Journal: The Journal of Clinical Investigation

Article Title: KRAS mutant–driven SUMOylation controls extracellular vesicle transmission to trigger lymphangiogenesis in pancreatic cancer

doi: 10.1172/JCI157644

Figure Lengend Snippet: ( A and B ) Representative H&E-stained and IHC images ( A ) and percentages of LYVE-1–positive lymphatic vessel density ( B ) in PDAC according to KRAS subtype ( KRAS WT , n = 15; KRAS G12C , n = 11; KRAS G12V , n = 64; KRAS G12D , n = 96). Scale bars: 50 μm (black) or 25 μm (red). The χ 2 test was used. ( C ) Sequencing evaluation of the KRAS G12D mutation. ( D ) HnRNPA1 expression in PDAC and normal pancreatic tissues was analyzed using data from TCGA database. The nonparametric Mann-Whitney U test was used. ( E and F ) Representative Western blotting images and quantification of hnRNPA1 expression in PDAC tissues and paired normal adjacent tissue (NAT) ( n = 186). The nonparametric Mann-Whitney U test was used. ( G ) qRT-PCR of hnRNPA1 expression in PDAC tissues ( n = 186) according to KRAS subtype. The nonparametric Mann-Whitney U test was used. ( H ) qRT-PCR of hnRNPA1 expression in LN-positive and LN-negative KRAS G12D PDAC tissues ( n = 186). The nonparametric Mann-Whitney U test was used. ( I and J ) Representative images and percentages of IHC staining for hnRNPA1 expression and LYVE-1–positive lymphatic vessel density in KRAS G12D PDAC. Scale bars: 50 μm. The χ 2 test was used. ( K and L ) TEM- ( K ) and NanoSight-characterized ( L ) EVs secreted by KRAS G12D PDAC cells. Scale bar: 100 nm. ( M and N ) Western blotting images and quantification of hnRNPA1 expression in EVs secreted by PDAC cells with different KRAS subtypes and HPDE cells. One-way ANOVA followed by Dunnett’s test was used. Data are presented as mean ± SD; 3 independent experiments were performed in K – N . The box-and-whisker plot in D represents medians with minimum and maximum values. The top and bottom of the box represent the first and third quartiles. * P < 0.05, ** P < 0.01.

Article Snippet: Human PDAC cell lines ( KRAS G12D : PANC-1, AsPC-1; KRAS G12V : Capan-2; KRAS G12C : Mia-PaCa2; KRAS WT : BxPC-3) were obtained from American Type Culture Collection (ATCC).

Techniques: Staining, Sequencing, Mutagenesis, Expressing, MANN-WHITNEY, Western Blot, Quantitative RT-PCR, Immunohistochemistry, Whisker Assay

Journal: The Journal of Clinical Investigation

Article Title: KRAS mutant–driven SUMOylation controls extracellular vesicle transmission to trigger lymphangiogenesis in pancreatic cancer

doi: 10.1172/JCI157644

Figure Lengend Snippet: ( A – C ) Representative images ( A ) and quantification of tube formation and migration ( B and C ) for HLECs treated with PBS or PDAC cell–secreted EVs. Scale bar: 100 μm. One-way ANOVA followed by Dunnett’s test was used. ( D and E ) Western blotting analysis of hnRNPA1 protein levels in PANC-1 cell–secreted EVs after hnRNPA1 silencing or overexpression. ( F and G ) Representative images and quantification of tube formation and migration by HLECs treated with PBS or indicated EVs. Scale bars: 100 μm. One-way ANOVA followed by Dunnett’s test was used. Data are presented as mean ± SD of 3 independent experiments. * P < 0.05, ** P < 0.01.

Article Snippet: Human PDAC cell lines ( KRAS G12D : PANC-1, AsPC-1; KRAS G12V : Capan-2; KRAS G12C : Mia-PaCa2; KRAS WT : BxPC-3) were obtained from American Type Culture Collection (ATCC).

Techniques: Migration, Western Blot, Over Expression

Journal: The Journal of Clinical Investigation

Article Title: KRAS mutant–driven SUMOylation controls extracellular vesicle transmission to trigger lymphangiogenesis in pancreatic cancer

doi: 10.1172/JCI157644

Figure Lengend Snippet: ( A ) Western blotting analysis of hnRNPA1 expression in PDAC cells and the corresponding EVs. ( B and C ) Western blotting assessment of hnRNPA1 expression in PANC-1 cells ( B ) and the corresponding EVs ( C ) after treatment with PBS or indicated inhibitors of PTMs. ( D ) IP assessment of SUMO2 binding to hnRNPA1 after 2-D08 treatment. IB, immunoblot. ( E ) Western blotting analysis of hnRNPA1 expression in EVs secreted by PANC-1 cells after SUMO2 silencing. ( F ) Schematic illustration of the hypothesis of KRAS G12D -induced SUMOylation of hnRNPA1. ( G ) Western blotting analysis of the KRAS downstream pathway in PANC-1 cells after treatment with MCP110. ( H – J ) qRT-PCR ( H and I ) and Western blotting analysis ( J ) of SUMOylation enzyme expression in PDAC cells after MCP110 treatment. The 2-tailed Student’s t test was used. ( K ) Co-IP assessment of SUMO2 binding to hnRNPA1 after SAE1 overexpression. ( L ) Western blotting analysis of hnRNPA1 expression in PANC-1 cell–secreted EVs after SAE1 overexpression. ( M – O ) Representative images ( M ) and quantification of tube formation ( N ) and migration ( O ) of HLECs treated with indicated EVs. Scale bars: 100 μm. One-way ANOVA followed by Dunnett’s test was used. Data are presented as mean ± SD of 3 independent experiments. * P < 0.05, ** P < 0.01.

Article Snippet: Human PDAC cell lines ( KRAS G12D : PANC-1, AsPC-1; KRAS G12V : Capan-2; KRAS G12C : Mia-PaCa2; KRAS WT : BxPC-3) were obtained from American Type Culture Collection (ATCC).

Techniques: Western Blot, Expressing, Binding Assay, Quantitative RT-PCR, Co-Immunoprecipitation Assay, Over Expression, Migration

Journal: The Journal of Clinical Investigation

Article Title: KRAS mutant–driven SUMOylation controls extracellular vesicle transmission to trigger lymphangiogenesis in pancreatic cancer

doi: 10.1172/JCI157644

Figure Lengend Snippet: ( A and B ) Co-IP assay followed by silver staining ( A ) and Western blotting analysis ( B ) for detecting SUMOylated-hnRNPA1–interacting proteins in PANC-1 cells with or without SAE1 knockdown. IB, immunoblot. ( C ) Co‑IP assays analyzing the interaction of hnRNPA1 and TSG101 mediated by SAE1-induced SUMOylation on hnRNPA1. ( D ) Representative immunofluorescence images of hnRNPA1 and TSG101 colocalization in PDAC cells. Scale bar: 5 μm. ( E and F ) Western blotting analysis of hnRNPA1 expression in PANC-1 cells ( E ) and corresponding EVs ( F ) after TSG101 knockdown. ( G and H ) Representative images and quantification of tube formation and migration of HLECs treated with indicated EVs. Scale bars: 100 μm. One-way ANOVA followed by Dunnett’s test was used. Data are presented as mean ± SD of 3 independent experiments. ** P < 0.01.

Article Snippet: Human PDAC cell lines ( KRAS G12D : PANC-1, AsPC-1; KRAS G12V : Capan-2; KRAS G12C : Mia-PaCa2; KRAS WT : BxPC-3) were obtained from American Type Culture Collection (ATCC).

Techniques: Co-Immunoprecipitation Assay, Silver Staining, Western Blot, Knockdown, Immunofluorescence, Expressing, Migration

Journal: The Journal of Clinical Investigation

Article Title: KRAS mutant–driven SUMOylation controls extracellular vesicle transmission to trigger lymphangiogenesis in pancreatic cancer

doi: 10.1172/JCI157644

Figure Lengend Snippet: ( A – C ) Representative images ( A ) for determination of LYVE-1–positive lymphatic vessel density and PROX1 expression in KRAS G12D PDAC according to EV-packaged-hnRNPA1 expression. The percentages of IHC staining for LYVE-1–positive lymphatic vessel density and the correlation between EV-packaged hnRNPA1 and PROX1 expression were analyzed ( B and C ). Scale bar: 50 μm. The χ 2 test was used. ( D and E ) ROC analysis of the diagnostic efficiency of serum EV-packaged hnRNPA1, CA19-9, CEA, and CA72-4 for KRAS G12D PDAC ( D ) or LN metastasis ( E ) of KRAS G12D PDAC. ( F ) Proposed model of KRAS signaling–induced SUMOylation of EV-packaged hnRNPA1 that mediates PROX1 mRNA stability for facilitating KRAS G12D PDAC LN metastasis. Data are presented as mean ± SD of 3 independent experiments. ** P < 0.01.

Article Snippet: Human PDAC cell lines ( KRAS G12D : PANC-1, AsPC-1; KRAS G12V : Capan-2; KRAS G12C : Mia-PaCa2; KRAS WT : BxPC-3) were obtained from American Type Culture Collection (ATCC).

Techniques: Expressing, Immunohistochemistry, Diagnostic Assay

Journal: bioRxiv

Article Title: Cancer-associated fibroblasts in pancreatic ductal adenocarcinoma determine response to SLC7A11 inhibition

doi: 10.1101/2020.07.12.199638

Figure Lengend Snippet: A) Quantitative real-time PCR analysis of SLC7A11 and SLC3A2 expression in total RNA extracts from normal pancreatic fibroblasts (isolated from n=8 patients with benign pancreatic conditions) and CAFs (isolated from n=10 PDAC patients). Bars show mean+s.e.m., circles indicate independent replicates (*p≤0.05, student t-test). B) Western blot of SLC7A11 in total protein extracts from PDAC cells and human CAFs (Cell lines 1-6). α-tubulin was used as a loading control. C) Immunoflourescence for DAPI, αSMA (CAF marker) and SLC7A11 in a human PDAC tissue specimen obtained through the Australian Pancreatic Cancer Genome Initiative (APGI). (D-G) Human PDA tissue microarrays obtained through the APGI (International Cancer Genome Consortium cohort) were stained for SLC7A11 by immunohistochemistry. D) Samples selected as references for scoring (0,1,2,3) for tumour and stromal compartments are shown (insets show magnified view of cells). Scores of 0-1 were classified as low SLC7A11 expression (“Tumour low ” and “Stroma low ”), scores of 2-3 were classified as high SLC7A11 expression (“Tumour high ” and “Stroma high ”). E-G) Kaplan-Meier survival curves showing the correlation between SLC7A11 expression in tumour cells (E), stroma (F), or a combination of both (G) with overall patient survival (days survived post-diagnosis). Patients that were deceased due to other causes or that were still alive were censored (shown as black ticks on each line graph). Total patient numbers for each group are indicated in the graph keys. Asterisks indicate significance based on (F) multivariate analysis (G) univariate Log-Rank test (*p≤0.05). H) Representative photos of Tumour low Stroma low , Tumour high Stroma low , Tumour low Stroma high , and Tumour high Stroma high groups. Scale bars in all photos = 100μm.

Article Snippet: Human PDAC cells (MiaPaCa-2, Panc-1, AsPC1 and HPAFII; American Tissue Culture Collection) were cultured as described ( - ).

Techniques: Real-time Polymerase Chain Reaction, Expressing, Isolation, Western Blot, Control, Marker, Staining, Immunohistochemistry, Biomarker Discovery

Journal: bioRxiv

Article Title: Cancer-associated fibroblasts in pancreatic ductal adenocarcinoma determine response to SLC7A11 inhibition

doi: 10.1101/2020.07.12.199638

Figure Lengend Snippet: A-B) Schematic diagram of 3D co-culture spheroid outgrowth assay and quantification of 3D co-culture spheroid outgrowth post-transfection with control-siRNA (ns-siRNA) or SLC7A11-siRNA pool. Labels: PDAC ns CAF ns = non-silencing controls; PDAC ns CAF slc = SLC7A11 knockdown in CAFs only; PDAC slc CAF ns = SLC7A11 knockdown in PDAC cells only; PDAC slc CAF slc = SLC7A11 knockdown in both PDAC cells and CAFs. Representative photos are shown above each bar with the core circled in white dashed lines and outgrowth in red dashed lines (bars in photos = 300μm). Circles indicate replicates, lines indicate mean±s.e.m., asterisks indicate significance (ns=not significant, *p≤0.05, **p≤0.01, ***p≤0.001; One-way ANOVA). C) Schematic diagram of 3D co-culture growth assay using stable shRNA cell lines and D) representative photos (bars in photos = 200μm) and quantification of 3D co-culture spheroid growth. Labels: PDAC scr CAF scr = scramble-shRNA controls; PDAC scr CAF slc = SLC7A11-shRNA seq 1 in CAFs only; PDAC slc CAF scr = SLC7A11-shRNA seq 1 in PDAC cells only; PDAC slc CAF slc = SLC7A11-shRNA seq 1 in both PDAC cells and CAFs. Circles indicate replicates, lines indicate mean±s.e.m., asterisks indicate significance (ns=not significant, *p≤0.05, **p≤0.01, ****p≤0.0001, n=4-6; One-way ANOVA). Replicate numbers in panel B refer to independent experiments performed using MiaPaCa-2 combined with CAF cells isolated from different PDAC patients. Replicate numbers in panels D refer to replicate spheroids performed using MiaPaCa-2 PDAC cells combined with an immortalised CAF line.

Article Snippet: Human PDAC cells (MiaPaCa-2, Panc-1, AsPC1 and HPAFII; American Tissue Culture Collection) were cultured as described ( - ).

Techniques: Co-Culture Assay, Transfection, Control, Knockdown, Growth Assay, shRNA, Isolation

Journal: bioRxiv

Article Title: Cancer-associated fibroblasts in pancreatic ductal adenocarcinoma determine response to SLC7A11 inhibition

doi: 10.1101/2020.07.12.199638

Figure Lengend Snippet: A) Quantification of Pancreatic Intraepithelial Neoplasia (PanINs) 1A-3 from KC mice (n=7) and KC mice with SLC7A11 conditional KO under Pdx1 -promoter (KC Slc7a11 fl/fl ; n=7) at 70 days of age (mean±s.e.m.). B) Kaplan-Meier analysis showing survival percentage of KPC (n=26) and KPC Slc7a11 fl/fl mice (n=24) mice. C) Representative photos of KPC and KPC Slc7a11 fl/fl tumour sections probed for αSMA (brown). The quantification of αSMA staining is shown in the graph (mean±s.e.m.), based on ImageJ analysis of representative regions from each tumour section (n=5 mice per group). Scale bars = 400μm. D) Representative photos of KPC and KPC Slc7a11 fl/fl tumour sections. The quantification of picrosirius red staining is shown in the bar graph (mean±s.e.m.), based on ImageJ analysis of representative regions from each tumour section. Scale bars = 400μm. Asterisks indicate significance (*p≤0.05; n=5 mice per group; student t-test). E-F) Live cell counts (mean±s.e.m.) of (E) KPC PDAC cells and (F) KPC CAFs 72h post-transfection with control-siRNA (ns-siRNA) or mouse SLC7A11-siRNA pool (SLC7A11 pool). Asterisks indicate significance (*p≤0.05, **p≤0.01; n=3; one-way ANOVA).

Article Snippet: Human PDAC cells (MiaPaCa-2, Panc-1, AsPC1 and HPAFII; American Tissue Culture Collection) were cultured as described ( - ).

Techniques: Staining, Transfection, Control

Journal: bioRxiv

Article Title: Cancer-associated fibroblasts in pancreatic ductal adenocarcinoma determine response to SLC7A11 inhibition

doi: 10.1101/2020.07.12.199638

Figure Lengend Snippet: All orthotopic tumours were co-injections of PDAC cells and CAFs. A) Orthotopic pancreatic tumours were treated with STAR nanoparticles + control-siRNA or SLC7A11 siRNA single sequence (SLC7A11 single seq) in the regimen shown. Representative photos of immunohistochemistry for SLC7A11 in tumour tissue at the model endpoint are shown. Graph shows optical density (staining intensity) calculated from average pixel intensity measurements from 3 representative images per tumour, using ImageJ. Circles indicate individual mice, lines indicate mean±s.e.m., asterisks indicate significance (*p≤0.05; One-way ANOVA). B) Treatment regimen for therapeutic model analysed in panels (C-D). Circles and triangles in all dot plots in panels C-D represent individual mice. C) Tumour volume at therapeutic model endpoint, as assessed by calliper measurement ex vivo (mean±s.e.m.). Asterisks indicate significance (*p≤0.05; One-way ANOVA). D) Representative photos of metastases confirmed by H&E staining following detection at model endpoint by ex vivo luminescence imaging of organs. Graph shows metastatic sites per mouse (mean±s.e.m.) for each treatment group. Scale bars in all figures = 200μm.

Article Snippet: Human PDAC cells (MiaPaCa-2, Panc-1, AsPC1 and HPAFII; American Tissue Culture Collection) were cultured as described ( - ).

Techniques: Control, Sequencing, Immunohistochemistry, Staining, Ex Vivo, Imaging

Journal: bioRxiv

Article Title: LYMTACs: Chimeric Small Molecules Repurpose Lysosomal Membrane Proteins for Target Protein Relocalization and Degradation

doi: 10.1101/2024.09.08.611923

Figure Lengend Snippet: A) Schematic of the LYMTAC concept. A LYMTAC is a heterobifunctional molecule composed of a POI ligand, a linker and an LMP ligand. LYMTACs co-opt short-lived lysosomal membrane proteins as effectors to deliver target proteins for lysosomal degradation. B) Cycloheximide (CHX) assay in FLAG-MTH1-RNF152 stably expressing HCT116 cells. Cells were pre-treated with DMSO, 200 nM Bafilomycin A1 (BafA1), or 1 µM ubiquitin E1 inhibitor (TAK-243) for 30 min, followed by co-treatment with 100 µg/mL CHX for 4 h. Cells were harvested at 0 h to include as a control. Cells were lysed and subjected to immunoblotting with MTH1 and vinculin antibodies. Data are representative of two independent experiments. C) Structure of LYMTAC-1 (promiscuous kinase inhibitor-PEG2-MTH1 ligand). D) Quantitative proteomics analysis of LYMTAC-1. HEK293T cells stably expressing FLAG-MTH1-RNF152 were treated with either DMSO or 500 nM LYMTAC-1 for 19 h and subjected to global proteomics analysis. Data are representative of three treatment replicates. E) LYMTAC-1 induced dose-dependent degradation of PTK2. HEK293T cells stably expressing FLAG-MTH1-RNF152 were treated with increasing concentrations of LYMTAC-1 for 19 h and subjected to immunoblotting. Data are representative of three independent experiments. F) LYMTAC-1 induced dose dependent degradation of EPHA2. Data are representative of three independent experiments. G-H) HEK293T cells stably expressing MTH1-RNF152 were pre-treated with BafA1 and MG-132 for 30 min, co-treated with DMSO or 500 nM LYMTAC-1 for 19 h, and subjected to immunoblotting with the indicated antibodies. Data are representative of two independent experiments.

Article Snippet: Human embryonic kidney 293T (HEK293T), AsPC1, and HCT116 cells were obtained from ATCC.

Techniques: Membrane, Stable Transfection, Expressing, Ubiquitin Proteomics, Control, Western Blot, Quantitative Proteomics

Journal: bioRxiv

Article Title: LYMTACs: Chimeric Small Molecules Repurpose Lysosomal Membrane Proteins for Target Protein Relocalization and Degradation

doi: 10.1101/2024.09.08.611923

Figure Lengend Snippet: A) Schematic of the chemical genetic system where the target protein is HiBiT-FKBP12 F36V -KRAS G12D , and the effector is MTH1-RNF152. B) Structure of LYMTAC-2 (FKBP12 F36V ligand-PEG2-MTH1 ligand). C) HiBiT cellular degradation assay in MTH1-RNF152 stably expressing, knock-in HCT116 (HiBiT-FKBP12 F36V -KRAS G12D: (HF-KRAS)) cells. Cells were treated with increasing concentrations of LYMTAC-2 for 24 h and HiBiT levels were measured using Nano-Glo® HiBiT lytic reagent. Data are representative of three independent experiments, reported as the mean ± S.E. D) HCT116 (HF-KRAS) cells stably expressing FLAG-MTH1-RNF152 were pre-treated with BafA1, MG-132, or TAK-243 for 30 min followed by co-treatment with DMSO or 500 nM LYMTAC-2 for 6 h and subjected to immunoblotting with KRAS and tubulin antibodies. Data are representative of three independent experiments. E) KRAS G12D ubiquitylation assay. HA-ubiquitin was transfected into HCT116, pre-treated with 200 nM BafA1 for 30 min, then co-treated with DMSO or 1 µM LYMTAC-2 for 4 h. Next, cells were lysed and subjected to immunoprecipitation with anti-HiBiT antibody. Whole cell lysate was used as INPUT. The respective blots are probed with HA, MTH1, HiBiT, and vinculin antibodies. Data are representative of three independent experiments.

Article Snippet: Human embryonic kidney 293T (HEK293T), AsPC1, and HCT116 cells were obtained from ATCC.

Techniques: Degradation Assay, Stable Transfection, Expressing, Knock-In, Western Blot, Ubiquitin Assay, Ubiquitin Proteomics, Transfection, Immunoprecipitation

Journal: bioRxiv

Article Title: LYMTACs: Chimeric Small Molecules Repurpose Lysosomal Membrane Proteins for Target Protein Relocalization and Degradation

doi: 10.1101/2024.09.08.611923

Figure Lengend Snippet: A) Schematic of complex formation between KRAS G12D and MTH1-RNF152 in the presence of KRAS-targeting LYMTAC. B) Structures of the pan-KRAS inhibitor and PROTAC C) Structures of LYMTAC-3 and LYMTAC-4. D) HiBiT cellular degradation assay in FLAG-MTH1-RNF152-stably expressing, knock-in HCT116 (HiBiT-FKBP12 F36V -KRAS G12D ) cells. Cells were treated with indicated doses of compounds for 24 h and subjected to Nano-Glo® HiBiT lytic assay. Data are representative of three independent experiments, reported as the mean ± S.E. E) Ternary complex formation assay, AsPC-1 cells stably expressing FLAG-MTH1-RNF152 were pre-treated with BafA1 for 30 min, followed by co-treatment with DMSO or 1 µM LYMTAC-3 or LYMTAC-4 for 4 h and subjected to immunoprecipitation with anti-FLAG antibody. Whole cell lysate was used as INPUT. The respective blots are probed with KRAS, MTH1, and vinculin antibodies. Data are representative of two independent experiments. F) AsPC-1 cells stably expressing FLAG-MTH1-RNF152 were treated with DMSO or 100 nM of the indicated compounds for 24 h and subjected to immunoblotting with KRAS, pERK, ERK, and vinculin antibodies. Data are representative of three independent experiments. G) AsPC-1 cells stably expressing FLAG-MTH1-RNF152 were treated with 100 nM KRAS inhibitor, 100 nM PROTAC, or 100 nM LYMTAC-4 for 6 h. For control experiments with LYMTAC-4, cells were pretreated with MTH1-ligand or BafA1 for 30 min, followed by co-treatment in the absence or presence of 100 nM LYMTAC-4 for 6 h, and subjected to immunoblotting with KRAS, p-ERK, ERK, and vinculin antibodies. Data are representative of two independent experiments. H) HEK293T cells stably expressing HiBiT-FKBP12-KRAS G12D and FLAG-MTH1-RNF152 were pre-treated with 1 µM TAK-243 for 30 min followed by DMSO or 1 µM LYMTAC-2 treatment for 6 h. The respective blots are probed with KRAS, p-ERK, ERK, and tubulin antibodies. Data are representative of two independent experiments.

Article Snippet: Human embryonic kidney 293T (HEK293T), AsPC1, and HCT116 cells were obtained from ATCC.

Techniques: Degradation Assay, Stable Transfection, Expressing, Knock-In, Tube Formation Assay, Immunoprecipitation, Western Blot, Control

Journal: bioRxiv

Article Title: LYMTACs: Chimeric Small Molecules Repurpose Lysosomal Membrane Proteins for Target Protein Relocalization and Degradation

doi: 10.1101/2024.09.08.611923

Figure Lengend Snippet: A) Schematic of Lysosome Membrane Proteins (LMPs). B) CHX assay in FLAG-MTH1-LAPTM4a-stably expressing HCT116 cells. Cells were pre-treated with DMSO, 200 nM Bafilomycin A1 (BafA1), or 1 µM E1 inhibitor (TAK-243) for 30 min, followed by co-treatment with CHX for 4 h. Cells were harvested at 0 h to include as a control. Cells were lysed and subjected to immunoblotting with MTH1 and vinculin antibodies. Data are representative of three independent experiments. C) CHX assay in FLAG-MTH1-LAPTM5-stably expressing HCT116 cells. Data are representative of three independent experiments. D) HiBiT cellular degradation assay in FLAG-MTH1-LAPTM4a- and FLAG-MTH1-LAPTM5-stably expressing HCT116 (knock-in-HiBiT-FKBP12 F36V -KRAS G12D: (HF-KRAS)) cells. Cells were treated with increasing concentrations of LYMTAC-2 for 24 h and HiBiT levels were measured using Nano-Glo® HiBiT lytic reagent. Data are representative of two independent experiments and reported as the mean ± S.E. E) HCT116 (HF-KRAS) cells stably expressing FLAG-MTH1-LAPTM5 were pre-treated with BafA1, 2 BTZ, or TAK-243 for 30 min followed by co-treatment with DMSO or 500 nM LYMTAC-2 for 6 h and subjected to immunoblotting with KRAS and tubulin antibodies. Data are representative of two independent experiments. F) KRAS ubiquitylation assay. HA-ubiquitin was transfected into HCT116 expressing FLAG-MTH1-LAPTM5, which were pre-treated with 200 nM BafA1 for 30 min followed by co-treatment with DMSO or 1 µM LYMTAC-2 treatment for 4 h. Next, cells were lysed and subjected to immunoprecipitation with anti-HiBiT antibody. Whole cell lysate was used as INPUT. The respective blots were probed with HA, MTH1, HiBiT, and vinculin antibodies. Data are representative of two independent experiments. G) AsPC-1 cells stably expressing FLAG-MTH1-LAPTM4a were pre-treated with either pan-KRAS inhibitor or MTH1 ligand, followed by DMSO or 100 nM LYMTAC-4 treatment for 6 h. Cells were lysed and subjected to immunoblotting with KRAS, pERK, and tubulin antibodies. Data are representative of two independent experiments.

Article Snippet: Human embryonic kidney 293T (HEK293T), AsPC1, and HCT116 cells were obtained from ATCC.

Techniques: Membrane, Stable Transfection, Expressing, Control, Western Blot, Degradation Assay, Knock-In, Ubiquitin Assay, Ubiquitin Proteomics, Transfection, Immunoprecipitation