ribose-5-phosphate; S7 P : seduoheptulose-7-phosphate; αKG: α-ketoglutarate; and P : pentose phosphate pathway. For all panels, data represent mean ± SD. *p

Techniques Used: Expressing, Western Blot, Plasmid Preparation

GOT2 loss in vitro slows glycolysis and can be rescued by exogenous electron acceptors. ( A ) Glycolytic rate assay showing the proton efflux rate (PER) of PaTu-8902 ishGOT2.1 −Dox (n=4) or +Dox (n=4). ( B–C ) Relative proliferation (B) and ATP levels (C) of PaTu-8902 ishGOT2.1 −Dox (n=3) or +Dox (n=3) cultured in normal Dulbecco’s modified Eagle medium (DMEM) or DMEM with 1 mM pyruvate (Pyr). ( D ) Immunoblots of GOT2 and VINCULIN loading control in PaTu-8902 (top) and MIAPaCa-2 (bottom) parental ( P ), empty vector (EV), or two sgRNAs targeting GOT2 (sg1, sg2). ( E ) Relative colony formation of PaTu-8902 (left) and MIAPaCa-2 (right) P, EV, sgGOT2.1, or sgGOT2.2 cultured in normal DMEM (−Pyr, n=3) or DMEM with 1 mM pyruvate (+Pyr, n=3), normalized to +Pyr for each condition. ( F ) Relative colony formation of PaTu-8902 (left) and MIAPaCa-2 (right) ishGOT2.1 −Dox (n=3) or +Dox (n=3) cultured in normal media (DMEM) or DMEM with 1 mM α-ketobutyrate (αKB), normalized to −Dox for each condition. ( G ) Relative colony formation of MIAPaCa-2 ishGOT2.1 −Dox (n=3) or +Dox (n=3) cultured in normal media (DMEM) or DMEM with 1 mM Pyr, αKB, or nicotinaminde mononucleotide (NMN), normalized to −Dox for each condition. For all panels, data represent mean ± SD. *p
Figure Legend Snippet: GOT2 loss in vitro slows glycolysis and can be rescued by exogenous electron acceptors. ( A ) Glycolytic rate assay showing the proton efflux rate (PER) of PaTu-8902 ishGOT2.1 −Dox (n=4) or +Dox (n=4). ( B–C ) Relative proliferation (B) and ATP levels (C) of PaTu-8902 ishGOT2.1 −Dox (n=3) or +Dox (n=3) cultured in normal Dulbecco’s modified Eagle medium (DMEM) or DMEM with 1 mM pyruvate (Pyr). ( D ) Immunoblots of GOT2 and VINCULIN loading control in PaTu-8902 (top) and MIAPaCa-2 (bottom) parental ( P ), empty vector (EV), or two sgRNAs targeting GOT2 (sg1, sg2). ( E ) Relative colony formation of PaTu-8902 (left) and MIAPaCa-2 (right) P, EV, sgGOT2.1, or sgGOT2.2 cultured in normal DMEM (−Pyr, n=3) or DMEM with 1 mM pyruvate (+Pyr, n=3), normalized to +Pyr for each condition. ( F ) Relative colony formation of PaTu-8902 (left) and MIAPaCa-2 (right) ishGOT2.1 −Dox (n=3) or +Dox (n=3) cultured in normal media (DMEM) or DMEM with 1 mM α-ketobutyrate (αKB), normalized to −Dox for each condition. ( G ) Relative colony formation of MIAPaCa-2 ishGOT2.1 −Dox (n=3) or +Dox (n=3) cultured in normal media (DMEM) or DMEM with 1 mM Pyr, αKB, or nicotinaminde mononucleotide (NMN), normalized to −Dox for each condition. For all panels, data represent mean ± SD. *p

Techniques Used: In Vitro, Cell Culture, Modification, Western Blot, Plasmid Preparation

CAF metabolism supports GOT2 KD pancreatic cancer cells in vitro. ( A ) Immunohistochemistry (IHC) for α-smooth muscle actin (αSMA) in PaTu-8902 ishGOT2.1 −Dox or +Dox flank xenografts. Scale bar is 200 µM. ( B ) Relative colony formation of PaTu-8902 ishGOT2.1 −Dox (n=3) or +Dox (n=3) cultured in normal media (Dulbecco’s modified Eagle medium, DMEM) or human cancer-associated fibroblasts (hCAF) conditioned media (CM), normalized to −Dox for each condition. ( C ) Relative colony formation of PaTu-8902 ishGOT2.1 −Dox (n=3) or +Dox (n=3) cultured in DMEM, hCAF CM, pancreatic ductal adenocarcinoma (PDA) CM, or tumor-educated macrophage (TEM) CM, normalized to −Dox for each condition. ( D ) Relative colony formation of PaTu-8902 ishGOT2.1 −Dox (n=3) or +Dox (n=3) cultured in DMEM, hCAF CM, or hCAF CM boiled, 3 kDa filtered, or subjected to freeze/thaw cycles, normalized to −Dox for each condition. ( E ) Heatmap of the relative abundances of metabolites significantly (p
Figure Legend Snippet: CAF metabolism supports GOT2 KD pancreatic cancer cells in vitro. ( A ) Immunohistochemistry (IHC) for α-smooth muscle actin (αSMA) in PaTu-8902 ishGOT2.1 −Dox or +Dox flank xenografts. Scale bar is 200 µM. ( B ) Relative colony formation of PaTu-8902 ishGOT2.1 −Dox (n=3) or +Dox (n=3) cultured in normal media (Dulbecco’s modified Eagle medium, DMEM) or human cancer-associated fibroblasts (hCAF) conditioned media (CM), normalized to −Dox for each condition. ( C ) Relative colony formation of PaTu-8902 ishGOT2.1 −Dox (n=3) or +Dox (n=3) cultured in DMEM, hCAF CM, pancreatic ductal adenocarcinoma (PDA) CM, or tumor-educated macrophage (TEM) CM, normalized to −Dox for each condition. ( D ) Relative colony formation of PaTu-8902 ishGOT2.1 −Dox (n=3) or +Dox (n=3) cultured in DMEM, hCAF CM, or hCAF CM boiled, 3 kDa filtered, or subjected to freeze/thaw cycles, normalized to −Dox for each condition. ( E ) Heatmap of the relative abundances of metabolites significantly (p

Techniques Used: In Vitro, Immunohistochemistry, Cell Culture, Modification, Transmission Electron Microscopy

MCT1 and LDHA inhibition dampen pyruvate rescue of GOT2 KD in vitro. ( A ) Relative Cancer Cell Line Encyclopedia (CCLE) mRNA expression of the indicated SLC16 family members in PaTu-8902 and MIAPaCa-2. ( B ) Immunoblot for MCT1 and VINCULIN loading control the indicated cell lines. ( C ) Single-cell RNA sequencing data from murine syngeneic orthotopic tumors showing expression of Slc16a1 (MCT1) and Slc16a3 (MCT4) in cancer-associated fibroblasts (CAF; marked by CDH11 expression) and epithelial (marked by Krt18 expression) populations. ( D,F ) Immunoblots for glutamate-oxaloacetate transaminase 2 (GOT2), MCT1, and VINCULIN in PaTu-8902 (D) and MIAPaCa-2 (F) ishGOT2.1 −Dox or +Dox expressing the indicated control (CTR) or constitutive MCT1-targeting shRNAs (sh1, sh2). ( E,G ) Relative proliferation of PaTu-8902 (E) and MIAPaCa-2 (G) ishGOT2.1 −Dox (n=3) or +Dox (n=3) expressing the indicated shRNAs and cultured in normal Dulbecco’s modified Eagle medium (DMEM; left), 0.25 mM pyruvate (middle), or human CAF (hCAF) CM (right), normalized to Day 0 for each condition. ( H ) Relative colony formation of MIAPaCa-2 ishGOT2.1 −Dox (n=3) or +Dox (n=3) cultured in normal media (DMEM) or the indicated doses of pyruvate, and treated with DMSO control or 100 nM AZD3965, normalized to −Dox for each condition. ( I ) Relative viability of MIAPaCa-2 cultured in normal media (DMEM), 0.25 mM pyruvate (left), or hCAF CM (right) and treated with DMSO vehicle control or 100 nM IACS-010759, alone or in combination with 100 nM AZD3965, normalized to −Dox for each condition. ( J ) Immunoblots for GOT2, LDHA, LDHB, and VINCULIN in PaTu-8902 and MIAPaCa-2 ishRNA. ( K ) Relative number of PaTu-8902 (left) and MIAPaCa-2 (right) ishGOT2.1 −Dox (n=3) or +Dox (n=3) cultured in normal DMEM, 0.25 mM pyruvate, or hCAF CM, and treated with DMSO vehicle control or 25 µM FX11, normalized to −Dox for each condition. For all panels, data represent mean ± SD. *p
Figure Legend Snippet: MCT1 and LDHA inhibition dampen pyruvate rescue of GOT2 KD in vitro. ( A ) Relative Cancer Cell Line Encyclopedia (CCLE) mRNA expression of the indicated SLC16 family members in PaTu-8902 and MIAPaCa-2. ( B ) Immunoblot for MCT1 and VINCULIN loading control the indicated cell lines. ( C ) Single-cell RNA sequencing data from murine syngeneic orthotopic tumors showing expression of Slc16a1 (MCT1) and Slc16a3 (MCT4) in cancer-associated fibroblasts (CAF; marked by CDH11 expression) and epithelial (marked by Krt18 expression) populations. ( D,F ) Immunoblots for glutamate-oxaloacetate transaminase 2 (GOT2), MCT1, and VINCULIN in PaTu-8902 (D) and MIAPaCa-2 (F) ishGOT2.1 −Dox or +Dox expressing the indicated control (CTR) or constitutive MCT1-targeting shRNAs (sh1, sh2). ( E,G ) Relative proliferation of PaTu-8902 (E) and MIAPaCa-2 (G) ishGOT2.1 −Dox (n=3) or +Dox (n=3) expressing the indicated shRNAs and cultured in normal Dulbecco’s modified Eagle medium (DMEM; left), 0.25 mM pyruvate (middle), or human CAF (hCAF) CM (right), normalized to Day 0 for each condition. ( H ) Relative colony formation of MIAPaCa-2 ishGOT2.1 −Dox (n=3) or +Dox (n=3) cultured in normal media (DMEM) or the indicated doses of pyruvate, and treated with DMSO control or 100 nM AZD3965, normalized to −Dox for each condition. ( I ) Relative viability of MIAPaCa-2 cultured in normal media (DMEM), 0.25 mM pyruvate (left), or hCAF CM (right) and treated with DMSO vehicle control or 100 nM IACS-010759, alone or in combination with 100 nM AZD3965, normalized to −Dox for each condition. ( J ) Immunoblots for GOT2, LDHA, LDHB, and VINCULIN in PaTu-8902 and MIAPaCa-2 ishRNA. ( K ) Relative number of PaTu-8902 (left) and MIAPaCa-2 (right) ishGOT2.1 −Dox (n=3) or +Dox (n=3) cultured in normal DMEM, 0.25 mM pyruvate, or hCAF CM, and treated with DMSO vehicle control or 25 µM FX11, normalized to −Dox for each condition. For all panels, data represent mean ± SD. *p

Techniques Used: Inhibition, In Vitro, Expressing, RNA Sequencing Assay, Western Blot, Cell Culture, Modification

Cytosolic reduction of pyruvate to lactate is necessary for GOT2 KD. ( A ) Relative proliferation of PaTu-8902 (left) and MIAPaCa-2 (right) ishGOT2.1 −Dox (n=3) or +Dox (n=3) cultured in 1 mM glucose Dulbecco’s modified Eagle medium (DMEM) with 1 mM pyruvate (Pyr) normalized to Day 0 for each condition. ( B–D ) Fractional labeling of intracellular citrate ( B ), aspartate ( C ), or alanine ( D ) from 13C3-pyruvate (1 mM) in PaTu-8902 and MIAPaCa-2 ishGOT2.1 −Dox (n=3) or +Dox (n=3) treated with DMSO vehicle control or 5 µM UK5099 (mitochondrial pyruvate carrier (MPC) inhibitor). Unlabeled controls presented at right. ( E ) Relative cell number of MIAPaCa-2 ishGOT2.1 −Dox (n=3) or +Dox (n=3) cultured in normal DMEM or DMEM with 1 mM Pyr, treated with DMSO vehicle control or 5 µM UK5099, and normalized to DMSO for each condition. ( F ) Relative colony formation of MIAPaCa-2 ishGOT2.1 −Dox (n=3) or +Dox (n=3) cultured in normal DMEM or DMEM with 1 mM Pyr or lactate (Lac), normalized to −Dox for each condition. ( G ) Relative colony formation of PaTu-8902 (left) and MIAPaCa-2 (right) ishGOT2.1 −Dox (n=3) or +Dox (n=3) cultured in normal DMEM or DMEM with 1 mM alanine, normalized to −Dox for each condition. ( H ) Relative colony formation of MIAPaCa-2 ishGOT2.1 −Dox (n=3) or +Dox (n=3) cultured in normal DMEM or DMEM with either 1 mM Pyr or 100 µM of the indicated combinations of adenine ( A ), guanine ( G ), thymidine ( T ), and cytidine ( C ), normalized to −Dox for each condition. For all panels, data represent mean ± SD. *p
Figure Legend Snippet: Cytosolic reduction of pyruvate to lactate is necessary for GOT2 KD. ( A ) Relative proliferation of PaTu-8902 (left) and MIAPaCa-2 (right) ishGOT2.1 −Dox (n=3) or +Dox (n=3) cultured in 1 mM glucose Dulbecco’s modified Eagle medium (DMEM) with 1 mM pyruvate (Pyr) normalized to Day 0 for each condition. ( B–D ) Fractional labeling of intracellular citrate ( B ), aspartate ( C ), or alanine ( D ) from 13C3-pyruvate (1 mM) in PaTu-8902 and MIAPaCa-2 ishGOT2.1 −Dox (n=3) or +Dox (n=3) treated with DMSO vehicle control or 5 µM UK5099 (mitochondrial pyruvate carrier (MPC) inhibitor). Unlabeled controls presented at right. ( E ) Relative cell number of MIAPaCa-2 ishGOT2.1 −Dox (n=3) or +Dox (n=3) cultured in normal DMEM or DMEM with 1 mM Pyr, treated with DMSO vehicle control or 5 µM UK5099, and normalized to DMSO for each condition. ( F ) Relative colony formation of MIAPaCa-2 ishGOT2.1 −Dox (n=3) or +Dox (n=3) cultured in normal DMEM or DMEM with 1 mM Pyr or lactate (Lac), normalized to −Dox for each condition. ( G ) Relative colony formation of PaTu-8902 (left) and MIAPaCa-2 (right) ishGOT2.1 −Dox (n=3) or +Dox (n=3) cultured in normal DMEM or DMEM with 1 mM alanine, normalized to −Dox for each condition. ( H ) Relative colony formation of MIAPaCa-2 ishGOT2.1 −Dox (n=3) or +Dox (n=3) cultured in normal DMEM or DMEM with either 1 mM Pyr or 100 µM of the indicated combinations of adenine ( A ), guanine ( G ), thymidine ( T ), and cytidine ( C ), normalized to −Dox for each condition. For all panels, data represent mean ± SD. *p

Techniques Used: Cell Culture, Modification, Labeling

MCT1 inhibition does not sensitize tumors to GOT2 KD in vivo. ( A,B ) Tumor volume (left) and weight (right) of PaTu-8902 (A) and tumor volume of MIAPaCa-2 (B) ishGOT2.1 flank xenografts in NOD scid gamma (NSG) mice fed normal chow (−Dox, n=6) or doxycycline chow (+Dox, n=6) expressing control (shNT) or shMCT1.1. ( C,D ) Immunoblots for GOT2, MCT1, and VINCULIN in representative tumors from PaTu-8902 (C) or MIAPaCa-2 (D) ishGOT2.1 −Dox (n=3) or +Dox (n=3) flank xenografts with shNT or shMCT1.1. Each lane represents an individual tumor. ( E ) Tumor volume (left) and weight (right) of PaTu-8902 flank xenografts in NSG mice fed normal chow (−Dox, n=6) or doxycycline chow (+Dox, n=6) expressing control (sgEV) or sgMCT1.1. ( F ) Immunoblot for GOT2, MCT1, MCT4, and VINCULIN in representative, independent tumors from PaTu-8902 ishGOT2.1 −Dox (n=3) or +Dox (n=3) flank xenografts with sgEV or sgMCT1.1. ( G ) Immunoblot for GOT2, MCT1, MCT4, and VINCULIN in PaTu-8902 ishGOT2.1 −Dox or +Dox cell lines with sgEV or sgMCT1.1. For (F, G) blots, arrow = MCT1 band, asterisk = non-specific band. ( H ) Tumor volume (left) and weight (right) of PaTu-8902 flank xenografts in NSG mice fed normal chow (−Dox, n=6) or doxycycline chow (+Dox, n=6) treated with PBS control or 100 mg/kg AZD3965. ( I ) Immunoblot for GOT2, MCT1, and VINCULIN in representative, independent tumors of PaTu-8902 ishGOT2.1 −Dox (n=3) or +Dox (n=3) flank xenografts. ( J ) Weights of mice treated in (H). For all panels, data represent mean ± SD. *p
Figure Legend Snippet: MCT1 inhibition does not sensitize tumors to GOT2 KD in vivo. ( A,B ) Tumor volume (left) and weight (right) of PaTu-8902 (A) and tumor volume of MIAPaCa-2 (B) ishGOT2.1 flank xenografts in NOD scid gamma (NSG) mice fed normal chow (−Dox, n=6) or doxycycline chow (+Dox, n=6) expressing control (shNT) or shMCT1.1. ( C,D ) Immunoblots for GOT2, MCT1, and VINCULIN in representative tumors from PaTu-8902 (C) or MIAPaCa-2 (D) ishGOT2.1 −Dox (n=3) or +Dox (n=3) flank xenografts with shNT or shMCT1.1. Each lane represents an individual tumor. ( E ) Tumor volume (left) and weight (right) of PaTu-8902 flank xenografts in NSG mice fed normal chow (−Dox, n=6) or doxycycline chow (+Dox, n=6) expressing control (sgEV) or sgMCT1.1. ( F ) Immunoblot for GOT2, MCT1, MCT4, and VINCULIN in representative, independent tumors from PaTu-8902 ishGOT2.1 −Dox (n=3) or +Dox (n=3) flank xenografts with sgEV or sgMCT1.1. ( G ) Immunoblot for GOT2, MCT1, MCT4, and VINCULIN in PaTu-8902 ishGOT2.1 −Dox or +Dox cell lines with sgEV or sgMCT1.1. For (F, G) blots, arrow = MCT1 band, asterisk = non-specific band. ( H ) Tumor volume (left) and weight (right) of PaTu-8902 flank xenografts in NSG mice fed normal chow (−Dox, n=6) or doxycycline chow (+Dox, n=6) treated with PBS control or 100 mg/kg AZD3965. ( I ) Immunoblot for GOT2, MCT1, and VINCULIN in representative, independent tumors of PaTu-8902 ishGOT2.1 −Dox (n=3) or +Dox (n=3) flank xenografts. ( J ) Weights of mice treated in (H). For all panels, data represent mean ± SD. *p

Techniques Used: Inhibition, In Vivo, Mouse Assay, Expressing, Western Blot

GOT2 knockdown (KD) impairs in vitro colony formation and proliferation of pancreatic cancer cell lines. ( A ) Immunoblots of glutamate-oxaloacetate transaminase 2 (GOT2) and VINCULIN loading control in pancreatic ductal adenocarcinoma (PDA) cell lines after 1 µg/mL doxycycline (Dox) induction of the indicated shRNAs (ishRNA) for 3 days. NT: non-targeting shRNA. Westerns for data in Figure 1D . ( B ) Immunoblot of GOT1, GOT2, and VINCULIN in PaTu-8902 ishRNA −Dox or +Dox after 3 days. ( C ) Representative images from colony formation assays in PDA ishRNA cell lines −Dox (n=3) or +Dox (n=3) for the data in Figure 1D . ( D ) Relative proliferation of PDA ishRNA cell lines −Dox (n=3) or +Dox (n=3), normalized to Day 0 cell number for each condition. ( E ) Immunoblots of GOT2 and VINCULIN in human cancer-associated fibroblast cell line human pancreatic stellate cells (hPSC) and human pancreatic nestin expressing cells (HPNE) ishRNA cells. ( F ) Relative proliferation of hPSC (left) and HPNE (right) ishRNA −Dox (n=3) or +Dox (n=3). For all panels, data represent mean ± SD. *p
Figure Legend Snippet: GOT2 knockdown (KD) impairs in vitro colony formation and proliferation of pancreatic cancer cell lines. ( A ) Immunoblots of glutamate-oxaloacetate transaminase 2 (GOT2) and VINCULIN loading control in pancreatic ductal adenocarcinoma (PDA) cell lines after 1 µg/mL doxycycline (Dox) induction of the indicated shRNAs (ishRNA) for 3 days. NT: non-targeting shRNA. Westerns for data in Figure 1D . ( B ) Immunoblot of GOT1, GOT2, and VINCULIN in PaTu-8902 ishRNA −Dox or +Dox after 3 days. ( C ) Representative images from colony formation assays in PDA ishRNA cell lines −Dox (n=3) or +Dox (n=3) for the data in Figure 1D . ( D ) Relative proliferation of PDA ishRNA cell lines −Dox (n=3) or +Dox (n=3), normalized to Day 0 cell number for each condition. ( E ) Immunoblots of GOT2 and VINCULIN in human cancer-associated fibroblast cell line human pancreatic stellate cells (hPSC) and human pancreatic nestin expressing cells (HPNE) ishRNA cells. ( F ) Relative proliferation of hPSC (left) and HPNE (right) ishRNA −Dox (n=3) or +Dox (n=3). For all panels, data represent mean ± SD. *p

Techniques Used: In Vitro, Western Blot, shRNA, Expressing

2) Product Images from "The Pancreatic Tumor Microenvironment Compensates for Loss of GOT2"

Article Title: The Pancreatic Tumor Microenvironment Compensates for Loss of GOT2

Journal: bioRxiv

doi: 10.1101/2020.08.07.238766

A) CCLE relative transcript expression levels of MCT family members in PDA cell lines. B) Western blot for MCT1, with vinculin loading control, in hCAFs or PaTu-8902 and MIAPaCa-2 cells. C) Single-cell RNA sequencing data from KPC syngeneic orthotopic tumors showing expression of Slc16a1 (MCT1) and Slc16a3 (MCT4) in CAF (CDH11) and epithelial (Krt18) populations. D, F) Western blots validating iDox-shGOT2.1 and constitutive MCT1 KD PaTu-8902 or MIAPaCa-2 cell lines, with vinculin loading controls. E,G ) Growth curves of PaTu-8902 or MIAPaCa-2 iDox-shGOT2.1, MCT1 KD cell lines cultured in DMEM, 0.25 mM pyruvate, or hCAF CM. H) Western blots for LDHA and LDHB expression PaTu-8902 or MIAPaCa-2 iDox-shGOT2.1 cell lines, with vinculin loading controls. I,J) Growth curves of PaTu-8902 or MIAPaCa-2 iDox-shGOT2.1 cell lines cultured in DMEM, 0.25 mM pyruvate, or hCAF CM and treated with 25 μM FX11.
Figure Legend Snippet: A) CCLE relative transcript expression levels of MCT family members in PDA cell lines. B) Western blot for MCT1, with vinculin loading control, in hCAFs or PaTu-8902 and MIAPaCa-2 cells. C) Single-cell RNA sequencing data from KPC syngeneic orthotopic tumors showing expression of Slc16a1 (MCT1) and Slc16a3 (MCT4) in CAF (CDH11) and epithelial (Krt18) populations. D, F) Western blots validating iDox-shGOT2.1 and constitutive MCT1 KD PaTu-8902 or MIAPaCa-2 cell lines, with vinculin loading controls. E,G ) Growth curves of PaTu-8902 or MIAPaCa-2 iDox-shGOT2.1, MCT1 KD cell lines cultured in DMEM, 0.25 mM pyruvate, or hCAF CM. H) Western blots for LDHA and LDHB expression PaTu-8902 or MIAPaCa-2 iDox-shGOT2.1 cell lines, with vinculin loading controls. I,J) Growth curves of PaTu-8902 or MIAPaCa-2 iDox-shGOT2.1 cell lines cultured in DMEM, 0.25 mM pyruvate, or hCAF CM and treated with 25 μM FX11.

Techniques Used: Expressing, Western Blot, RNA Sequencing Assay, Cell Culture

GOT2 is not required for PDA tumor growth in vivo. A) Growth of PaTu-8902 iDox-shNT, shGOT2.1, or shGOT2.2 subcutaneous xenografts in NSG mice (n=6 tumors per group). Arrows indicate administration of doxycycline chow. B) Growth across a panel of subcutaneous PDA iDox-shGOT2.1 subcutaneous xenografts in NSG mice (n=6 tumors per group). Arrows indicate administration of doxycycline chow. C) Western blot for expression of GOT2 and Vinculin loading control in PaTu-8902 iDox-shNT or shGOT2.1 subcutaneous xenografts. D) Representative images of Ki67 staining in tissue slices from PaTu-8902 iDox-shNT or shGOT2.1 subcutaneous xenografts, 10x. E) Quantification of nuclei positive for Ki67 in tissue slices from (B) (n=6 tumors per group). F-H) Metabolites significantly changed between PaTu-8902 iDox-shGOT2.1 +DOX (n=6) and –DOX (n=6) ((p
Figure Legend Snippet: GOT2 is not required for PDA tumor growth in vivo. A) Growth of PaTu-8902 iDox-shNT, shGOT2.1, or shGOT2.2 subcutaneous xenografts in NSG mice (n=6 tumors per group). Arrows indicate administration of doxycycline chow. B) Growth across a panel of subcutaneous PDA iDox-shGOT2.1 subcutaneous xenografts in NSG mice (n=6 tumors per group). Arrows indicate administration of doxycycline chow. C) Western blot for expression of GOT2 and Vinculin loading control in PaTu-8902 iDox-shNT or shGOT2.1 subcutaneous xenografts. D) Representative images of Ki67 staining in tissue slices from PaTu-8902 iDox-shNT or shGOT2.1 subcutaneous xenografts, 10x. E) Quantification of nuclei positive for Ki67 in tissue slices from (B) (n=6 tumors per group). F-H) Metabolites significantly changed between PaTu-8902 iDox-shGOT2.1 +DOX (n=6) and –DOX (n=6) ((p

Techniques Used: In Vivo, Mouse Assay, Western Blot, Expressing, Staining

A) Representative αSMA staining in tissue slices from Fig.2A . B) Relative colony formation of PaTu-8902 iDox-shNT, shGOT2.1, or shGOT2.2 cells cultured in DMEM or hCAF CM, with images of representative wells. C) Relative colony formation of PaTu-8902 iDox-shGOT2.1 cells cultured in DMEM or hCAF CM, tumor educated macrophage (TEM) CM, or PaTu-8902 CM. D) Relative colony formation of PA-TU8902 iDox-shGOT2.1 cells cultured in DMEM or fresh hCAF CM, boiled CM, CM passed through a 3 kD filter, or CM subjected to freeze/thaw cycles, with images of representative wells. E) Relative abundances of metabolites significantly (p
Figure Legend Snippet: A) Representative αSMA staining in tissue slices from Fig.2A . B) Relative colony formation of PaTu-8902 iDox-shNT, shGOT2.1, or shGOT2.2 cells cultured in DMEM or hCAF CM, with images of representative wells. C) Relative colony formation of PaTu-8902 iDox-shGOT2.1 cells cultured in DMEM or hCAF CM, tumor educated macrophage (TEM) CM, or PaTu-8902 CM. D) Relative colony formation of PA-TU8902 iDox-shGOT2.1 cells cultured in DMEM or fresh hCAF CM, boiled CM, CM passed through a 3 kD filter, or CM subjected to freeze/thaw cycles, with images of representative wells. E) Relative abundances of metabolites significantly (p

Techniques Used: Staining, Cell Culture, Transmission Electron Microscopy

A,C) Graphs depicting endpoint tumor volumes (left) or tumor weights (right) of PaTu-8902 or MIAPaCa-2 iDox-shGOT2.1 subcutaneous xenografts treated with 2 mg/kg FX11. B,D) Western blots confirming GOT2 KD and showing LDHA expression, with vinculin loading controls, with protein lysates from xenografts in A) and C).
Figure Legend Snippet: A,C) Graphs depicting endpoint tumor volumes (left) or tumor weights (right) of PaTu-8902 or MIAPaCa-2 iDox-shGOT2.1 subcutaneous xenografts treated with 2 mg/kg FX11. B,D) Western blots confirming GOT2 KD and showing LDHA expression, with vinculin loading controls, with protein lysates from xenografts in A) and C).

Techniques Used: Western Blot, Expressing

A) Relative confluence over time of PaTu-8902 iDox-shGOT2.1 cells cultured in DMEM or 1 mM pyruvate. B) Western blot for GOT2 expression in parental PDA cells, or cells expressing sgEV, sgGOT2.1, or sgGOT2.2, with Vinculin loading control. C) Relative colony formation of MIAPaCa-2 GOT2 KO cultured in DMEM or 1 mM pyruvate, with images of representative wells. D) Relative colony formation of MIAPaCa-2 iDox-shGOT2.1 cells cultured in DMEM, 10 mM aspartate, 4 mM αKG, or both Asp/αKG, with images of representative wells. E) Relative abundance of aspartate in DMEM or media containing 10 mM aspartate, as measured by LC/MS. F) Relative abundance of aspartate in PaTu-8902 or MIAPaCa-2 cells after culture in DMEM or 10 mM aspartate, as measured by LC/MS. G) Relative colony formation of MIAPaCa-2 iDox-shGOT2.1 cells cultured in DMEM or 100 μM of the indicated combinations of adenine (A), guanine (G), thymine (T), and cytosine (C), with images of representative wells. H) Relative colony formation of PaTu-8902 or MIAPaCa-2 iDox-shGOT2.1 cells cultured in DMEM or 1 mM alanine, with images of representative wells. I) Growth curves of PaTu-8902 or MIAPaCa-2 iDox-shGOT2.1 cells in media containing 1 mM unlabelled glucose and 1 mM U13C-Pyruvate. J-K) Labelling patterns of intracellular citrate (J), aspartate (K), or alanine (L) in PaTu-8902 or MIAPaCa-2 iDox-shGOT2.1 cells and treated with UK5099. Bars represent mean ± SD, *p
Figure Legend Snippet: A) Relative confluence over time of PaTu-8902 iDox-shGOT2.1 cells cultured in DMEM or 1 mM pyruvate. B) Western blot for GOT2 expression in parental PDA cells, or cells expressing sgEV, sgGOT2.1, or sgGOT2.2, with Vinculin loading control. C) Relative colony formation of MIAPaCa-2 GOT2 KO cultured in DMEM or 1 mM pyruvate, with images of representative wells. D) Relative colony formation of MIAPaCa-2 iDox-shGOT2.1 cells cultured in DMEM, 10 mM aspartate, 4 mM αKG, or both Asp/αKG, with images of representative wells. E) Relative abundance of aspartate in DMEM or media containing 10 mM aspartate, as measured by LC/MS. F) Relative abundance of aspartate in PaTu-8902 or MIAPaCa-2 cells after culture in DMEM or 10 mM aspartate, as measured by LC/MS. G) Relative colony formation of MIAPaCa-2 iDox-shGOT2.1 cells cultured in DMEM or 100 μM of the indicated combinations of adenine (A), guanine (G), thymine (T), and cytosine (C), with images of representative wells. H) Relative colony formation of PaTu-8902 or MIAPaCa-2 iDox-shGOT2.1 cells cultured in DMEM or 1 mM alanine, with images of representative wells. I) Growth curves of PaTu-8902 or MIAPaCa-2 iDox-shGOT2.1 cells in media containing 1 mM unlabelled glucose and 1 mM U13C-Pyruvate. J-K) Labelling patterns of intracellular citrate (J), aspartate (K), or alanine (L) in PaTu-8902 or MIAPaCa-2 iDox-shGOT2.1 cells and treated with UK5099. Bars represent mean ± SD, *p

Techniques Used: Cell Culture, Western Blot, Expressing, Liquid Chromatography with Mass Spectroscopy

GOT2 KD disturbs redox homeostasis, which is restored by extracellular pyruvate. A) Relative ratio of NADH/NAD+ in PaTu-8902 iDox-shGOT2.1 cells, as determined by LC/MS. B) Schematic summarizing the metabolomics data following GOT2 KD depicting the effects of an increase in NADH levels on glycolysis. C) Log2 fold change of glycolytic and pentose phosphate pathway (PPP) intermediates in PaTu-8902 iDox-shGOT2.1 cells. D) Relative colony formation of PaTu-8902 iDox-shGOT2.1 cells cultured in DMEM or 1 mM αKB, with images of representative wells. E) Relative colony formation of PaTu-8902 iDox-shGOT2.1 cells expressing cytosolic or mitochondrial LbNOX. F-H) Relative viability of PaTu-8902 cells cultured in DMEM, hCAF CM, or 1 mM pyruvate and treated with the indicated doses of rotenone (F), phenformin (G), or IACS-010759 (H). Bars represent mean ± SD, *p
Figure Legend Snippet: GOT2 KD disturbs redox homeostasis, which is restored by extracellular pyruvate. A) Relative ratio of NADH/NAD+ in PaTu-8902 iDox-shGOT2.1 cells, as determined by LC/MS. B) Schematic summarizing the metabolomics data following GOT2 KD depicting the effects of an increase in NADH levels on glycolysis. C) Log2 fold change of glycolytic and pentose phosphate pathway (PPP) intermediates in PaTu-8902 iDox-shGOT2.1 cells. D) Relative colony formation of PaTu-8902 iDox-shGOT2.1 cells cultured in DMEM or 1 mM αKB, with images of representative wells. E) Relative colony formation of PaTu-8902 iDox-shGOT2.1 cells expressing cytosolic or mitochondrial LbNOX. F-H) Relative viability of PaTu-8902 cells cultured in DMEM, hCAF CM, or 1 mM pyruvate and treated with the indicated doses of rotenone (F), phenformin (G), or IACS-010759 (H). Bars represent mean ± SD, *p

Techniques Used: Liquid Chromatography with Mass Spectroscopy, Cell Culture, Expressing

A,C) Graphs depicting endpoint tumor volumes (left) or tumor weights (right) of PaTu-8902 or MIAPaCa-2 iDox-shGOT2.1, MCT1.1 KD subcutaneous xenografts in NSG mice. B,D) Western blots confirming GOT2 KD and MCT1 KD, with vinculin loading controls, with protein lysates from xenografts in A) and C). E) Graphs depicting endpoint tumor volumes (left) or tumor weights (right) of PaTu-8902 iDox-shGOT2.1, sgMCT1.1 subcutaneous xenografts in NSG mice. F) Western blots confirming GOT2 KD and MCT1 KO along with MCT4 expression, with vinculin loading controls, with protein lysates from xenografts in E). G) Graphs depicting endpoint tumor volumes (left) or tumor weights (right) of PaTu-8902 iDox-shGOT2.1 subcutaneous xenografts treated with 100 mg/kg AZD3965 via daily oral gavage. H) Western blots confirming GOT2 KD and showing MCT1 expression, with vinculin loading controls, with protein lysates from xenografts in G). I) Weights of mice fed normal or doxycycline chow and treated with vehicle or AZD3965.
Figure Legend Snippet: A,C) Graphs depicting endpoint tumor volumes (left) or tumor weights (right) of PaTu-8902 or MIAPaCa-2 iDox-shGOT2.1, MCT1.1 KD subcutaneous xenografts in NSG mice. B,D) Western blots confirming GOT2 KD and MCT1 KD, with vinculin loading controls, with protein lysates from xenografts in A) and C). E) Graphs depicting endpoint tumor volumes (left) or tumor weights (right) of PaTu-8902 iDox-shGOT2.1, sgMCT1.1 subcutaneous xenografts in NSG mice. F) Western blots confirming GOT2 KD and MCT1 KO along with MCT4 expression, with vinculin loading controls, with protein lysates from xenografts in E). G) Graphs depicting endpoint tumor volumes (left) or tumor weights (right) of PaTu-8902 iDox-shGOT2.1 subcutaneous xenografts treated with 100 mg/kg AZD3965 via daily oral gavage. H) Western blots confirming GOT2 KD and showing MCT1 expression, with vinculin loading controls, with protein lysates from xenografts in G). I) Weights of mice fed normal or doxycycline chow and treated with vehicle or AZD3965.

Techniques Used: Mouse Assay, Western Blot, Expressing

Blocking pyruvate uptake in vitro sensitizes PDA cells to redox disruption. A) Relative colony formation of MIAPaCa-2 iDox-shGOT2.1 cells treated with 100 nM AZD3965 and cultured in DMEM, 0.25 mM pyruvate, or hCAF CM, with images of representative wells. B) Relative colony formation of MIAPaCa-2 iDox-GOT2.1 cells treated with 100 nM AZD3965 and cultured in DMEM or the indicated doses of pyruvate, with images of representative wells. C) Western blot for GOT2 and MCT1, with Vinculin loading control, in PaTu-8902 iDox-shGOT2.1, MCT1 KO cells. D) Relative colony formation of PaTu-8902 iDox-shGOT2.1, MCT1 KO cells cultured in DMEM or with the indicated doses of pyruvate, with images or representative wells. E) Relative viability of MIAPaCa-2 cells treated with 100 nM IACS-010759 and 100 nM AZD3965 and cultured in DMEM, 1 mM pyruvate, or hCAF CM. F) Growth of PDA GOT2 KD cells cultured in 0.25 mM pyruvate or hCAF CM and treated with 25 μM FX11. G) Working model depicting the redox imbalance induced by GOT2 knockdown or complex I inhibition, which is then corrected through uptake of pyruvate released from CAFs or circulating through the vasculature. Bars represent mean ± SD, *p
Figure Legend Snippet: Blocking pyruvate uptake in vitro sensitizes PDA cells to redox disruption. A) Relative colony formation of MIAPaCa-2 iDox-shGOT2.1 cells treated with 100 nM AZD3965 and cultured in DMEM, 0.25 mM pyruvate, or hCAF CM, with images of representative wells. B) Relative colony formation of MIAPaCa-2 iDox-GOT2.1 cells treated with 100 nM AZD3965 and cultured in DMEM or the indicated doses of pyruvate, with images of representative wells. C) Western blot for GOT2 and MCT1, with Vinculin loading control, in PaTu-8902 iDox-shGOT2.1, MCT1 KO cells. D) Relative colony formation of PaTu-8902 iDox-shGOT2.1, MCT1 KO cells cultured in DMEM or with the indicated doses of pyruvate, with images or representative wells. E) Relative viability of MIAPaCa-2 cells treated with 100 nM IACS-010759 and 100 nM AZD3965 and cultured in DMEM, 1 mM pyruvate, or hCAF CM. F) Growth of PDA GOT2 KD cells cultured in 0.25 mM pyruvate or hCAF CM and treated with 25 μM FX11. G) Working model depicting the redox imbalance induced by GOT2 knockdown or complex I inhibition, which is then corrected through uptake of pyruvate released from CAFs or circulating through the vasculature. Bars represent mean ± SD, *p

Techniques Used: Blocking Assay, In Vitro, Cell Culture, Western Blot, Inhibition

Pyruvate compensates for GOT2 KD in vitro. A) Relative colony formation of MIAPaCa-2 iDox-shNT, shGOT2.1, or shGOT2.2 cells cultured in DMEM or 1 mM pyruvate, with images of representative wells. B) Relative colony formation of MIAPaCa-2 iDox-shGOT2.1 cells cultured in DMEM or the indicated doses of pyruvate, with images of representative wells. C) Relative ATP levels over time in PaTu-8902 iDox-shGOT2.1 cells cultured in DMEM or 1 mM pyruvate. D) Relative colony formation of PaTu-8902 parental WT, sgEV, sgGOT2.1, or sgGOT2.2 cells cultured in DMEM or 1 mM pyruvate, with images of representative wells. E) Relative viability of MIAPaCa-2 iDox-shGOT2.1 cells after treatment with 5 μM UK5099 and culture in DMEM, 1 mM pyruvate, or hCAF CM. F) Relative colony formation of MIAPaCa-2 iDox-shGOT2.1 cells cultured in DMEM, 1 mM pyruvate, or 1 mM lactate. G) Schematic depicting the labelling patterns following incubation for 16 hours with 1 mM U13C-Pyruvate and treatment with 5 μM UK5099. Lactate dehydrogenase (LDH), mitochondrial pyruvate carrier (MPC), glutamate-pyruvate transaminase (GPT). H,I) Labelling patterns of intracellular pyruvate (H) or intracellular lactate (I) in PaTu-8902 or MIAPaCa-2 iDox-shGOT2.1 and treated with 5 μM UK5099. Bars represent mean ± SD, *p
Figure Legend Snippet: Pyruvate compensates for GOT2 KD in vitro. A) Relative colony formation of MIAPaCa-2 iDox-shNT, shGOT2.1, or shGOT2.2 cells cultured in DMEM or 1 mM pyruvate, with images of representative wells. B) Relative colony formation of MIAPaCa-2 iDox-shGOT2.1 cells cultured in DMEM or the indicated doses of pyruvate, with images of representative wells. C) Relative ATP levels over time in PaTu-8902 iDox-shGOT2.1 cells cultured in DMEM or 1 mM pyruvate. D) Relative colony formation of PaTu-8902 parental WT, sgEV, sgGOT2.1, or sgGOT2.2 cells cultured in DMEM or 1 mM pyruvate, with images of representative wells. E) Relative viability of MIAPaCa-2 iDox-shGOT2.1 cells after treatment with 5 μM UK5099 and culture in DMEM, 1 mM pyruvate, or hCAF CM. F) Relative colony formation of MIAPaCa-2 iDox-shGOT2.1 cells cultured in DMEM, 1 mM pyruvate, or 1 mM lactate. G) Schematic depicting the labelling patterns following incubation for 16 hours with 1 mM U13C-Pyruvate and treatment with 5 μM UK5099. Lactate dehydrogenase (LDH), mitochondrial pyruvate carrier (MPC), glutamate-pyruvate transaminase (GPT). H,I) Labelling patterns of intracellular pyruvate (H) or intracellular lactate (I) in PaTu-8902 or MIAPaCa-2 iDox-shGOT2.1 and treated with 5 μM UK5099. Bars represent mean ± SD, *p

Techniques Used: In Vitro, Cell Culture, Incubation

A) Relative abundances of glycolytic intermediates in PaTu-8902 iDox-shGOT2.1 subcutaneous xenografts. B) Proton efflux rate (PER) of PaTu-8902 iDox-shGOT2.1 cells cultured +DOX for 3 days, as determined by the Glycolytic Rate Assay. C) Relative colony formation of MIAPaCa-2 iDox-shGOT2.1 cells cultured in DMEM or 1 mM αKB, with images of representative wells. D) Western blot for Flag-tagged, doxycycline-inducible LbNOX or GOT2, with Vinculin loading control, in PaTu-8902 or MIAPaCa-2 iDox-shGOT2.1 cells. E) Relative colony formation of MIAPaCa-2 iDox-shGOT2.1 cells expressing cytosolic or mitochondrial LbNOX. F) Relative colony formation of MIAPaCa-2 iDox-shGOT2.1 cells cultured in DMEM, 1 mM pyruvate, αKB, or NMN, with images of representative wells. Bars represent mean ± SD, *p
Figure Legend Snippet: A) Relative abundances of glycolytic intermediates in PaTu-8902 iDox-shGOT2.1 subcutaneous xenografts. B) Proton efflux rate (PER) of PaTu-8902 iDox-shGOT2.1 cells cultured +DOX for 3 days, as determined by the Glycolytic Rate Assay. C) Relative colony formation of MIAPaCa-2 iDox-shGOT2.1 cells cultured in DMEM or 1 mM αKB, with images of representative wells. D) Western blot for Flag-tagged, doxycycline-inducible LbNOX or GOT2, with Vinculin loading control, in PaTu-8902 or MIAPaCa-2 iDox-shGOT2.1 cells. E) Relative colony formation of MIAPaCa-2 iDox-shGOT2.1 cells expressing cytosolic or mitochondrial LbNOX. F) Relative colony formation of MIAPaCa-2 iDox-shGOT2.1 cells cultured in DMEM, 1 mM pyruvate, αKB, or NMN, with images of representative wells. Bars represent mean ± SD, *p

Techniques Used: Cell Culture, Western Blot, Expressing

GOT2 KD impairs in vitro PDA colony formation. A) Schematic depicting the metabolic roles of GOT2 in PDA. B) Western blot of GOT2 expression with Vinculin loading control in PaTu-8902 iDox-shNT, shGOT2.1, or shGOT2.2 cells. C) Images of representative wells showing PaTu-8902 colony formation after GOT2 KD. D) Heatmap summarizing the relative colony formation after GOT2 KD across a panel of PDA cell lines, normalized to -DOX for each hairpin. E-H) Metabolites significantly changed between +DOX (n=3) and –DOX (n=3) (p
Figure Legend Snippet: GOT2 KD impairs in vitro PDA colony formation. A) Schematic depicting the metabolic roles of GOT2 in PDA. B) Western blot of GOT2 expression with Vinculin loading control in PaTu-8902 iDox-shNT, shGOT2.1, or shGOT2.2 cells. C) Images of representative wells showing PaTu-8902 colony formation after GOT2 KD. D) Heatmap summarizing the relative colony formation after GOT2 KD across a panel of PDA cell lines, normalized to -DOX for each hairpin. E-H) Metabolites significantly changed between +DOX (n=3) and –DOX (n=3) (p

Techniques Used: In Vitro, Western Blot, Expressing

A) Western blots of GOT2 expression with Vinculin loading control in PDA cells expressing iDox-shNT, shGOT2.1, or shGOT2.2. B) Western blot for GOT1 and GOT2 with Vinculin loading control in PaTu-8902 iDox-shGOT2.1 cells. C) Images of representative wells showing colony formation in a panel of PDA cells expressing iDox-shNT, shGOT2.1, or shGOT2.2. D) Western blots of GOT2 expression with Vinculin loading control in HPNE or hPSC cell lines expressing iDox-shNT, shGOT2.1, or shGOT2.2. E) Relative proliferation of HPNE or hPSC cell lines expressing iDox-shNT, shGOT2.1, or shGOT2.2. F,G) Metabolites significantly changed between +DOX (n=3) and –DOX (n=3) (p
Figure Legend Snippet: A) Western blots of GOT2 expression with Vinculin loading control in PDA cells expressing iDox-shNT, shGOT2.1, or shGOT2.2. B) Western blot for GOT1 and GOT2 with Vinculin loading control in PaTu-8902 iDox-shGOT2.1 cells. C) Images of representative wells showing colony formation in a panel of PDA cells expressing iDox-shNT, shGOT2.1, or shGOT2.2. D) Western blots of GOT2 expression with Vinculin loading control in HPNE or hPSC cell lines expressing iDox-shNT, shGOT2.1, or shGOT2.2. E) Relative proliferation of HPNE or hPSC cell lines expressing iDox-shNT, shGOT2.1, or shGOT2.2. F,G) Metabolites significantly changed between +DOX (n=3) and –DOX (n=3) (p

Techniques Used: Western Blot, Expressing

3) Product Images from "Rapid and direct control of target protein levels with VHL-recruiting dTAG molecules"

Article Title: Rapid and direct control of target protein levels with VHL-recruiting dTAG molecules

Journal: Nature Communications

doi: 10.1038/s41467-020-18377-w

dTAG V -1 is an in vivo-compatible degrader of FKBP12 F36V -tagged proteins. a Immunoblot analysis of PATU-8902 FKBP12 F36V -KRAS G12V ; KRAS −/− clone treated with DMSO, dTAG V -1, or dTAG V -1-NEG for the indicated time-course. b Immunoblot analysis of 293T WT FKBP12 F36V -KRAS G12V or 293T VHL-/- FKBP12 F36V -KRAS G12V cells treated with DMSO or the indicated dTAG molecules for 24 h. Data in a , b are representative of n = 3 independent experiments. c DMSO-normalized antiproliferation of PATU-8902 LACZ-FKBP12 F36V or FKBP12 F36V -KRAS G12V ; KRAS -/- clones treated with dTAG V -1 or dTAG V -1-NEG for 120 h. Cells were cultured as ultra-low adherent 3D-spheroid suspensions. Data are presented as mean ± s.d. of n = 4 biologically independent samples and are representative of n = 3 independent experiments. d Bioluminescent imaging to evaluate degradation of luciferase-FKBP12 F36V in mice was performed daily as follows: day 0 to establish baseline signal, day 1–3 to monitor luciferase-FKBP12 F36V signal 4 h after vehicle or dTAG molecule treatment (T), day 4 to monitor duration of luciferase-FKBP12 F36V signal 28 h after third and final vehicle or dTAG molecule treatment. Total flux for each mouse is depicted. Data are presented from vehicle ( n = 5 biologically independent mice at day 0–4), dTAG-13 ( n = 5 biologically independent mice at day 0–3; n = 4 biologically independent mice at day 4) or dTAG V -1 ( n = 5 biologically independent mice at day 0–4) treated mice. P values are derived from a two-tailed Welch’s t -test (* P
Figure Legend Snippet: dTAG V -1 is an in vivo-compatible degrader of FKBP12 F36V -tagged proteins. a Immunoblot analysis of PATU-8902 FKBP12 F36V -KRAS G12V ; KRAS −/− clone treated with DMSO, dTAG V -1, or dTAG V -1-NEG for the indicated time-course. b Immunoblot analysis of 293T WT FKBP12 F36V -KRAS G12V or 293T VHL-/- FKBP12 F36V -KRAS G12V cells treated with DMSO or the indicated dTAG molecules for 24 h. Data in a , b are representative of n = 3 independent experiments. c DMSO-normalized antiproliferation of PATU-8902 LACZ-FKBP12 F36V or FKBP12 F36V -KRAS G12V ; KRAS -/- clones treated with dTAG V -1 or dTAG V -1-NEG for 120 h. Cells were cultured as ultra-low adherent 3D-spheroid suspensions. Data are presented as mean ± s.d. of n = 4 biologically independent samples and are representative of n = 3 independent experiments. d Bioluminescent imaging to evaluate degradation of luciferase-FKBP12 F36V in mice was performed daily as follows: day 0 to establish baseline signal, day 1–3 to monitor luciferase-FKBP12 F36V signal 4 h after vehicle or dTAG molecule treatment (T), day 4 to monitor duration of luciferase-FKBP12 F36V signal 28 h after third and final vehicle or dTAG molecule treatment. Total flux for each mouse is depicted. Data are presented from vehicle ( n = 5 biologically independent mice at day 0–4), dTAG-13 ( n = 5 biologically independent mice at day 0–3; n = 4 biologically independent mice at day 4) or dTAG V -1 ( n = 5 biologically independent mice at day 0–4) treated mice. P values are derived from a two-tailed Welch’s t -test (* P

Techniques Used: In Vivo, Clone Assay, Cell Culture, Imaging, Luciferase, Mouse Assay, Derivative Assay, Two Tailed Test

dTAG V -1 is an exclusively selective degrader of FKBP12 F36V -tagged proteins. a Schematic depiction of the dTAG system using VHL-recruiting dTAG molecules. VHL-recruiting dTAG molecules promote ternary complex formation between the FKBP12 F36V -tagged target protein and E3 ubiquitin ligase complex, inducing target protein ubiquitination and degradation. b Chemical structures of dTAG V -1 and dTAG V -1-NEG. c DMSO-normalized ratio of Nluc/Fluc signal of 293FT FKBP12 WT -Nluc or FKBP12 F36V -Nluc cells treated with dTAG V -1 or dTAG V -1-NEG for 24 h. Data are presented as mean ± s.d. of n = 4 biologically independent samples and are representative of n = 3 independent experiments. d Protein abundance after treatment of PATU-8902 LACZ-FKBP12 F36V clone with 500 nM dTAG V -1 for 4 h compared to DMSO treatment. Volcano plots depict fold change abundance relative to DMSO versus P value derived from a two-tailed Student’s t -test. Fold change values and significance designations derived from a two-tailed Student’s t -test and a permutation-based FDR estimation are provided in Supplementary Data 2 . Data are from n = 3 biologically independent samples. e Immunoblot analysis of PATU-8902 LACZ-FKBP12 F36V clone co-treated with DMSO, THAL-SNS-032, and/or dTAG V -1 as indicated for 24 h. Data is representative of n = 3 independent experiments. Source data for c and e are provided as a Source Data file.
Figure Legend Snippet: dTAG V -1 is an exclusively selective degrader of FKBP12 F36V -tagged proteins. a Schematic depiction of the dTAG system using VHL-recruiting dTAG molecules. VHL-recruiting dTAG molecules promote ternary complex formation between the FKBP12 F36V -tagged target protein and E3 ubiquitin ligase complex, inducing target protein ubiquitination and degradation. b Chemical structures of dTAG V -1 and dTAG V -1-NEG. c DMSO-normalized ratio of Nluc/Fluc signal of 293FT FKBP12 WT -Nluc or FKBP12 F36V -Nluc cells treated with dTAG V -1 or dTAG V -1-NEG for 24 h. Data are presented as mean ± s.d. of n = 4 biologically independent samples and are representative of n = 3 independent experiments. d Protein abundance after treatment of PATU-8902 LACZ-FKBP12 F36V clone with 500 nM dTAG V -1 for 4 h compared to DMSO treatment. Volcano plots depict fold change abundance relative to DMSO versus P value derived from a two-tailed Student’s t -test. Fold change values and significance designations derived from a two-tailed Student’s t -test and a permutation-based FDR estimation are provided in Supplementary Data 2 . Data are from n = 3 biologically independent samples. e Immunoblot analysis of PATU-8902 LACZ-FKBP12 F36V clone co-treated with DMSO, THAL-SNS-032, and/or dTAG V -1 as indicated for 24 h. Data is representative of n = 3 independent experiments. Source data for c and e are provided as a Source Data file.

Techniques Used: Derivative Assay, Two Tailed Test

4) Product Images from "Rapid and direct control of target protein levels with VHL-recruiting dTAG molecules"

Article Title: Rapid and direct control of target protein levels with VHL-recruiting dTAG molecules

Journal: Nature Communications

doi: 10.1038/s41467-020-18377-w

dTAG V -1 is an in vivo-compatible degrader of FKBP12 F36V -tagged proteins. a Immunoblot analysis of PATU-8902 FKBP12 F36V -KRAS G12V ; KRAS −/− clone treated with DMSO, dTAG V -1, or dTAG V -1-NEG for the indicated time-course. b Immunoblot analysis of 293T WT FKBP12 F36V -KRAS G12V or 293T VHL-/- FKBP12 F36V -KRAS G12V cells treated with DMSO or the indicated dTAG molecules for 24 h. Data in a , b are representative of n = 3 independent experiments. c DMSO-normalized antiproliferation of PATU-8902 LACZ-FKBP12 F36V or FKBP12 F36V -KRAS G12V ; KRAS -/- clones treated with dTAG V -1 or dTAG V -1-NEG for 120 h. Cells were cultured as ultra-low adherent 3D-spheroid suspensions. Data are presented as mean ± s.d. of n = 4 biologically independent samples and are representative of n = 3 independent experiments. d Bioluminescent imaging to evaluate degradation of luciferase-FKBP12 F36V in mice was performed daily as follows: day 0 to establish baseline signal, day 1–3 to monitor luciferase-FKBP12 F36V signal 4 h after vehicle or dTAG molecule treatment (T), day 4 to monitor duration of luciferase-FKBP12 F36V signal 28 h after third and final vehicle or dTAG molecule treatment. Total flux for each mouse is depicted. Data are presented from vehicle ( n = 5 biologically independent mice at day 0–4), dTAG-13 ( n = 5 biologically independent mice at day 0–3; n = 4 biologically independent mice at day 4) or dTAG V -1 ( n = 5 biologically independent mice at day 0–4) treated mice. P values are derived from a two-tailed Welch’s t -test (* P
Figure Legend Snippet: dTAG V -1 is an in vivo-compatible degrader of FKBP12 F36V -tagged proteins. a Immunoblot analysis of PATU-8902 FKBP12 F36V -KRAS G12V ; KRAS −/− clone treated with DMSO, dTAG V -1, or dTAG V -1-NEG for the indicated time-course. b Immunoblot analysis of 293T WT FKBP12 F36V -KRAS G12V or 293T VHL-/- FKBP12 F36V -KRAS G12V cells treated with DMSO or the indicated dTAG molecules for 24 h. Data in a , b are representative of n = 3 independent experiments. c DMSO-normalized antiproliferation of PATU-8902 LACZ-FKBP12 F36V or FKBP12 F36V -KRAS G12V ; KRAS -/- clones treated with dTAG V -1 or dTAG V -1-NEG for 120 h. Cells were cultured as ultra-low adherent 3D-spheroid suspensions. Data are presented as mean ± s.d. of n = 4 biologically independent samples and are representative of n = 3 independent experiments. d Bioluminescent imaging to evaluate degradation of luciferase-FKBP12 F36V in mice was performed daily as follows: day 0 to establish baseline signal, day 1–3 to monitor luciferase-FKBP12 F36V signal 4 h after vehicle or dTAG molecule treatment (T), day 4 to monitor duration of luciferase-FKBP12 F36V signal 28 h after third and final vehicle or dTAG molecule treatment. Total flux for each mouse is depicted. Data are presented from vehicle ( n = 5 biologically independent mice at day 0–4), dTAG-13 ( n = 5 biologically independent mice at day 0–3; n = 4 biologically independent mice at day 4) or dTAG V -1 ( n = 5 biologically independent mice at day 0–4) treated mice. P values are derived from a two-tailed Welch’s t -test (* P

Techniques Used: In Vivo, Clone Assay, Cell Culture, Imaging, Luciferase, Mouse Assay, Derivative Assay, Two Tailed Test

dTAG V -1 is an exclusively selective degrader of FKBP12 F36V -tagged proteins. a Schematic depiction of the dTAG system using VHL-recruiting dTAG molecules. VHL-recruiting dTAG molecules promote ternary complex formation between the FKBP12 F36V -tagged target protein and E3 ubiquitin ligase complex, inducing target protein ubiquitination and degradation. b Chemical structures of dTAG V -1 and dTAG V -1-NEG. c DMSO-normalized ratio of Nluc/Fluc signal of 293FT FKBP12 WT -Nluc or FKBP12 F36V -Nluc cells treated with dTAG V -1 or dTAG V -1-NEG for 24 h. Data are presented as mean ± s.d. of n = 4 biologically independent samples and are representative of n = 3 independent experiments. d Protein abundance after treatment of PATU-8902 LACZ-FKBP12 F36V clone with 500 nM dTAG V -1 for 4 h compared to DMSO treatment. Volcano plots depict fold change abundance relative to DMSO versus P value derived from a two-tailed Student’s t -test. Fold change values and significance designations derived from a two-tailed Student’s t -test and a permutation-based FDR estimation are provided in Supplementary Data 2 . Data are from n = 3 biologically independent samples. e Immunoblot analysis of PATU-8902 LACZ-FKBP12 F36V clone co-treated with DMSO, THAL-SNS-032, and/or dTAG V -1 as indicated for 24 h. Data is representative of n = 3 independent experiments. Source data for c and e are provided as a Source Data file.
Figure Legend Snippet: dTAG V -1 is an exclusively selective degrader of FKBP12 F36V -tagged proteins. a Schematic depiction of the dTAG system using VHL-recruiting dTAG molecules. VHL-recruiting dTAG molecules promote ternary complex formation between the FKBP12 F36V -tagged target protein and E3 ubiquitin ligase complex, inducing target protein ubiquitination and degradation. b Chemical structures of dTAG V -1 and dTAG V -1-NEG. c DMSO-normalized ratio of Nluc/Fluc signal of 293FT FKBP12 WT -Nluc or FKBP12 F36V -Nluc cells treated with dTAG V -1 or dTAG V -1-NEG for 24 h. Data are presented as mean ± s.d. of n = 4 biologically independent samples and are representative of n = 3 independent experiments. d Protein abundance after treatment of PATU-8902 LACZ-FKBP12 F36V clone with 500 nM dTAG V -1 for 4 h compared to DMSO treatment. Volcano plots depict fold change abundance relative to DMSO versus P value derived from a two-tailed Student’s t -test. Fold change values and significance designations derived from a two-tailed Student’s t -test and a permutation-based FDR estimation are provided in Supplementary Data 2 . Data are from n = 3 biologically independent samples. e Immunoblot analysis of PATU-8902 LACZ-FKBP12 F36V clone co-treated with DMSO, THAL-SNS-032, and/or dTAG V -1 as indicated for 24 h. Data is representative of n = 3 independent experiments. Source data for c and e are provided as a Source Data file.

Techniques Used: Derivative Assay, Two Tailed Test

5) Product Images from "Rapid and direct control of target protein levels with VHL-recruiting dTAG molecules"

Article Title: Rapid and direct control of target protein levels with VHL-recruiting dTAG molecules

Journal: bioRxiv

doi: 10.1101/2020.03.13.980946

dTAG V -1 is an exclusively selective, in vivo -compatible degrader of FKBP12 F36V -tagged proteins. ( a ) Schematic depiction of the dTAG system using VHL-recruiting dTAG molecules. VHL-recruiting dTAG molecules promote ternary complex formation between the FKBP12 F36V -tagged target protein and E3 ubiquitin ligase complex, inducing target protein ubiquitination and degradation. ( b ) Chemical structures of dTAG V -1 and dTAG V -1-NEG. ( c ) DMSO-normalized ratio of Nluc/Fluc signal of 293FT FKBP12 WT -Nluc or FKBP12 F36V -Nluc cells treated with dTAG V -1 or dTAG V -1-NEG molecules for 24 h. Data are presented as mean ± s.d. of n = 4 biologically independent samples and are representative of n = 3 independent experiments. ( d ) Protein abundance after treatment of PATU-8902 LACZ-FKBP12 F36V clone with 500 nM dTAG V -1 for 4 h compared to DMSO treatment. Volcano plots depict fold change abundance relative to DMSO versus P value. Significance designations derived from a permutation-based FDR estimation (q
Figure Legend Snippet: dTAG V -1 is an exclusively selective, in vivo -compatible degrader of FKBP12 F36V -tagged proteins. ( a ) Schematic depiction of the dTAG system using VHL-recruiting dTAG molecules. VHL-recruiting dTAG molecules promote ternary complex formation between the FKBP12 F36V -tagged target protein and E3 ubiquitin ligase complex, inducing target protein ubiquitination and degradation. ( b ) Chemical structures of dTAG V -1 and dTAG V -1-NEG. ( c ) DMSO-normalized ratio of Nluc/Fluc signal of 293FT FKBP12 WT -Nluc or FKBP12 F36V -Nluc cells treated with dTAG V -1 or dTAG V -1-NEG molecules for 24 h. Data are presented as mean ± s.d. of n = 4 biologically independent samples and are representative of n = 3 independent experiments. ( d ) Protein abundance after treatment of PATU-8902 LACZ-FKBP12 F36V clone with 500 nM dTAG V -1 for 4 h compared to DMSO treatment. Volcano plots depict fold change abundance relative to DMSO versus P value. Significance designations derived from a permutation-based FDR estimation (q

Techniques Used: In Vivo, Derivative Assay

6) Product Images from "Rapid and direct control of target protein levels with VHL-recruiting dTAG molecules"

Article Title: Rapid and direct control of target protein levels with VHL-recruiting dTAG molecules

Journal: bioRxiv

doi: 10.1101/2020.03.13.980946

dTAG V -1 is an exclusively selective, in vivo -compatible degrader of FKBP12 F36V -tagged proteins. ( a ) Schematic depiction of the dTAG system using VHL-recruiting dTAG molecules. VHL-recruiting dTAG molecules promote ternary complex formation between the FKBP12 F36V -tagged target protein and E3 ubiquitin ligase complex, inducing target protein ubiquitination and degradation. ( b ) Chemical structures of dTAG V -1 and dTAG V -1-NEG. ( c ) DMSO-normalized ratio of Nluc/Fluc signal of 293FT FKBP12 WT -Nluc or FKBP12 F36V -Nluc cells treated with dTAG V -1 or dTAG V -1-NEG molecules for 24 h. Data are presented as mean ± s.d. of n = 4 biologically independent samples and are representative of n = 3 independent experiments. ( d ) Protein abundance after treatment of PATU-8902 LACZ-FKBP12 F36V clone with 500 nM dTAG V -1 for 4 h compared to DMSO treatment. Volcano plots depict fold change abundance relative to DMSO versus P value. Significance designations derived from a permutation-based FDR estimation (q
Figure Legend Snippet: dTAG V -1 is an exclusively selective, in vivo -compatible degrader of FKBP12 F36V -tagged proteins. ( a ) Schematic depiction of the dTAG system using VHL-recruiting dTAG molecules. VHL-recruiting dTAG molecules promote ternary complex formation between the FKBP12 F36V -tagged target protein and E3 ubiquitin ligase complex, inducing target protein ubiquitination and degradation. ( b ) Chemical structures of dTAG V -1 and dTAG V -1-NEG. ( c ) DMSO-normalized ratio of Nluc/Fluc signal of 293FT FKBP12 WT -Nluc or FKBP12 F36V -Nluc cells treated with dTAG V -1 or dTAG V -1-NEG molecules for 24 h. Data are presented as mean ± s.d. of n = 4 biologically independent samples and are representative of n = 3 independent experiments. ( d ) Protein abundance after treatment of PATU-8902 LACZ-FKBP12 F36V clone with 500 nM dTAG V -1 for 4 h compared to DMSO treatment. Volcano plots depict fold change abundance relative to DMSO versus P value. Significance designations derived from a permutation-based FDR estimation (q

Techniques Used: In Vivo, Derivative Assay

7) Product Images from "Rapid and direct control of target protein levels with VHL-recruiting dTAG molecules"

Article Title: Rapid and direct control of target protein levels with VHL-recruiting dTAG molecules

Journal: bioRxiv

doi: 10.1101/2020.03.13.980946

dTAG V -1 is an exclusively selective, in vivo -compatible degrader of FKBP12 F36V -tagged proteins. ( a ) Schematic depiction of the dTAG system using VHL-recruiting dTAG molecules. VHL-recruiting dTAG molecules promote ternary complex formation between the FKBP12 F36V -tagged target protein and E3 ubiquitin ligase complex, inducing target protein ubiquitination and degradation. ( b ) Chemical structures of dTAG V -1 and dTAG V -1-NEG. ( c ) DMSO-normalized ratio of Nluc/Fluc signal of 293FT FKBP12 WT -Nluc or FKBP12 F36V -Nluc cells treated with dTAG V -1 or dTAG V -1-NEG molecules for 24 h. Data are presented as mean ± s.d. of n = 4 biologically independent samples and are representative of n = 3 independent experiments. ( d ) Protein abundance after treatment of PATU-8902 LACZ-FKBP12 F36V clone with 500 nM dTAG V -1 for 4 h compared to DMSO treatment. Volcano plots depict fold change abundance relative to DMSO versus P value. Significance designations derived from a permutation-based FDR estimation (q
Figure Legend Snippet: dTAG V -1 is an exclusively selective, in vivo -compatible degrader of FKBP12 F36V -tagged proteins. ( a ) Schematic depiction of the dTAG system using VHL-recruiting dTAG molecules. VHL-recruiting dTAG molecules promote ternary complex formation between the FKBP12 F36V -tagged target protein and E3 ubiquitin ligase complex, inducing target protein ubiquitination and degradation. ( b ) Chemical structures of dTAG V -1 and dTAG V -1-NEG. ( c ) DMSO-normalized ratio of Nluc/Fluc signal of 293FT FKBP12 WT -Nluc or FKBP12 F36V -Nluc cells treated with dTAG V -1 or dTAG V -1-NEG molecules for 24 h. Data are presented as mean ± s.d. of n = 4 biologically independent samples and are representative of n = 3 independent experiments. ( d ) Protein abundance after treatment of PATU-8902 LACZ-FKBP12 F36V clone with 500 nM dTAG V -1 for 4 h compared to DMSO treatment. Volcano plots depict fold change abundance relative to DMSO versus P value. Significance designations derived from a permutation-based FDR estimation (q

Techniques Used: In Vivo, Derivative Assay

Similar Products

  • Logo
  • About
  • News
  • Press Release
  • Team
  • Advisors
  • Partners
  • Contact
  • Bioz Stars
  • Bioz vStars
  • 90
    DSMZ human pancreatic adenocarcinoma patu 8902 cell line
    The effect of lactic acidosis on redox homeostasis of cancer cells. <t>PaTu-8902</t> cells were treated with the experimental media for 72 h and ( A ) relative antioxidant capacity, ( B ) ratio of reduced (GSH) to oxidized (GSSG) glutathione in the cells, ( C ) relative concentration of NADPH, ( D ) oxidation of dihydroethidium, and ( E ) oxidation of 2′,7′-dichlorodihydrofluorescein measured as a relative fluorescence were determined. C, control; A, acidosis; LA, lactic acidosis; L, high lactate (* p
    Human Pancreatic Adenocarcinoma Patu 8902 Cell Line, supplied by DSMZ, used in various techniques. Bioz Stars score: 90/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/human pancreatic adenocarcinoma patu 8902 cell line/product/DSMZ
    Average 90 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    human pancreatic adenocarcinoma patu 8902 cell line - by Bioz Stars, 2022-11
    90/100 stars
      Buy from Supplier

    94
    DSMZ patu 8902
    dTAG V -1 is an in vivo-compatible degrader of FKBP12 F36V -tagged proteins. a Immunoblot analysis of <t>PATU-8902</t> FKBP12 F36V -KRAS G12V ; KRAS −/− clone treated with DMSO, dTAG V -1, or dTAG V -1-NEG for the indicated time-course. b Immunoblot analysis of 293T WT FKBP12 F36V -KRAS G12V or 293T VHL-/- FKBP12 F36V -KRAS G12V cells treated with DMSO or the indicated dTAG molecules for 24 h. Data in a , b are representative of n = 3 independent experiments. c DMSO-normalized antiproliferation of PATU-8902 LACZ-FKBP12 F36V or FKBP12 F36V -KRAS G12V ; KRAS -/- clones treated with dTAG V -1 or dTAG V -1-NEG for 120 h. Cells were cultured as ultra-low adherent 3D-spheroid suspensions. Data are presented as mean ± s.d. of n = 4 biologically independent samples and are representative of n = 3 independent experiments. d Bioluminescent imaging to evaluate degradation of luciferase-FKBP12 F36V in mice was performed daily as follows: day 0 to establish baseline signal, day 1–3 to monitor luciferase-FKBP12 F36V signal 4 h after vehicle or dTAG molecule treatment (T), day 4 to monitor duration of luciferase-FKBP12 F36V signal 28 h after third and final vehicle or dTAG molecule treatment. Total flux for each mouse is depicted. Data are presented from vehicle ( n = 5 biologically independent mice at day 0–4), dTAG-13 ( n = 5 biologically independent mice at day 0–3; n = 4 biologically independent mice at day 4) or dTAG V -1 ( n = 5 biologically independent mice at day 0–4) treated mice. P values are derived from a two-tailed Welch’s t -test (* P
    Patu 8902, supplied by DSMZ, used in various techniques. Bioz Stars score: 94/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/patu 8902/product/DSMZ
    Average 94 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    patu 8902 - by Bioz Stars, 2022-11
    94/100 stars
      Buy from Supplier

    Image Search Results


    The effect of lactic acidosis on redox homeostasis of cancer cells. PaTu-8902 cells were treated with the experimental media for 72 h and ( A ) relative antioxidant capacity, ( B ) ratio of reduced (GSH) to oxidized (GSSG) glutathione in the cells, ( C ) relative concentration of NADPH, ( D ) oxidation of dihydroethidium, and ( E ) oxidation of 2′,7′-dichlorodihydrofluorescein measured as a relative fluorescence were determined. C, control; A, acidosis; LA, lactic acidosis; L, high lactate (* p

    Journal: International Journal of Molecular Sciences

    Article Title: Inhibition of Mitochondrial Metabolism Leads to Selective Eradication of Cells Adapted to Acidic Microenvironment

    doi: 10.3390/ijms221910790

    Figure Lengend Snippet: The effect of lactic acidosis on redox homeostasis of cancer cells. PaTu-8902 cells were treated with the experimental media for 72 h and ( A ) relative antioxidant capacity, ( B ) ratio of reduced (GSH) to oxidized (GSSG) glutathione in the cells, ( C ) relative concentration of NADPH, ( D ) oxidation of dihydroethidium, and ( E ) oxidation of 2′,7′-dichlorodihydrofluorescein measured as a relative fluorescence were determined. C, control; A, acidosis; LA, lactic acidosis; L, high lactate (* p

    Article Snippet: Human pancreatic adenocarcinoma PaTu-8902 cell line (DSMZ, Germany), human cervix carcinoma HeLa cell line, human hepatoblastoma Hep G2 cell line, and human dermal fibroblasts HDF (all ATCC, Manassas, VA, USA) were cultured in the Eagle’s Minimum Essential Medium (EMEM) supplemented with 0.5 mM glutamine and 5% (v /v ) fetal bovine serum in 5% CO2 atmosphere at 37 °C.

    Techniques: Concentration Assay, Fluorescence

    The effect of lactic acidosis on cancer cell metabolism. PaTu-8902 cells were treated with the experimental media for 72 h and ( A ) concentration of total cellular ATP, ( B ) mitochondrial membrane potential, ( C ) oxygen consumption, the changes in ( D ) lactate and ( E ) glucose levels in the culture media, and ( F ) mass of cellular phospholipids were determined. C, control; A, acidosis; LA, lactic acidosis; L, high lactate (* p

    Journal: International Journal of Molecular Sciences

    Article Title: Inhibition of Mitochondrial Metabolism Leads to Selective Eradication of Cells Adapted to Acidic Microenvironment

    doi: 10.3390/ijms221910790

    Figure Lengend Snippet: The effect of lactic acidosis on cancer cell metabolism. PaTu-8902 cells were treated with the experimental media for 72 h and ( A ) concentration of total cellular ATP, ( B ) mitochondrial membrane potential, ( C ) oxygen consumption, the changes in ( D ) lactate and ( E ) glucose levels in the culture media, and ( F ) mass of cellular phospholipids were determined. C, control; A, acidosis; LA, lactic acidosis; L, high lactate (* p

    Article Snippet: Human pancreatic adenocarcinoma PaTu-8902 cell line (DSMZ, Germany), human cervix carcinoma HeLa cell line, human hepatoblastoma Hep G2 cell line, and human dermal fibroblasts HDF (all ATCC, Manassas, VA, USA) were cultured in the Eagle’s Minimum Essential Medium (EMEM) supplemented with 0.5 mM glutamine and 5% (v /v ) fetal bovine serum in 5% CO2 atmosphere at 37 °C.

    Techniques: Concentration Assay

    The effect of lactic acidosis on uptake and localization of photosensitizers in cancer cells. Localization of ( A ) pheophorbide a , ( B ) Mo cluster, and ( C ) 5,10,15,20-tetrakis(4-isopropylphosphinatophenyl)porphyrin (TIPPP) in HeLa cells treated with the experimental media for 72 h. The photosensitizers (red emission) were co-localized with ( A , B ) MitoTracker TM Green FM or ( C ) LysoTracker TM Green DND-26 (both green emission) to visualize the subcellular localization. Uptake of ( D) pheophorbide a , ( E ) Mo cluster, and ( F ) TIPPP in PaTu-8902 cells treated with the experimental media for 72 h measured by flow cytometry. C, control; A, acidosis; LA, lactic acidosis; L, high lactate.

    Journal: International Journal of Molecular Sciences

    Article Title: Inhibition of Mitochondrial Metabolism Leads to Selective Eradication of Cells Adapted to Acidic Microenvironment

    doi: 10.3390/ijms221910790

    Figure Lengend Snippet: The effect of lactic acidosis on uptake and localization of photosensitizers in cancer cells. Localization of ( A ) pheophorbide a , ( B ) Mo cluster, and ( C ) 5,10,15,20-tetrakis(4-isopropylphosphinatophenyl)porphyrin (TIPPP) in HeLa cells treated with the experimental media for 72 h. The photosensitizers (red emission) were co-localized with ( A , B ) MitoTracker TM Green FM or ( C ) LysoTracker TM Green DND-26 (both green emission) to visualize the subcellular localization. Uptake of ( D) pheophorbide a , ( E ) Mo cluster, and ( F ) TIPPP in PaTu-8902 cells treated with the experimental media for 72 h measured by flow cytometry. C, control; A, acidosis; LA, lactic acidosis; L, high lactate.

    Article Snippet: Human pancreatic adenocarcinoma PaTu-8902 cell line (DSMZ, Germany), human cervix carcinoma HeLa cell line, human hepatoblastoma Hep G2 cell line, and human dermal fibroblasts HDF (all ATCC, Manassas, VA, USA) were cultured in the Eagle’s Minimum Essential Medium (EMEM) supplemented with 0.5 mM glutamine and 5% (v /v ) fetal bovine serum in 5% CO2 atmosphere at 37 °C.

    Techniques: Flow Cytometry

    The effect of lactic acidosis on cancer cell sensitivity to radio-, chemo-, and phototherapy. PaTu-8902 cells were cultured in experimental media for 72 h and then treated with ( A ) X-ray irradiation, ( B ) doxorubicin, ( C ) pharmacological ascorbate, and ( D – F ) photodynamic therapy using pheophorbide a , Mo cluster, and 5,10,15,20-tetrakis(4-isopropylphosphinatophenyl)porphyrin (TIPPP), respectively. The proliferation of irradiated cells after reseeding and subsequent 72 h incubation and viability of the rest after 24 h were measured by resazurin assay. C, control; A, acidosis; LA, lactic acidosis; L, high lactate (* p

    Journal: International Journal of Molecular Sciences

    Article Title: Inhibition of Mitochondrial Metabolism Leads to Selective Eradication of Cells Adapted to Acidic Microenvironment

    doi: 10.3390/ijms221910790

    Figure Lengend Snippet: The effect of lactic acidosis on cancer cell sensitivity to radio-, chemo-, and phototherapy. PaTu-8902 cells were cultured in experimental media for 72 h and then treated with ( A ) X-ray irradiation, ( B ) doxorubicin, ( C ) pharmacological ascorbate, and ( D – F ) photodynamic therapy using pheophorbide a , Mo cluster, and 5,10,15,20-tetrakis(4-isopropylphosphinatophenyl)porphyrin (TIPPP), respectively. The proliferation of irradiated cells after reseeding and subsequent 72 h incubation and viability of the rest after 24 h were measured by resazurin assay. C, control; A, acidosis; LA, lactic acidosis; L, high lactate (* p

    Article Snippet: Human pancreatic adenocarcinoma PaTu-8902 cell line (DSMZ, Germany), human cervix carcinoma HeLa cell line, human hepatoblastoma Hep G2 cell line, and human dermal fibroblasts HDF (all ATCC, Manassas, VA, USA) were cultured in the Eagle’s Minimum Essential Medium (EMEM) supplemented with 0.5 mM glutamine and 5% (v /v ) fetal bovine serum in 5% CO2 atmosphere at 37 °C.

    Techniques: Cell Culture, Irradiation, Incubation, Resazurin Assay

    Inhibition of mitochondrial metabolism. PaTu-8902 cells were treated with the experimental media containing the indicated amount of ( A ) tetracycline or ( B ) CPI-613 for 72 h, and the viability was measured. ( C ) Oxidation of 2′,7′-dichlorodihydrofluorescein was measured as a relative fluorescence in PaTu-8902 treated with 50 µM CPI-613 for 4 and 24 h. ( D ) HeLa, ( E ) HepG2, and ( F ) HDF cells were treated with the experimental media containing the indicated amount of CPI-613 for 72 h, and the viability was measured. ( G ) PaTu-8902 cells were treated with experimental media containing 50 µM CPI-613, 50 µM tetracycline, or their combination for 2 weeks, and the viability was measured by resazurin assay. C, control; A, acidosis; LA, lactic acidosis; L, high lactate (* p

    Journal: International Journal of Molecular Sciences

    Article Title: Inhibition of Mitochondrial Metabolism Leads to Selective Eradication of Cells Adapted to Acidic Microenvironment

    doi: 10.3390/ijms221910790

    Figure Lengend Snippet: Inhibition of mitochondrial metabolism. PaTu-8902 cells were treated with the experimental media containing the indicated amount of ( A ) tetracycline or ( B ) CPI-613 for 72 h, and the viability was measured. ( C ) Oxidation of 2′,7′-dichlorodihydrofluorescein was measured as a relative fluorescence in PaTu-8902 treated with 50 µM CPI-613 for 4 and 24 h. ( D ) HeLa, ( E ) HepG2, and ( F ) HDF cells were treated with the experimental media containing the indicated amount of CPI-613 for 72 h, and the viability was measured. ( G ) PaTu-8902 cells were treated with experimental media containing 50 µM CPI-613, 50 µM tetracycline, or their combination for 2 weeks, and the viability was measured by resazurin assay. C, control; A, acidosis; LA, lactic acidosis; L, high lactate (* p

    Article Snippet: Human pancreatic adenocarcinoma PaTu-8902 cell line (DSMZ, Germany), human cervix carcinoma HeLa cell line, human hepatoblastoma Hep G2 cell line, and human dermal fibroblasts HDF (all ATCC, Manassas, VA, USA) were cultured in the Eagle’s Minimum Essential Medium (EMEM) supplemented with 0.5 mM glutamine and 5% (v /v ) fetal bovine serum in 5% CO2 atmosphere at 37 °C.

    Techniques: Inhibition, Fluorescence, Resazurin Assay

    The effect of adaptation time on cell sensitivity to oxidative insult. PaTu-8902 cells were treated with experimental media for ( A ) 6 h, ( B ) 14 days, and ( C , D ) 72 h followed by 4 and 48 h washout with the control medium, respectively. Then, the cells were treated with the indicated concentrations of hydrogen peroxide and their viability was measured by resazurin assay. C, control; A, acidosis; LA, lactic acidosis; L, high lactate (* p

    Journal: International Journal of Molecular Sciences

    Article Title: Inhibition of Mitochondrial Metabolism Leads to Selective Eradication of Cells Adapted to Acidic Microenvironment

    doi: 10.3390/ijms221910790

    Figure Lengend Snippet: The effect of adaptation time on cell sensitivity to oxidative insult. PaTu-8902 cells were treated with experimental media for ( A ) 6 h, ( B ) 14 days, and ( C , D ) 72 h followed by 4 and 48 h washout with the control medium, respectively. Then, the cells were treated with the indicated concentrations of hydrogen peroxide and their viability was measured by resazurin assay. C, control; A, acidosis; LA, lactic acidosis; L, high lactate (* p

    Article Snippet: Human pancreatic adenocarcinoma PaTu-8902 cell line (DSMZ, Germany), human cervix carcinoma HeLa cell line, human hepatoblastoma Hep G2 cell line, and human dermal fibroblasts HDF (all ATCC, Manassas, VA, USA) were cultured in the Eagle’s Minimum Essential Medium (EMEM) supplemented with 0.5 mM glutamine and 5% (v /v ) fetal bovine serum in 5% CO2 atmosphere at 37 °C.

    Techniques: Resazurin Assay

    The effect of lactic acidosis on cell sensitivity to oxidative insult. ( A ) Viability of PaTu-8902 cells cultured in media of different pH and concentrations of lactate anion (NaL) for 72 h and then treated with 500 µM hydrogen peroxide. Viability of ( B ) PaTu-8902, ( C ) HeLa, ( D ) Hep G2, and ( E ) noncancerous HDF cells cultured in experimental media for 72 h and then treated with indicated concentrations of hydrogen peroxide. Viability was measured by resazurin assay. C, control; A, acidosis; LA, lactic acidosis; L, high lactate (* p

    Journal: International Journal of Molecular Sciences

    Article Title: Inhibition of Mitochondrial Metabolism Leads to Selective Eradication of Cells Adapted to Acidic Microenvironment

    doi: 10.3390/ijms221910790

    Figure Lengend Snippet: The effect of lactic acidosis on cell sensitivity to oxidative insult. ( A ) Viability of PaTu-8902 cells cultured in media of different pH and concentrations of lactate anion (NaL) for 72 h and then treated with 500 µM hydrogen peroxide. Viability of ( B ) PaTu-8902, ( C ) HeLa, ( D ) Hep G2, and ( E ) noncancerous HDF cells cultured in experimental media for 72 h and then treated with indicated concentrations of hydrogen peroxide. Viability was measured by resazurin assay. C, control; A, acidosis; LA, lactic acidosis; L, high lactate (* p

    Article Snippet: Human pancreatic adenocarcinoma PaTu-8902 cell line (DSMZ, Germany), human cervix carcinoma HeLa cell line, human hepatoblastoma Hep G2 cell line, and human dermal fibroblasts HDF (all ATCC, Manassas, VA, USA) were cultured in the Eagle’s Minimum Essential Medium (EMEM) supplemented with 0.5 mM glutamine and 5% (v /v ) fetal bovine serum in 5% CO2 atmosphere at 37 °C.

    Techniques: Cell Culture, Resazurin Assay

    Synergic effects of lactic acidosis and metabolic manipulations. ( A ) The viability of PaTu-8902 cells treated with the experimental media lacking glucose for 72 h and challenged with the indicated concentrations of hydrogen peroxide. ( B ) The viability of PaTu-8902 cells was treated with experimental media and carbonyl cyanide- p -trifluoromethoxyphenylhydrazone (FCCP) for 72 h and challenged with indicated concentrations of hydrogen peroxide. Viability was measured by resazurin assay. C, control; A, acidosis; LA, lactic acidosis; L, high lactate (* p

    Journal: International Journal of Molecular Sciences

    Article Title: Inhibition of Mitochondrial Metabolism Leads to Selective Eradication of Cells Adapted to Acidic Microenvironment

    doi: 10.3390/ijms221910790

    Figure Lengend Snippet: Synergic effects of lactic acidosis and metabolic manipulations. ( A ) The viability of PaTu-8902 cells treated with the experimental media lacking glucose for 72 h and challenged with the indicated concentrations of hydrogen peroxide. ( B ) The viability of PaTu-8902 cells was treated with experimental media and carbonyl cyanide- p -trifluoromethoxyphenylhydrazone (FCCP) for 72 h and challenged with indicated concentrations of hydrogen peroxide. Viability was measured by resazurin assay. C, control; A, acidosis; LA, lactic acidosis; L, high lactate (* p

    Article Snippet: Human pancreatic adenocarcinoma PaTu-8902 cell line (DSMZ, Germany), human cervix carcinoma HeLa cell line, human hepatoblastoma Hep G2 cell line, and human dermal fibroblasts HDF (all ATCC, Manassas, VA, USA) were cultured in the Eagle’s Minimum Essential Medium (EMEM) supplemented with 0.5 mM glutamine and 5% (v /v ) fetal bovine serum in 5% CO2 atmosphere at 37 °C.

    Techniques: Resazurin Assay

    dTAG V -1 is an in vivo-compatible degrader of FKBP12 F36V -tagged proteins. a Immunoblot analysis of PATU-8902 FKBP12 F36V -KRAS G12V ; KRAS −/− clone treated with DMSO, dTAG V -1, or dTAG V -1-NEG for the indicated time-course. b Immunoblot analysis of 293T WT FKBP12 F36V -KRAS G12V or 293T VHL-/- FKBP12 F36V -KRAS G12V cells treated with DMSO or the indicated dTAG molecules for 24 h. Data in a , b are representative of n = 3 independent experiments. c DMSO-normalized antiproliferation of PATU-8902 LACZ-FKBP12 F36V or FKBP12 F36V -KRAS G12V ; KRAS -/- clones treated with dTAG V -1 or dTAG V -1-NEG for 120 h. Cells were cultured as ultra-low adherent 3D-spheroid suspensions. Data are presented as mean ± s.d. of n = 4 biologically independent samples and are representative of n = 3 independent experiments. d Bioluminescent imaging to evaluate degradation of luciferase-FKBP12 F36V in mice was performed daily as follows: day 0 to establish baseline signal, day 1–3 to monitor luciferase-FKBP12 F36V signal 4 h after vehicle or dTAG molecule treatment (T), day 4 to monitor duration of luciferase-FKBP12 F36V signal 28 h after third and final vehicle or dTAG molecule treatment. Total flux for each mouse is depicted. Data are presented from vehicle ( n = 5 biologically independent mice at day 0–4), dTAG-13 ( n = 5 biologically independent mice at day 0–3; n = 4 biologically independent mice at day 4) or dTAG V -1 ( n = 5 biologically independent mice at day 0–4) treated mice. P values are derived from a two-tailed Welch’s t -test (* P

    Journal: Nature Communications

    Article Title: Rapid and direct control of target protein levels with VHL-recruiting dTAG molecules

    doi: 10.1038/s41467-020-18377-w

    Figure Lengend Snippet: dTAG V -1 is an in vivo-compatible degrader of FKBP12 F36V -tagged proteins. a Immunoblot analysis of PATU-8902 FKBP12 F36V -KRAS G12V ; KRAS −/− clone treated with DMSO, dTAG V -1, or dTAG V -1-NEG for the indicated time-course. b Immunoblot analysis of 293T WT FKBP12 F36V -KRAS G12V or 293T VHL-/- FKBP12 F36V -KRAS G12V cells treated with DMSO or the indicated dTAG molecules for 24 h. Data in a , b are representative of n = 3 independent experiments. c DMSO-normalized antiproliferation of PATU-8902 LACZ-FKBP12 F36V or FKBP12 F36V -KRAS G12V ; KRAS -/- clones treated with dTAG V -1 or dTAG V -1-NEG for 120 h. Cells were cultured as ultra-low adherent 3D-spheroid suspensions. Data are presented as mean ± s.d. of n = 4 biologically independent samples and are representative of n = 3 independent experiments. d Bioluminescent imaging to evaluate degradation of luciferase-FKBP12 F36V in mice was performed daily as follows: day 0 to establish baseline signal, day 1–3 to monitor luciferase-FKBP12 F36V signal 4 h after vehicle or dTAG molecule treatment (T), day 4 to monitor duration of luciferase-FKBP12 F36V signal 28 h after third and final vehicle or dTAG molecule treatment. Total flux for each mouse is depicted. Data are presented from vehicle ( n = 5 biologically independent mice at day 0–4), dTAG-13 ( n = 5 biologically independent mice at day 0–3; n = 4 biologically independent mice at day 4) or dTAG V -1 ( n = 5 biologically independent mice at day 0–4) treated mice. P values are derived from a two-tailed Welch’s t -test (* P

    Article Snippet: Cell lines The following cell lines were employed in this study: 293T (source: ATCC #CRL-3216, media: DMEM with 10% FBS and 1% Penicillin–Streptomycin), 293FT (source: Thermo Fisher Scientific #R70007, media: DMEM with 10% FBS and 1% Penicillin–Streptomycin), PATU-8902 (source: DSMZ #ACC-179, media: DMEM with 10% FBS and 1% Penicillin–Streptomycin), MV4;11 (source: ATCC #CRL-9591, media: RPMI with 10% FBS and 1% Penicillin–Streptomycin) and EWS502 (source: kindly provided by Dr. Stephen L. Lessnick of Nationwide Children’s Hospital and established by Dr. Jonathan A. Fletcher of Harvard Medical School, media: RPMI with 15% FBS and 1% Penicillin–Streptomycin-l -Glutamine).

    Techniques: In Vivo, Clone Assay, Cell Culture, Imaging, Luciferase, Mouse Assay, Derivative Assay, Two Tailed Test

    dTAG V -1 is an exclusively selective degrader of FKBP12 F36V -tagged proteins. a Schematic depiction of the dTAG system using VHL-recruiting dTAG molecules. VHL-recruiting dTAG molecules promote ternary complex formation between the FKBP12 F36V -tagged target protein and E3 ubiquitin ligase complex, inducing target protein ubiquitination and degradation. b Chemical structures of dTAG V -1 and dTAG V -1-NEG. c DMSO-normalized ratio of Nluc/Fluc signal of 293FT FKBP12 WT -Nluc or FKBP12 F36V -Nluc cells treated with dTAG V -1 or dTAG V -1-NEG for 24 h. Data are presented as mean ± s.d. of n = 4 biologically independent samples and are representative of n = 3 independent experiments. d Protein abundance after treatment of PATU-8902 LACZ-FKBP12 F36V clone with 500 nM dTAG V -1 for 4 h compared to DMSO treatment. Volcano plots depict fold change abundance relative to DMSO versus P value derived from a two-tailed Student’s t -test. Fold change values and significance designations derived from a two-tailed Student’s t -test and a permutation-based FDR estimation are provided in Supplementary Data 2 . Data are from n = 3 biologically independent samples. e Immunoblot analysis of PATU-8902 LACZ-FKBP12 F36V clone co-treated with DMSO, THAL-SNS-032, and/or dTAG V -1 as indicated for 24 h. Data is representative of n = 3 independent experiments. Source data for c and e are provided as a Source Data file.

    Journal: Nature Communications

    Article Title: Rapid and direct control of target protein levels with VHL-recruiting dTAG molecules

    doi: 10.1038/s41467-020-18377-w

    Figure Lengend Snippet: dTAG V -1 is an exclusively selective degrader of FKBP12 F36V -tagged proteins. a Schematic depiction of the dTAG system using VHL-recruiting dTAG molecules. VHL-recruiting dTAG molecules promote ternary complex formation between the FKBP12 F36V -tagged target protein and E3 ubiquitin ligase complex, inducing target protein ubiquitination and degradation. b Chemical structures of dTAG V -1 and dTAG V -1-NEG. c DMSO-normalized ratio of Nluc/Fluc signal of 293FT FKBP12 WT -Nluc or FKBP12 F36V -Nluc cells treated with dTAG V -1 or dTAG V -1-NEG for 24 h. Data are presented as mean ± s.d. of n = 4 biologically independent samples and are representative of n = 3 independent experiments. d Protein abundance after treatment of PATU-8902 LACZ-FKBP12 F36V clone with 500 nM dTAG V -1 for 4 h compared to DMSO treatment. Volcano plots depict fold change abundance relative to DMSO versus P value derived from a two-tailed Student’s t -test. Fold change values and significance designations derived from a two-tailed Student’s t -test and a permutation-based FDR estimation are provided in Supplementary Data 2 . Data are from n = 3 biologically independent samples. e Immunoblot analysis of PATU-8902 LACZ-FKBP12 F36V clone co-treated with DMSO, THAL-SNS-032, and/or dTAG V -1 as indicated for 24 h. Data is representative of n = 3 independent experiments. Source data for c and e are provided as a Source Data file.

    Article Snippet: Cell lines The following cell lines were employed in this study: 293T (source: ATCC #CRL-3216, media: DMEM with 10% FBS and 1% Penicillin–Streptomycin), 293FT (source: Thermo Fisher Scientific #R70007, media: DMEM with 10% FBS and 1% Penicillin–Streptomycin), PATU-8902 (source: DSMZ #ACC-179, media: DMEM with 10% FBS and 1% Penicillin–Streptomycin), MV4;11 (source: ATCC #CRL-9591, media: RPMI with 10% FBS and 1% Penicillin–Streptomycin) and EWS502 (source: kindly provided by Dr. Stephen L. Lessnick of Nationwide Children’s Hospital and established by Dr. Jonathan A. Fletcher of Harvard Medical School, media: RPMI with 15% FBS and 1% Penicillin–Streptomycin-l -Glutamine).

    Techniques: Derivative Assay, Two Tailed Test