Journal: Scientific Reports

Article Title: A scDb-based trivalent bispecific antibody for T-cell-mediated killing of HER3-expressing cancer cells

doi: 10.1038/s41598-021-93351-0

Figure Lengend Snippet: Binding properties of scDb and scDb-scFv. Binding to ( a ) LIM1215, ( b ) MCF-7, ( c ) BT-474, ( d ) FaDu, ( e ) SW-620, ( f ) HCT116, ( g ) HT1080, ( h ) MDA-MB-231, ( i ) WM1791c and ( j ) CD3-expressing Jurkat cells was analyzed in flow cytometry. Bound protein was detected using a PE-labeled anti-His mAb. Mean ± SD, n = 3.

Article Snippet: LIM1215 were obtained from Merck KGaA (10092301-1VL), SW-620 cells and MDA-MB-231 cells were obtained from CLS Cell Lines Service GmbH (300466), respectively, and cultured in RPMI-1640 (Thermo Fisher Scientific, 11875), 10% FBS (Pan Biotech, P30-3309).

Techniques: Binding Assay, Expressing, Flow Cytometry, Labeling

Journal: Scientific Reports

Article Title: A scDb-based trivalent bispecific antibody for T-cell-mediated killing of HER3-expressing cancer cells

doi: 10.1038/s41598-021-93351-0

Figure Lengend Snippet: Effect of scDb and scDb-scFv on cytotoxic potential of PBMCs. Target cells (( a ) LIM1215, ( b ) MCF-7, ( c ) BT-474, ( d ) FaDu, ( e ) SW-620, ( f ) HCT116, ( g ) HT1080, ( h ) MDA-MB-231 or ( i ) WM1791c cells) were incubated with a serial dilution of scDb or scDb-scFv and PBMCs in an effector:target cell ratio (E:T) of 10:1. After 3 days, cell viability was determined using crystal violet staining. Mean ± SD. On the low expressing cell lines SW-620, HCT116, HT1080, MDA-MB-231 and WM1791c three independent experiments (n = 3) were performed using PBMCs from one donor. On the cell lines LIM1215, MCF-7, BT-474 and FaDu three independent experiments for each donor using PBMCs from two different donors were performed (n = 6). In total, PBMCs from 5 different donors were used. ( j ) Potency of scDb and scDb-scFv versus HER3 expression.

Article Snippet: LIM1215 were obtained from Merck KGaA (10092301-1VL), SW-620 cells and MDA-MB-231 cells were obtained from CLS Cell Lines Service GmbH (300466), respectively, and cultured in RPMI-1640 (Thermo Fisher Scientific, 11875), 10% FBS (Pan Biotech, P30-3309).

Techniques: Incubation, Serial Dilution, Staining, Expressing

Journal: OncoTargets and therapy

Article Title: miR-486-5p attenuates tumor growth and lymphangiogenesis by targeting neuropilin-2 in colorectal carcinoma

doi: 10.2147/OTT.S103460

Figure Lengend Snippet: NRP2 is a target of miR-486-5p. Notes: ( A ) Effect of miR-486-5p on NRP2 expression as determined by a luciferase reporter assay. The data were normalized by determining the firefly and Renilla luciferase activities measured at 72 hours posttransfection; SW620 and HT-29 cells were transfected with miR-486-5p mimic (miR-486-5p group), mimic control (NC group), and blank control (blank group). ( B ) Transfection of miR-486-5p mimics to SW620 and HT-29 cells increases the expression of miR-486-5p detected by qRT-PCR. ( C ) NRP2 protein expression level was significantly decreased in miR-486-5p-transfected SW620 and HT-29 cells. ( D ) Relative NRP2 protein levels in figure ( C ) were quantified using the gray scale quantification analysis. ** P <0.01. Abbreviations: GAPDH, glyceraldehyde 3-phosphate dehydrogenase; NRP2, neuropilin-2; qRT-PCR, quantitative real-time polymerase chain reaction; NC, nontarget control.

Article Snippet: Human CRC cell lines SW620 and HT-29 were purchased from Boster Company (Boster, Wuhan, People’s Republic of China).

Techniques: Expressing, Luciferase, Reporter Assay, Transfection, Quantitative RT-PCR, Real-time Polymerase Chain Reaction

Journal: OncoTargets and therapy

Article Title: miR-486-5p attenuates tumor growth and lymphangiogenesis by targeting neuropilin-2 in colorectal carcinoma

doi: 10.2147/OTT.S103460

Figure Lengend Snippet: miR-486-5p inhibited xenograft tumor growth by targeting NRP2 in vivo. Notes: ( A ) Groups of nude mice were implanted subcutaneously with SW620/PBS cells (Blank group), SW620/mimic-control cells (NC group), or SW620/miR-486-5p cells (miR-486-5p group), Tumor volumes were recorded at the indicated times. ( B ) The tumor mass was determined when the mice were sacrificed. ( C ) NRP2 expression in tumors was examined by Western blot. ( D ) Quantitative analysis of the NRP2 protein level using a gray scan analysis. One representative tumor mass group from three independent experiments with similar results is shown. * P <0.05, ** P <0.01. Abbreviations: GAPDH, glyceraldehyde 3-phosphate dehydrogenase; NRP2, neuropilin-2; PBS, phosphate-buffered saline; NC, nontarget control.

Article Snippet: Human CRC cell lines SW620 and HT-29 were purchased from Boster Company (Boster, Wuhan, People’s Republic of China).

Techniques: In Vivo, Expressing, Western Blot

Journal: Oncotarget

Article Title: MiR-675-5p supports hypoxia induced epithelial to mesenchymal transition in colon cancer cells

doi: 10.18632/oncotarget.14464

Figure Lengend Snippet: ( A ) Real time-PCR for miR-675-5p in SW480 and SW620 cells. All data were normalized for U6 and ΔΔct was expressed as relative amount of miRNAs. Values are presented as the mean ± SD (of three independent experiments). SW620 vs SW480 ** p < 0.001. ( B ) Real time-PCR for miR-675-5p and EMT master genes: Snail, Slug and E-cadherin in SW480 and SW620 cells. Data were normalized for β-actin, while U6 was used for miRNA normalization. ΔΔct is expressed as Fold of induction (FOI) with respect to expression in control samples. Data are the mean ± SD of three independent experiments. SW480 miRNA inhibitor vs SW480 scramble control: * p < 0.05; *** p < 0.0001; SW620 miRNA inhibitor vs SW620 scramble control * p < 0.05; ** p < 0.001; *** p < 0.0001. ( C ) Immunofluorescences and median focal plane in confocal analysis for E-CADHERIN, ZO-1and SNAIL in SW480 and SW620 cells treated with miR-675-5p inhibitor or scramble control, in blue the nuclear staining with DAPI. ( D ) Migration assay: Phase contrast micrographs (10×) showing the migration of SW480 and SW620 cells pre-treated with miR-675-5p inhibitor or scramble control. Right panel: Quantification of migration by counting the number of migrated cells (violet) per field ( n = 6); * p < 0.05.

Article Snippet: SW620 cells were grown at a density of 100.000 cells/well in a 6 wells plate, and transfected by using Attractene Transfection Reagent (cat. number.1051531, Quiagen) for 6 hours with 0.2 mg/ml H19 siRNA (SR319206B Origene Technologies) or scramble negative control (SR30004 Origene Technologies) at the same dose following manufacturer's indications.

Techniques: Real-time Polymerase Chain Reaction, Expressing, Staining, Migration

Journal: Oncotarget

Article Title: MiR-675-5p supports hypoxia induced epithelial to mesenchymal transition in colon cancer cells

doi: 10.18632/oncotarget.14464

Figure Lengend Snippet: Real time-PCR for HIF1α ( A ) and VEGF-A ( B ) in SW480 and SW620 cells treated with miR-675-5p inhibitor or scramble control. Data were normalized for β-actin and ΔΔct is expressed as FOI with respect to expression in control samples. SW480 miRNA inhibitor vs SW480 control * p < 0.05; SW620 miRNA inhibitor vs SW620 control * p < 0.05 ( C ) Real time-PCR for HIF1α in SW480 and SW620 cells treated with miR-675-5p inhibitor or scramble control after 6 hours of hypoxia. Data were normalized for β-actin and ΔΔct is expressed as FOI with respect to expression in normoxia. Values are presented as the mean ± SD. SW620 inhibitor vs SW620 ctr ** p < 0.001. ELISA assay for HIF-1α ( D ) and VEGFa ( E ) performed in SW480 and SW620 cells, transfected with miRNA inhibitor or scramble control, after 6 hours of hypoxia. Data are expressed as Absorbance (ABS) values at 450 nm. SW620 miRNA inhibitor vs SW620 scramble control ** p < 0.001. Real time-PCR for hypoxia targets genes and EMT regulator genes (VEGF, SNAIL, SLUG) in SW480 cells ( F ) and SW620 cells ( G ) treated with miR-675-5p inhibitor or scramble control, after 6 hours of hypoxia. Data were normalized for β-actin and ΔΔct is expressed as FOI with respect to expression in control samples. SW620 inhibitor vs SW620 control ** p < 0.001. ( H ) Real time-PCR for lncH19 and HIF1α in SW620 cells treated with siRNA H19 or scramble control and exposed to hypoxia for 6 hours. Data were normalized for β-actin and ΔΔct is expressed as FOI with respect to expression in control samples. SW620 siH19 vs SW620 control * p < 0.05;. ( I ) Real time-PCR for Snail and Slug in SW620 cells transfected with siRNA H19 or scrambled control and exposed to hypoxia for 6 hours. Data were normalized for β-actin and ΔΔct is expressed as FOI with respect to expression in control samples. ** p < 0.001. Data are the mean ± SD of three independent experiments.

Article Snippet: SW620 cells were grown at a density of 100.000 cells/well in a 6 wells plate, and transfected by using Attractene Transfection Reagent (cat. number.1051531, Quiagen) for 6 hours with 0.2 mg/ml H19 siRNA (SR319206B Origene Technologies) or scramble negative control (SR30004 Origene Technologies) at the same dose following manufacturer's indications.

Techniques: Real-time Polymerase Chain Reaction, Expressing, Enzyme-linked Immunosorbent Assay, Transfection

Journal: Oncotarget

Article Title: MiR-675-5p supports hypoxia induced epithelial to mesenchymal transition in colon cancer cells

doi: 10.18632/oncotarget.14464

Figure Lengend Snippet: ( A ) ELISA assay for HIF-1α performed in SW620 cells nuclear extracts after mimic or scramble-control transfection. Data are expressed as Absorbance (ABS) values at 450nm. SW620 mimic vs SW620 scramble control ** p < 0.001. Data are the mean ± SD of three independent experiments. ( B ) Immunofluorescences and median focal plane of confocal analysis for HIF1α (red) in SW620 cells treated with miR-675-5p mimic or scrambled control, in blue the nuclear staining with DAPI. ( C ) Real-time PCR for HIF1α targets gene (Snail, Slug, VEGF, VEGFR-2) from SW620 cells after miR-675-5p mimic or scramble control transfection. Data were normalized for β-actin and ΔΔct is expressed as FOI of indicated genes after mimic transfection with respect to scramble control. ( D ) ELISA assay for VEGF levels in supernatants from SW620 cell lines 18 hours after mimic and scramble control transfection. Data are expressed as pg/ml of soluble VEGF. SW620 mimic vs SW620 scramble control *** p < 0,0001. Data are the mean ± SD of three independent experiments. ( E ) Migration assay: Phase contrast micrographs (10×) showing the migration of SW620 cells pre-treated with mimic miR-675-5p and scramble control. Right panel: Quantification of motility established by counting the number of migrated cells (violet) per field; SW620 mimic vs SW620 scramble control * p < 0.05.

Article Snippet: SW620 cells were grown at a density of 100.000 cells/well in a 6 wells plate, and transfected by using Attractene Transfection Reagent (cat. number.1051531, Quiagen) for 6 hours with 0.2 mg/ml H19 siRNA (SR319206B Origene Technologies) or scramble negative control (SR30004 Origene Technologies) at the same dose following manufacturer's indications.

Techniques: Enzyme-linked Immunosorbent Assay, Transfection, Staining, Real-time Polymerase Chain Reaction, Migration

Journal: Oncotarget

Article Title: MiR-675-5p supports hypoxia induced epithelial to mesenchymal transition in colon cancer cells

doi: 10.18632/oncotarget.14464

Figure Lengend Snippet: ( A ) Left: predicted seed sequence for miR-675-5p on DDB2 gene. Right: Real-time PCR for DDB-2 gene from SW620 cells after 18 hours of miR-675-5p mimic or miR-675-5p inhibitor or scramble control transfection. Data were normalized for β-actin and ΔΔct is expressed as FOI with respect to scramble control. SW620 mimic and inhibitor vs SW620 ** p < 0,001. Data are the mean ± SD of three independent experiments. ( B ) Western blot for DDB2, HIF1α and β-actin in SW620 cells after 18 hours of miR-675-5p scramble control or inhibitor transfection.

Article Snippet: SW620 cells were grown at a density of 100.000 cells/well in a 6 wells plate, and transfected by using Attractene Transfection Reagent (cat. number.1051531, Quiagen) for 6 hours with 0.2 mg/ml H19 siRNA (SR319206B Origene Technologies) or scramble negative control (SR30004 Origene Technologies) at the same dose following manufacturer's indications.

Techniques: Sequencing, Real-time Polymerase Chain Reaction, Transfection, Western Blot

Journal: Journal of Korean Medical Science

Article Title: Dual-Blocking of PI3K and mTOR Improves Chemotherapeutic Effects on SW620 Human Colorectal Cancer Stem Cells by Inducing Differentiation

doi: 10.3346/jkms.2016.31.3.360

Figure Lengend Snippet: Stemness properties of sorted SW620 CD133+ cells. ( A ) The mRNA expression of stemness and differentiation marker in SW620 CD133+ and CD133–cells were measured by RT-PCR. β-Actin was used as a loading control. For comparisons, the relative value for markers of CD133–cells was considered to be “1”. ( B ) Self-renewal ability of sorted cells was analyzed by sphere formation assay. Pictures were taken at ×40 magnification. Scale bar = 100 µm. Data are expressed as the mean ± standard error of the mean (SEM) of three independent experiments performed (* P < 0.001).

Article Snippet: The SW620 human colorectal cancer cell line was purchased from the Korean cell line bank (Seoul, Korea).

Techniques: Expressing, Marker, Reverse Transcription Polymerase Chain Reaction, Control, Tube Formation Assay

Journal: Journal of Korean Medical Science

Article Title: Dual-Blocking of PI3K and mTOR Improves Chemotherapeutic Effects on SW620 Human Colorectal Cancer Stem Cells by Inducing Differentiation

doi: 10.3346/jkms.2016.31.3.360

Figure Lengend Snippet: The drug sensitivity of inhibited SW620 CD133+ cells. ( A ) Inhibited SW620 CD133+ cells were treated with paclitaxel (100 nM) for 24 hours and cell viability was assessed by CCK-8 assay. For comparisons cell viability of DMSO treated CD133+ cells was 100%. ( B ) Self-renewal ability of inhibited SW620 CD133+ cells treated with paclitaxel and self-renewal ability was assessed by sphere formation assay. ( C ) Differentiation marker (CEA) expression of inhibited CSCs treated with paclitaxel. The protein expression of CEA was assessed by IF. For the comparison, relative intensity of CTCF for CEA of DMSO treated CD133+ cells was defined as “1”. All data are expressed as the mean ± standard error of the mean (SEM) of three independent experiments performed (* P < 0.05, † P < 0.01, ‡ P < 0.001, “N/S” means “statistically not significant”). Pictures were taken at ×400 magnification. Scale bar = 10 μm.

Article Snippet: The SW620 human colorectal cancer cell line was purchased from the Korean cell line bank (Seoul, Korea).

Techniques: CCK-8 Assay, Tube Formation Assay, Marker, Expressing, Comparison

Journal: Journal of Korean Medical Science

Article Title: Dual-Blocking of PI3K and mTOR Improves Chemotherapeutic Effects on SW620 Human Colorectal Cancer Stem Cells by Inducing Differentiation

doi: 10.3346/jkms.2016.31.3.360

Figure Lengend Snippet: Xenograft tumorigenecity assay of inhibitors treated SW620 CD133+ cells before and after injection of anti-cancer drug. ( A ) Comparison of tumorigenecity of inhibitor treated SW620 CD133+ cells in BALB/ c nude mice. Balb/ c nude mice were subcutaneously injected with sorted SW620 CD133+ cells. The left flank was injected with untreated CD133+ cells, while the right flank was injected with CD133+ cells treated with each inhibitor. Anti-cancer drug (paclitaxel) was injected since 28 days. ( B ) Tumor volume (mm 3 ) of xenografts from Balb/ c mice. The change in tumor volume was checked for each group. DMSO treated group was used as a control. Data are expressed as the mean ± standard error of the mean (SEM) of mice for each group.

Article Snippet: The SW620 human colorectal cancer cell line was purchased from the Korean cell line bank (Seoul, Korea).

Techniques: Injection, Comparison, Control

Journal: Journal of Experimental & Clinical Cancer Research : CR

Article Title: EGFR inhibition augments the therapeutic efficacy of the NAT10 inhibitor Remodelin in Colorectal cancer

doi: 10.1186/s13046-025-03277-y

Figure Lengend Snippet: Effects of the NAT10 inhibitor Remodelin on CRC proliferation. A . WB analysis of NAT10 expression in a panel of CRC cell lines treated with Remodelin. B . CCK-8 assay of SW48 and SW620 cells treated with different concentrations of Remodelin. C . The correlation between the basal NAT10 expression content and Remodelin efficacy. D , E . EdU and colony formation assays were performed to verify the effect of Remodelin on CRC cell proliferation. The data are representative of three independent experiments and presented as the mean ± SD. * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001, determined using two-tailed unpaired Student’s t- tests

Article Snippet: Human CRC cell lines, including CACO2, SW48, DLD1, SW480, SW620, and HCT-116, were purchased from Procell Life Science & Technology Company (Wuhan, China).

Techniques: Expressing, CCK-8 Assay, Two Tailed Test

Journal: Journal of Experimental & Clinical Cancer Research : CR

Article Title: EGFR inhibition augments the therapeutic efficacy of the NAT10 inhibitor Remodelin in Colorectal cancer

doi: 10.1186/s13046-025-03277-y

Figure Lengend Snippet: Identification of downstream targets of NAT10 in CRC cells. A . Heat map showing differentially expressed genes identified by RNA-seq in NAT10-knockdown cells relative to control cells. Green and red indicate low and high mRNA expression levels, respectively. B . Pathway enrichment and GO enrichment analysis of significantly downregulated genes. C . GO enrichment analysis of the potential NAT10 binding RNAs. D . ac4C peak mainly appeared in coding sequences (CDS) of SW620 cells. E . Pathway enrichment and GO enrichment analysis of acRIP-Seq RNA sequence data. F . Relative protein levels of PI3K, p-PI3K, AKT, p-AKT, mTOR and p-mTOR in CRC cells following NAT10 knockdown or overexpression. G . GO enrichment analysis of genes that overlapped with those identified in RIP-seq and acRIP-seq analysis. H . Overlap of genes identified using RNA-seq, RIP-seq, and acRIP-seq

Article Snippet: Human CRC cell lines, including CACO2, SW48, DLD1, SW480, SW620, and HCT-116, were purchased from Procell Life Science & Technology Company (Wuhan, China).

Techniques: RNA Sequencing Assay, Knockdown, Control, Expressing, Binding Assay, Sequencing, Over Expression

Journal: Journal of Experimental & Clinical Cancer Research : CR

Article Title: EGFR inhibition augments the therapeutic efficacy of the NAT10 inhibitor Remodelin in Colorectal cancer

doi: 10.1186/s13046-025-03277-y

Figure Lengend Snippet: NAT10 interacts with UBR5 and is ubiquitinated by ubiquitin ligase. A . RT-qPCR analysis of NAT10 expression in CRC cell lines. B . CQ (20 µM) was used to inhibit lysosomal degradation and MG132 (100 nM) was employed to repress proteasomal degradation in SW620 cells. C . Half-life analysis of NAT10 in SW480 and SW620 cells treated with 50 µg/mL cycloheximide (CHX) for the indicated times. D . Ubiquitination assay was performed on the indicated cells. E . Co-IP was performed to determine the relationship between NAT10 and UBR5 expression. F . Knockdown of UBR5 in DLD1 and SW620 cells was confirmed by WB. G . WB analysis of NAT10 in indicated cells treated or not treated with 5-Fu (500 µM). H, I. HA-NAT10 (WT or indicated K-R mutants) was co-expressed with MYC-Ub and Flag-UBR5 plasmids. After MG132 (100 nM, 8 h) treatment, IP was performed with HA antibody, followed by immunoblotting with indicated antibodies. The data are representative of three independent experiments and presented as the mean ± SD. * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001, using two-tailed unpaired Student’s t -tests

Article Snippet: Human CRC cell lines, including CACO2, SW48, DLD1, SW480, SW620, and HCT-116, were purchased from Procell Life Science & Technology Company (Wuhan, China).

Techniques: Quantitative RT-PCR, Expressing, Ubiquitin Assay, Co-Immunoprecipitation Assay, Knockdown, Western Blot, Two Tailed Test

Journal: Clinical and Translational Medicine

Article Title: Extracellular vesicle expansion of PMIS‐miR‐210 expression inhibits colorectal tumour growth via apoptosis and an XIST/NME1 regulatory mechanism

doi: 10.1002/ctm2.1037

Figure Lengend Snippet: miR‐210 expression and inhibition profiles in colorectal tumour cells. (A) The metastatic colorectal cancer (CRC) cell line SW620 has the highest level of miR‐210 transcripts compared to primary CRC SW480 cells (both cell lines originated from the same patient at different stages of cancer progression) and normal colon epithelia (CCD841). (B) PMIS‐miR‐210 inhibits miR‐210 expression in SW620 cells, but does not inhibit other miRs. (C and D) RNA sequencing from PMIS‐miR‐210 SW620 cells revealed different sets of genes regulated by miR‐210 compared to PMIS‐miR‐210 ‐excised murine xenograft tumours. Only 120 regulated genes were similar in the two sets of RNA‐seq data. (E) These genes were further analyzed for their gene ontology pathways and histone modifying genes including H3K27ac and H3K4me3, which were upregulated. * p < .05; ** p < .01

Article Snippet: RNA sequencing was performed using SW620 PMIS‐EV and SW620 PMIS‐miR‐210 cell lines, and tumours generated from those cell lines by LC sciences (Houston, TX, USA) and analyzed using TruSeq Stranded RNA‐seq software.

Techniques: Expressing, Inhibition, RNA Sequencing

Journal: Clinical and Translational Medicine

Article Title: Extracellular vesicle expansion of PMIS‐miR‐210 expression inhibits colorectal tumour growth via apoptosis and an XIST/NME1 regulatory mechanism

doi: 10.1002/ctm2.1037

Figure Lengend Snippet: Small colorectal tumours are inhibited or ablated by injection of PMIS‐miR‐210 . (A) Transduced PMIS‐miR‐210 or PMIS‐EV SW620 cells (5 × 10 5 ) were transplanted into the flanks of Foxn1 nu/j nude mice and allowed to grow for 4 weeks, excised and analyzed. Five of the eight PMIS‐miR‐210 SW620 implanted cells failed to grow tumours compared to controls, which all grew large tumours. (B and C) SW620 cells (5 × 10 5 ) were transplanted into nude mice and allowed to grow for 7–10 days and form solid tumours. After 7–10 days, different doses (2.5, 5 or 10 μg) of PMIS‐miR‐210 or PMIS‐EV plasmid DNA were directly injected into the tumour. After 35–40 days, the tumours were removed and analyzed; interestingly, in three of the five mice (at different DNA concentrations) no tumours were found in the PMIS‐miR‐210 ‐injected tumours. (D and E) SW620 cells were transduced with a luciferase construct (5 × 10 5 ) to allow for live imaging of tumour formation and after 7–10 days of growth, tumours were injected with 10 μg of PMIS‐miR‐210 or PMIS‐EV plasmid DNA. After 35–40 days, the tumours were imaged, removed and quantitated. In two PMIS‐miR‐210 ‐injected mice, the tumours were not detectable by BLI or visible by dissection.

Article Snippet: RNA sequencing was performed using SW620 PMIS‐EV and SW620 PMIS‐miR‐210 cell lines, and tumours generated from those cell lines by LC sciences (Houston, TX, USA) and analyzed using TruSeq Stranded RNA‐seq software.

Techniques: Injection, Plasmid Preparation, Transduction, Luciferase, Construct, Imaging, Dissection

Journal: Clinical and Translational Medicine

Article Title: Extracellular vesicle expansion of PMIS‐miR‐210 expression inhibits colorectal tumour growth via apoptosis and an XIST/NME1 regulatory mechanism

doi: 10.1002/ctm2.1037

Figure Lengend Snippet: Large colorectal tumours are significantly reduced in NSG xenograft mice with PMIS‐miR‐210 injections. (A) SW620 cells were transduced with a luciferase construct and (1 × 10 6 ) cells (2× more than Figure ) were transplanted into each flank of NSG mice and allowed to grow and imaged after 7–10 days. (B) After 7–10 days, SW620 tumours were injected with 5 μg of PMIS‐miR‐210 or PMIS‐EV plasmid DNA and 5 days later another dose of 5 μg plasmid DNA was administered to the tumours. Capecitabine was given to the mice as a control treatment to compare the efficacy of PMIS‐miR‐210 treatments. After 35–40 days, the mice were imaged for tumour growth. (C and D) Tumours 35–40 days after treatments were removed and analyzed and tumour weights were recorded, each mouse had two tumours, one on each flank. N = 8 per group, except for capecitabine treatment ( N = 4); Student's t ‐tests conducted between groups

Article Snippet: RNA sequencing was performed using SW620 PMIS‐EV and SW620 PMIS‐miR‐210 cell lines, and tumours generated from those cell lines by LC sciences (Houston, TX, USA) and analyzed using TruSeq Stranded RNA‐seq software.

Techniques: Transduction, Luciferase, Construct, Injection, Plasmid Preparation, Control

Journal: Clinical and Translational Medicine

Article Title: Extracellular vesicle expansion of PMIS‐miR‐210 expression inhibits colorectal tumour growth via apoptosis and an XIST/NME1 regulatory mechanism

doi: 10.1002/ctm2.1037

Figure Lengend Snippet: Large colorectal tumours are significantly reduced in Foxn1 nu/j xenograft mice with PMIS‐miR‐210 injections. (A) SW620 cells were transduced with a luciferase construct and (1 × 10 6 ) cells were transplanted into each flank of Foxn1 nu/j mice and allowed to grow and imaged after 7–10 days. (B) After 7–10 days, SW620 tumours were injected with 5 μg of PMIS‐miR‐210 or PMIS‐EV plasmid DNA and 5 days later another dose of 5 μg plasmid DNA was administered to the tumours. Capecitabine was given to the mice as a control treatment to compare the efficacy of PMIS‐miR‐210 treatments. After 35–40 days, the mice were imaged for tumour growth. (C and D) Tumours 35–40 days after treatments were removed and analyzed and tumour weights were recorded, each mouse had two tumours, one on each flank. N = 8 per group; N = 4 for capecitabine treatment; Student's t ‐tests conducted between groups

Article Snippet: RNA sequencing was performed using SW620 PMIS‐EV and SW620 PMIS‐miR‐210 cell lines, and tumours generated from those cell lines by LC sciences (Houston, TX, USA) and analyzed using TruSeq Stranded RNA‐seq software.

Techniques: Transduction, Luciferase, Construct, Injection, Plasmid Preparation, Control

Journal: Clinical and Translational Medicine

Article Title: Extracellular vesicle expansion of PMIS‐miR‐210 expression inhibits colorectal tumour growth via apoptosis and an XIST/NME1 regulatory mechanism

doi: 10.1002/ctm2.1037

Figure Lengend Snippet: PMIS‐miR‐210 is widely expressed in the injected tumours. (A) PMIS‐EV and PMIS‐miR‐210 injected SW620 tumours from Foxn1 nu/j (from Figure ) mice were sectioned and stained for GFP, and Ki67 expression. GFP is expressed from both PMIS‐EV and PMIS‐miR‐210 expression constructs. (B and C) The relative intensities of GFP and Ki67 immunofluorescence were calculated using ImageJ and values shown in the graphs. Four independent sections for each of the two tumours per group were used, N = 4 each. (D) Serial sections from the tumours were probed for cleaved caspase 3 (CC3) expression to demonstrate active apoptosis. (E) CC3 expression measured by immunofluorescence was significantly increased in SW620 tumours expressing PMIS‐miR‐210 . N = 3; * p < .05

Article Snippet: RNA sequencing was performed using SW620 PMIS‐EV and SW620 PMIS‐miR‐210 cell lines, and tumours generated from those cell lines by LC sciences (Houston, TX, USA) and analyzed using TruSeq Stranded RNA‐seq software.

Techniques: Injection, Staining, Expressing, Construct, Immunofluorescence

Journal: Clinical and Translational Medicine

Article Title: Extracellular vesicle expansion of PMIS‐miR‐210 expression inhibits colorectal tumour growth via apoptosis and an XIST/NME1 regulatory mechanism

doi: 10.1002/ctm2.1037

Figure Lengend Snippet: The tumour suppressor NME1 is regulated by miR‐210 . (A) RNA‐seq from SW620 cells and tumours were specifically analyzed for XIST , NME1 , BRCA1 , FGFRL1 , BRK1 and MYC expression with and without PMIS‐miR‐210 expression. (B) Transcript levels of the genes in panel A were validated by qPCR in SW620 cells with and without PMIS‐miR‐210 expression. N = 3. (C) NME1 protein levels were increased in PMIS‐miR‐210 ‐transduced SW620 cells compared to PMIS‐EV and untreated cell controls. (D and E) NME1 expression measured by immunofluorescence was significantly increased in SW620 tumours expressing PMIS‐miR‐210 . N = 6; * p < .05

Article Snippet: RNA sequencing was performed using SW620 PMIS‐EV and SW620 PMIS‐miR‐210 cell lines, and tumours generated from those cell lines by LC sciences (Houston, TX, USA) and analyzed using TruSeq Stranded RNA‐seq software.

Techniques: RNA Sequencing, Expressing, Immunofluorescence

Journal: Clinical and Translational Medicine

Article Title: Extracellular vesicle expansion of PMIS‐miR‐210 expression inhibits colorectal tumour growth via apoptosis and an XIST/NME1 regulatory mechanism

doi: 10.1002/ctm2.1037

Figure Lengend Snippet: miR‐210 regulates XIST transcript levels. (A) RNA‐seq and qPCR showing that miR‐210 levels were inhibited over 80% in SW620 cells and XIST transcript were increased 143‐fold in PMIS‐miR‐210 ‐transduced SW620 cells. (B) miR‐210 expression increases in metastatic colorectal SW620 cells. (C) Demonstration of an inverse relationship between miR‐210 and XIST expression. Increasing amounts of transfected miR‐210 expression vector resulted in decreasing amounts of endogenous XIST transcripts. Increasing amounts of transfected PMIS‐miR‐210 correlate with decreased miR‐210 and increased XIST transcripts. The mouse (Mu) and Human (Hu) XIST transcripts contain miR‐210 binding sites. (D) Dual luciferase reporter assays in 293 cells with a Luc‐XIST reporter, 2.5 μg (contains miR‐210 binding sequence) and Luc‐XIST mut reporter 2.5 μg ( miR‐210 binding sequences mutated). miR‐210 , miR‐200c , scrambled and empty vector (EV) expression constructs (5 μg) were transfected in 293 cells. Luciferase was measured after 48 h post transfection. N = 3, * p < .05; ** p < .01

Article Snippet: RNA sequencing was performed using SW620 PMIS‐EV and SW620 PMIS‐miR‐210 cell lines, and tumours generated from those cell lines by LC sciences (Houston, TX, USA) and analyzed using TruSeq Stranded RNA‐seq software.

Techniques: RNA Sequencing, Expressing, Transfection, Plasmid Preparation, Binding Assay, Luciferase, Sequencing, Construct

Journal: Clinical and Translational Medicine

Article Title: Extracellular vesicle expansion of PMIS‐miR‐210 expression inhibits colorectal tumour growth via apoptosis and an XIST/NME1 regulatory mechanism

doi: 10.1002/ctm2.1037

Figure Lengend Snippet: Epigenetic regulation of NME1 by miR‐210 and XIST . (A) Analyses of epigenetic regulators activating NME1 expression revealed that H3K27Ac and H3K4me3 both marked the NME1 transcription start site (TSS) and identified NME1 as an actively transcribed gene. (B) ChIP‐qPCR experiments demonstrate in SW620 cells transduced with PMIS‐miR‐210 that both H3K27Ac and H3K4me3 marks were increased at the proximal promoter. As a control, Brk1 was not affected, N = 4. (C and D) ChIP‐qPCR experiments in SW620 cells demonstrate that XIST overexpression increased H3K27ac and H3K4me3 chromatin marks at the NME1 TSS but not at the NME1 3′UTR, used as a control, N = 5 (D). SW620 cells transfected with PMIS‐miR‐210 increased both epigenetic marks at the NME1 TSS (C) but not at the NME1 3′UTR (D). SW620 cells co‐transfected with PMIS‐miR‐210 and shXIST , decreased the epigenetic marks compared to PMIS‐miR‐210 alone, indicating that miR‐210 inhibition of XIST plays a role in NME1 epigenetic regulation. N = 5, * p < .05; ** p < .01

Article Snippet: RNA sequencing was performed using SW620 PMIS‐EV and SW620 PMIS‐miR‐210 cell lines, and tumours generated from those cell lines by LC sciences (Houston, TX, USA) and analyzed using TruSeq Stranded RNA‐seq software.

Techniques: Expressing, ChIP-qPCR, Transduction, Control, Over Expression, Transfection, Inhibition

Journal: Clinical and Translational Medicine

Article Title: Extracellular vesicle expansion of PMIS‐miR‐210 expression inhibits colorectal tumour growth via apoptosis and an XIST/NME1 regulatory mechanism

doi: 10.1002/ctm2.1037

Figure Lengend Snippet: Cell‐to‐cell transfer of PMIS‐miR‐210 by extracellular vesicles (ECVs). (A) Transmission electron microscopy (TEM) photo of ECVs secreted from SW620 and SW620 PMIS‐miR‐210 cells. (B) Isolated ECVs were probed for CD63 and CD9 to prove their identity. (C) ECVs isolated from PMIS‐miR‐210 ‐transduced SW620 and 293 cells were probed by qPCR for PMIS‐miR‐210 transcripts. SW620 PMIS‐miR‐210 cells secreted high levels of PMIS‐miR‐210 in ECVs compared to controls and higher levels than 293 PMIS‐miR‐210 cells. (D) Transwell experiments demonstrate that SW620 cells transduced with PMIS‐miR‐210 (donor cells) could transfect and express PMIS‐miR‐210 in SW620 recipient cells. A ratio of 1/1 (2 × 10 5 donor cells to 2 × 10 5 recipient cells) showed that the recipient cells expressed the PMIS‐miR‐210 transcript after 72 h. Furthermore, miR‐210 expression was decreased in the recipient cells at a 1/1 ratio. The concentration of donor cells was decreased by half at 1 × 10 5 (0.5/1), while the recipient cell concentration was maintained at 2 × 10 5 and the recipient cells showed less expression of PMIS‐miR‐210 compared to the 1/1 ratio. miR‐210 expression was decreased but less than the 1/1 ratio. A ratio of 0.25 donor/1 recipient cell did not show a significant effect. As controls, the donor cells express high levels of PMIS‐miR‐210 and decreased levels of miR‐210 , while the recipient cells alone did not express PMIS‐miR‐210 . (E) Identical experiments show that the long non‐coding RNA (LncRNA X‐inactive specific transcript) XIST levels were increased in the recipient cells at 1/1 and 0.5/1 ratios. XIST is highly expressed in the SW620 PMIS‐miR‐210 ‐transduced cells but not in the SW620 cells. (F) The tumour suppressor NME1 is also increased in the recipient cells at 1/1 and 0.5/1 ratios. As a control, we show that P21 was not affected as it is not a target of miR‐210 . NME1 transcripts are increased in the SW620 PMIS‐miR‐210 ‐transduced donor cells. N = 3 for transwell experiments; * p < .05; ** p < .01

Article Snippet: RNA sequencing was performed using SW620 PMIS‐EV and SW620 PMIS‐miR‐210 cell lines, and tumours generated from those cell lines by LC sciences (Houston, TX, USA) and analyzed using TruSeq Stranded RNA‐seq software.

Techniques: Transmission Assay, Electron Microscopy, Isolation, Transduction, Expressing, Concentration Assay, Control

Journal: Clinical and Translational Medicine

Article Title: Extracellular vesicle expansion of PMIS‐miR‐210 expression inhibits colorectal tumour growth via apoptosis and an XIST/NME1 regulatory mechanism

doi: 10.1002/ctm2.1037

Figure Lengend Snippet: miR‐210 controls cytoplasmic and nuclear accumulation of XIST . (A) Immunofluorescence of XIST transcripts in SW620 cells and PMIS‐miR‐210 ‐transduced SW620 cells by in situ hybridization. The nuclei are stained with DAPI (blue). (B) miR‐210 and XIST transcripts are decreased in SW620 cells after treatment with actinomycin D, measured by qPCR and C t values plotted. However, XIST transcripts are relatively stable in PMIS‐miR‐210 ‐transduced SW620 cells, while miR‐210 transcripts are rapidly degraded in the presence of actinomycin D. β‐Tubulin was used as a control and rapidly degraded after 3 days of actinomycin treatment. N = 3; representative experiment shown

Article Snippet: RNA sequencing was performed using SW620 PMIS‐EV and SW620 PMIS‐miR‐210 cell lines, and tumours generated from those cell lines by LC sciences (Houston, TX, USA) and analyzed using TruSeq Stranded RNA‐seq software.

Techniques: Immunofluorescence, In Situ Hybridization, Staining, Control

Journal: Nature Metabolism

Article Title: Formate overflow drives toxic folate trapping in MTHFD1 inhibited cancer cells

doi: 10.1038/s42255-023-00771-5

Figure Lengend Snippet: a , 1C metabolism flux between mitochondria and cytosol/nucleus. b – e , Dose–response curves of SW620 cells treated for 96 h with TH9619 ( b ), TH9975 ( c ), DS18561882 ( d ) or SHIN1 ( e ) in the presence of 50 μM thymidine, 1 mM sodium formate or vehicle (cultured in RPMI-FBS), means ± s.d. ( n = 3). f , [U- 13 C]serine-derived formate release rate of SW620 WT, MTHFD2 −/− and SHMT1 −/− cells treated for 24 h with the indicated concentrations of TH9619 and TH9975 or 50 nM MTX (cultured in RPMI-FBS), means ± s.d. ( n = 3 for control and TH9619, n = 2 for TH9975, n = 1 for MTX); one-way ANOVA (analysis of variance) with Tukey’s multiple comparisons test. g , Quantification of mitochondrial membrane potential in SW620 WT cells treated with 1 µM of TH9619 or TH9975 or 0.5 µM MTX for 48 h. FCCP was used as positive control. Data are displayed as means ± s.d. ( n = 4, n = 5 for FCCP); one-way ANOVA with Dunnett’s test for multiple comparisons. h , i , CETSA analysis of MTHFD2 stabilization in SW620 WT cells treated for 3 h with 10 µM TH9619 or vehicle. MTHFD2 stabilization was assessed by western blot by normalizing remaining MTHFD2 signal to HSP60 in the mitochondrial fractions ( h ) or lamin A/C in the nuclear fractions ( i ). j , Representative immunoblots of one out of two independent experiments. k , DARTS analysis of MTHFD1 stabilization in SW620 lysates that were incubated with 10 µM TH9619 or vehicle for 30 min, followed by protein digestion at increasing pronase concentration and assessment of MTHFD1 degradation by western blot. MTHFD1 signal was normalized to SOD1. Shown is a representative out of three independent experiments. l , Mean pIC 50 (−log of half-maximum inhibitory concentration) values for indicated MTHFD1/2 inhibitors assessed for their inhibitory activity against MTHFD1(DC) WT or MTHFD1(DC) mutant (Q100A), mean (from left to right n = 4, 9, 2, 19, 5, 15, 5, 6, 4, 8); unpaired, two-tailed t -test. m , CRISPR-Select cassette for MTHFD1-Q100A was delivered to SW620 cells. The graph shows the ratio of MTHFD1-Q100A versus WT on day 2 and day 25 of 10 μM TH9619 treatment normalized to day 2 value, means ± s.d. ( n = 4); paired, two-tailed t -test.

Article Snippet: SW620 MTHFD2 −/− and SW620 SHTM1 −/− cells were purchased from Synthego using single-guide RNA sequences targeting exon 2 (5′-CGCCAACCAGGAUCACACUC-3′ for MTHFD2 −/− ) or exon 4 (5′-UCAGGUGGCCCCCAUCCGGA-3′ for SHMT1 −/− ).

Techniques: Cell Culture, Derivative Assay, Control, Membrane, Positive Control, Western Blot, Incubation, Concentration Assay, Activity Assay, Mutagenesis, Two Tailed Test, CRISPR

Journal: Nature Metabolism

Article Title: Formate overflow drives toxic folate trapping in MTHFD1 inhibited cancer cells

doi: 10.1038/s42255-023-00771-5

Figure Lengend Snippet: ( a ) Chemical structures of MTHFD1/2 inhibitors TH9619 and TH9975 and IC 50 values for inhibition of MTHFD1(DC) and MTHFD2 enzymatic activities for each inhibitor. ( b ) Enzymatic activity of TYMS, SHMT1 and SHMT2 upon incubation with 100 µM TH9975 compared to their known inhibitors lometrexol (LMTX) and raltitrexed (RLTX), means ± SD (n = 3). ( c , d ) Expression of the indicated proteins was analyzed by Western Blot in SW620 WT, MTHFD2 -/- and SHMT1 -/- cells. β-actin served as loading control. Immunoblots are representative of 3 independent repeats. Signal intensity was quantified and normalized to loading control and global mean, means ± SD (n = 3). ( e ) Verification of the purity of mitochondrial and nuclear fractions for CETSA analysis. Shown are representative immunoblots of one out of two independent experiments. ( f , g ) Serine and ATP levels normalized to packed cell volume (PCV) in SW620 WT cells treated for 24 h with 10 µM DS18561882, mean ± SD (n = 3); paired, two-tailed t-test. ( h ) [U- 13 C]serine-derived MID of ATP in SW620 WT cells treated for 24 h with 10 µM DS18561882, mean ± SD (n = 3); one-way ANOVA with Šídák’s multiple comparison test for M + 0. ( i ) [U- 13 C]serine-derived release rate of formate M + 1 isotopologue from SW620 WT cells treated for 24 h with 10 µM DS18561882, mean ± SD (n = 3); paired, two-tailed t-test. ( j , k ) DARTS analysis of MTHFD1 stabilization in SW620 cells upon TH9619 ( j ) or DS18561882 ( k ) treatment. Intact cells ( j ) or cell lysates ( k ) were incubated with 10 µM TH9619 or vehicle for 3 h ( j ) or 30 min ( k ), followed by protein digestion at increasing pronase concentration and assessment of MTHFD1 degradation by Western blot. MTHFD1 intensities were normalized to SOD1; one representative immunoblot of two independent experiments. ( l ) Design of MTHFD1/2 inhibitor resistant MTHFD1 protein based on the crystal structure of structurally similar MTHFD2 bound to LY345899 (PDB ID: 5TC4). Highlighted is the residue Q100 that was mutated to alanine (Q100A) and tested in downstream biochemical assays. ( m ) Enzymatic activity of purified MTHFD1(DC) WT and MTHFD1(DC) mutant (Q100A), means ± SD (n = 3).

Article Snippet: SW620 MTHFD2 −/− and SW620 SHTM1 −/− cells were purchased from Synthego using single-guide RNA sequences targeting exon 2 (5′-CGCCAACCAGGAUCACACUC-3′ for MTHFD2 −/− ) or exon 4 (5′-UCAGGUGGCCCCCAUCCGGA-3′ for SHMT1 −/− ).

Techniques: Inhibition, Activity Assay, Incubation, Expressing, Western Blot, Control, Two Tailed Test, Derivative Assay, Comparison, Concentration Assay, Residue, Purification, Mutagenesis

Journal: Nature Metabolism

Article Title: Formate overflow drives toxic folate trapping in MTHFD1 inhibited cancer cells

doi: 10.1038/s42255-023-00771-5

Figure Lengend Snippet: ( a ) Dose-response curves of HCT116 cells treated for 96 h with TH9619, TH9975, methotrexate (MTX) or 5-fluorouracil (5-FU) and 50 μM thymidine, 250 μM hypoxanthine or vehicle (cultured in RPMI-FBS), means ± SD (n = 3). ( b ) Dose-response curves of SW620 cells treated for 96 h with TH9619, TH9975, MTX or 5-FU and 50 μM thymidine, 1 μM folinic acid or vehicle (cultured in RPMI-FBS), means ± SD (n = 3). ( c ) Dose-response curves of SW620 cells treated for 96 h with TH9619, TH9975, MTX or 5-FU and 50 μM thymidine, 250 μM folic acid or vehicle (cultured in RPMI-FBS), means ± SD (n = 3). ( d ) [U- 13 C]serine- derived MID of ADP in SW620 WT, MTHFD2 -/- and SHMT1 -/- cells treated for 24 h with the indicated concentrations of TH9619 and TH9975 in μM or 50 nM MTX (cultured in RPMI-FBS), means ± SD (n = 3 for untreated and TH9619, n = 2 for TH9975, n = 1 for MTX); one-way ANOVA with Tukey’s multiple comparisons test for M + 0. P -values indicate comparisons to the untreated control of each cell line. ( e ) ADP levels normalized to packed cell volume (PCV) in SW620 WT, MTHFD2 -/- and SHMT1 -/- cells treated for 24 h with the indicated concentrations of TH9619 and TH9975 in μM (cultured in RPMI-FBS), mean ± SD (n = 3 for untreated and TH9619, n = 2 for TH9975); one-way ANOVA with Dunnett’s test for multiple comparisons. P -values indicate comparisons to the untreated control.

Article Snippet: SW620 MTHFD2 −/− and SW620 SHTM1 −/− cells were purchased from Synthego using single-guide RNA sequences targeting exon 2 (5′-CGCCAACCAGGAUCACACUC-3′ for MTHFD2 −/− ) or exon 4 (5′-UCAGGUGGCCCCCAUCCGGA-3′ for SHMT1 −/− ).

Techniques: Cell Culture, Derivative Assay, Control

Journal: Nature Metabolism

Article Title: Formate overflow drives toxic folate trapping in MTHFD1 inhibited cancer cells

doi: 10.1038/s42255-023-00771-5

Figure Lengend Snippet: a – f , Dose–response curves of SW620 WT cells treated for 96 h with TH9619 ( a ), TH9975 ( b ), DS18561882 ( c ), SHIN1 ( d ), MTX ( e ) or 5-FU ( f ) in the presence of 50 μM thymidine, 250 μM hypoxanthine or vehicle (cultured in RPMI-FBS), means ± s.d. ( n = 3). g , 1C metabolism labelling derived from [U- 13 C] serine tracer with red dots indicating 13 C atoms in individual metabolites. 5,10-CH 2 -THF, 5,10-methylenetetrahydrofolate; 10-CHO-THF, 10-formyl tetrahydrofolate; MTHFD1/1L/2/2L, methylenetetrahydrofolate dehydrogenase 1/1L/2/2L. h , 1C metabolism labelling derived from [2,3,3- 2 H]serine tracer with coloured dots indicating cytosol and mitochondrial-derived 2 H atoms in individual metabolites. i , [U- 13 C]serine-derived MID of ATP in SW620 WT, MTHFD2 −/− and SHMT1 −/− cells treated for 24 h with the indicated concentrations of TH9619 and TH9975 in μM or 50 nM MTX (cultured in RPMI-FBS), mean ± s.d. ( n = 3 for untreated and TH9619, n = 2 for TH9975, n = 1 for MTX); one-way ANOVA with Tukey’s multiple comparisons test for M + 0. P values indicate comparisons to untreated control of each cell line. j , ATP level normalized to PCV in SW620 WT, MTHFD2 −/− and SHMT1 −/− cells treated for 24 h with the indicated concentrations of TH9619 and TH9975 in μM (cultured in RPMI-FBS), means ± s.d. ( n = 3 for untreated and TH9619, n = 2 for TH9975); one-way ANOVA with Dunnett’s test for multiple comparisons. P values indicate comparisons to the untreated control. k , Ratio of the [2,3,3- 2 H]serine-derived M + 1 to M + 2 isotopologues of dTMP in SW620 WT and MTHFD2 −/− cells treated for 24 h with 1 μM TH9619 and TH9975 (cultured in RPMI-FBS), means ± s.d. ( n = 3 for untreated and TH9619, n = 2 for TH9975); one-way ANOVA with Tukey’s multiple comparisons test. l , Relative enrichment of [2,3,3- 2 H]serine-derived M + 1 to M + 2 isotopologues of dTMP in SW620 WT and MTHFD2 −/− cells treated for 24 h with 1 μM TH9619 and TH9975 (cultured in RPMI-FBS), means ± s.d. ( n = 3 for untreated and TH9619, n = 2 for TH9975); one-way ANOVA with Tukey’s multiple comparisons test for D + 1 isotopologue of dTMP.

Article Snippet: SW620 MTHFD2 −/− and SW620 SHTM1 −/− cells were purchased from Synthego using single-guide RNA sequences targeting exon 2 (5′-CGCCAACCAGGAUCACACUC-3′ for MTHFD2 −/− ) or exon 4 (5′-UCAGGUGGCCCCCAUCCGGA-3′ for SHMT1 −/− ).

Techniques: Cell Culture, Derivative Assay, Control

Journal: Nature Metabolism

Article Title: Formate overflow drives toxic folate trapping in MTHFD1 inhibited cancer cells

doi: 10.1038/s42255-023-00771-5

Figure Lengend Snippet: a – d , Cell viability dose–response curves of SW620 WT cells treated for 96 h with TH9619 ( a ), TH9975 ( b ), MTX ( c ) or DS18561882 ( d ), and cultured in RPMI-FBS, RPMI-dFBS or RPMI-dFBS supplemented with 50 μM hypoxanthine, means ± s.d. ( n = 8 independent cell cultures for TH9619 and TH9975, n = 4 for MTX and DS18561882). e , Titration of hypoxanthine at indicated doses in SW620 WT cells treated for 96 h with TH9619 (cultured in RPMI-dFBS). Data are displayed as means ( n = 2 independent cell cultures). f , g , Dose–response curves of SW620 WT and MTHFD2 −/− cells treated for 96 h with TH9619 and cultured in RPMI-FBS ( f ) or RPMI-dFBS ( g ), means ± s.d. ( n = 3 independent cell cultures). h – k , Cell viability dose–response curves of SW620 WT cells treated for 96 h with TH9619 ( h ), TH9975 ( i ), MTX ( j ) or DS18561882 ( k ) and 50 μM thymidine, 50 μM hypoxanthine or vehicle (cultured in RPMI-dFBS), means ( n = 2 independent cell cultures). l – o , Cell viability dose–response curves of SW620 MTHFD2 −/− cells treated for 96 h with TH9619 ( l ), TH9975 ( m ), MTX ( n ) or DS18561882 ( o ) and 50 μM thymidine, 50 μM hypoxanthine or vehicle (cultured in RPMI-dFBS), means ± s.d. ( n = 4 independent cell cultures for TH9619 and TH9975, n = 2 for MTX and DS18561882).

Article Snippet: SW620 MTHFD2 −/− and SW620 SHTM1 −/− cells were purchased from Synthego using single-guide RNA sequences targeting exon 2 (5′-CGCCAACCAGGAUCACACUC-3′ for MTHFD2 −/− ) or exon 4 (5′-UCAGGUGGCCCCCAUCCGGA-3′ for SHMT1 −/− ).

Techniques: Cell Culture, Titration

Journal: Nature Metabolism

Article Title: Formate overflow drives toxic folate trapping in MTHFD1 inhibited cancer cells

doi: 10.1038/s42255-023-00771-5

Figure Lengend Snippet: ( a ) HX concentration in HI-FBS and dialyzed FBS was determined by titration with HX standard of known concentration. Linear regression of the standard curve was used to calculate y-intercept and corresponding HX concentration in nM in pure HI-FBS or dialyzed FBS. ( b ) Dose-response curves of SW620 WT cells treated for 96 h with TH9619, TH9975, methotrexate (MTX) or DS18561882 and 50 μM adenine, 50 μM adenosine, 50 μM guanosine or vehicle (cultured in RPMI-dFBS), means (n = 2 independent cell cultures). ( c ) Dose-response curves of SW620 WT cells treated for 96 h with TH9619, TH9975, MTX or DS18561882 and 50 μM thymidine, 1 mM sodium formate or vehicle (cultured in RPMI-dFBS), means (n = 2 independent cell cultures). ( d ) Dose-response curves of SW620 MTHFD2 -/- cells treated as in ( c ). Means ± SD (n = 4 independent cell cultures for TH9619 and TH9975, n = 2 for MTX and DS18561882). ( e ) Cell viability dose-response curves of PA-TU-8988T cells treated for 96 h with TH9619, TH9975 or MTX and 50 μM thymidine, 50 μM hypoxanthine, 1 mM sodium formate or vehicle (cultured in DMEM-dFBS), means (n = 2 independent cell cultures). ( f ) Dose-response curves of SW620 WT cells treated for 96 h with TH9619 or TH9975 and increasing doses of thymidine. Cells were cultured in media containing dFBS and 10 or 50 µM hypoxanthine, means ± SD (n = 4).

Article Snippet: SW620 MTHFD2 −/− and SW620 SHTM1 −/− cells were purchased from Synthego using single-guide RNA sequences targeting exon 2 (5′-CGCCAACCAGGAUCACACUC-3′ for MTHFD2 −/− ) or exon 4 (5′-UCAGGUGGCCCCCAUCCGGA-3′ for SHMT1 −/− ).

Techniques: Concentration Assay, Titration, Cell Culture

Journal: Nature Metabolism

Article Title: Formate overflow drives toxic folate trapping in MTHFD1 inhibited cancer cells

doi: 10.1038/s42255-023-00771-5

Figure Lengend Snippet: a , [U- 13 C]serine- derived MID of ATP in SW620 WT and MTHFD2 −/− cells treated for 24 h with 1 μM TH9619 or TH9975, 50 μM hypoxanthine and 50 μM thymidine (cultured in RPMI-dFBS), means ± s.d. ( n = 3); one-way ANOVA with Šídák’s multiple comparisons test for M + 0. P values indicate comparisons to DMSO controls of each cell line. b , ATP levels normalized to PCV in SW620 WT and MTHFD2 −/− cells treated for 24 h with 1 μM TH9619 or TH9975, 50 μM hypoxanthine and 50 μM thymidine (cultured in RPMI-dFBS), means ± s.d. ( n = 3); one-way ANOVA with Šídák’s multiple comparisons test. c , Relative enrichment of [2,3,3- 2 H]serine-derived M + 1 to M + 2 isotopologues of dTMP in SW620 WT and MTHFD2 −/− cells treated for 24 h with 1 μM TH9619 and TH9975 (cultured in RPMI-dFBS), means ± s.d. ( n = 3); one-way ANOVA with Tukey’s multiple comparisons test. d , dTMP levels normalized to PCV in SW620 WT and MTHFD2 −/− cells treated for 24 h with 1 μM TH9619 or TH9975, 50 μM hypoxanthine and 50 μM thymidine (cultured in RPMI-dFBS), means ± s.d. ( n = 3); one-way ANOVA with Šídák’s multiple comparisons test. e , [U- 13 C]serine-derived MID of dTMP in SW620 WT and MTHFD2 −/− cells treated for 24 h with 1 μM TH9619 or TH9975, 50 μM hypoxanthine and 50 μM thymidine (cultured in RPMI-dFBS), means ± s.d. ( n = 3); one-way ANOVA with Šídák’s multiple comparisons test for M + 0. P values indicate comparisons to respective DMSO controls of each cell line. f , dUMP levels normalized to PCV in SW620 WT and MTHFD2 −/− cells treated for 24 h with 1 μM TH9619 or TH9975, 50 μM hypoxanthine and 50 μM thymidine (cultured in RPMI-dFBS), means ± s.d. ( n = 3); one-way ANOVA with Šídák’s multiple comparisons test. P values indicate comparisons to respective DMSO controls of each cell line.

Article Snippet: SW620 MTHFD2 −/− and SW620 SHTM1 −/− cells were purchased from Synthego using single-guide RNA sequences targeting exon 2 (5′-CGCCAACCAGGAUCACACUC-3′ for MTHFD2 −/− ) or exon 4 (5′-UCAGGUGGCCCCCAUCCGGA-3′ for SHMT1 −/− ).

Techniques: Derivative Assay, Cell Culture

Journal: Nature Metabolism

Article Title: Formate overflow drives toxic folate trapping in MTHFD1 inhibited cancer cells

doi: 10.1038/s42255-023-00771-5

Figure Lengend Snippet: ( a – d ) [U- 13 C]serine- derived MID of GMP ( a ) and IMP ( c ) and levels of GMP ( b ) and IMP ( d ) normalized to packed cell volume (PCV) in SW620 WT and MTHFD2 -/- cells treated for 24 h with 1 μM TH9619 or TH9975, 50 μM hypoxanthine and 50 μM thymidine (cultured in RPMI-dFBS), means ± SD (n = 3); one-way ANOVA with Šídák’s multiple comparisons test. For panels a and c, statistical analysis was performed for M + 0. P -values indicate comparisons to respective DMSO controls.

Article Snippet: SW620 MTHFD2 −/− and SW620 SHTM1 −/− cells were purchased from Synthego using single-guide RNA sequences targeting exon 2 (5′-CGCCAACCAGGAUCACACUC-3′ for MTHFD2 −/− ) or exon 4 (5′-UCAGGUGGCCCCCAUCCGGA-3′ for SHMT1 −/− ).

Techniques: Derivative Assay, Cell Culture

Journal: Nature Metabolism

Article Title: Formate overflow drives toxic folate trapping in MTHFD1 inhibited cancer cells

doi: 10.1038/s42255-023-00771-5

Figure Lengend Snippet: a , b , HCT116 ( a ) and SW620 ( b ) cells were grown as spheroid cultures in three dimensions. Proliferation was measured as spheroid area from day 0 to 5 on 1 µM TH9619 or vehicle, means ± s.d. ( n = 3 for HCT116 and n = 2 for SW620). Representative pictures shown for both cell lines. Scale bars, 500 μm. c , In the folate trapping model, hypoxanthine is converted to IMP, GMP or AMP by purine salvage, causing feedback inhibition on de novo purine synthesis. Simultaneously, TH9619 inhibits MTHFD1(DC), which together blocks consumption of 10-CHO-THF, creating a folate trap and preventing thymidylate synthesis due to exhaustion of THF. d , 10-CHO-THF levels normalized to PCV in SW620 WT and MTHFD2 −/− cells treated for 24 h with 1 μM TH9619 and 50 μM hypoxanthine (cultured in RPMI-dFBS), means ± s.d. ( n = 3); one-way ANOVA with Tukey’s multiple comparisons test. e , LC–MS chromatograms for one representative experiment of d . RT, retention time.

Article Snippet: SW620 MTHFD2 −/− and SW620 SHTM1 −/− cells were purchased from Synthego using single-guide RNA sequences targeting exon 2 (5′-CGCCAACCAGGAUCACACUC-3′ for MTHFD2 −/− ) or exon 4 (5′-UCAGGUGGCCCCCAUCCGGA-3′ for SHMT1 −/− ).

Techniques: Inhibition, Cell Culture, Liquid Chromatography with Mass Spectroscopy

Journal: Nature Metabolism

Article Title: Formate overflow drives toxic folate trapping in MTHFD1 inhibited cancer cells

doi: 10.1038/s42255-023-00771-5

Figure Lengend Snippet: ( a – d ) Dose-response curves of SW620 cells treated for 96 h with TH9619, TH9975, methotrexate (MTX) or DS18561882 (cultured in RPMI-dFBS, RPMI-dFBS with 50 μM hypoxanthine or HPLM-dFBs), one representative experiment displayed as means (n = 2 independent cell cultures). ( e ) Hypothesis that hypoxanthine converts to uric acid to inhibit UMPS. ( f ) Dose-response curves of SW620 cells treated for 96 h with TH9619, 50 μM hypoxanthine, 350 μM uric acid or vehicle (cultured in RPMI-dFBS), means ± SD (n = 3). ( g ) Dose-response curves of SW620 cells treated for 96 h with TH9619, pyrazofurin or 50 μM hypoxanthine (cultured in RPMI-dFBS), means ± SD (n = 3 independent cell cultures). ( h ) Dose-response curves of SW620 cells treated for 96 h with TH9619, 50 μM hypoxanthine, 1 mM orotic acid, 500 μM deoxyuridine (dU) or vehicle (cultured in RPMI-dFBS), means ± SD (n = 3). ( i ) Dose-response curves of SW620 cells treated for 96 h with TH9619, 50 μM hypoxanthine (Hx), 10 μM allopurinol, 10 μM febuxostat or vehicles (cultured in RPMI-dFBS), means ± SD (n = 3). ( j ) Dose-response curves of SW620 cells treated for 96 h with TH9619 and 50 μM hypoxanthine, 100 μM H 2 O 2 or vehicle (cultured in RPMI-dFBS), means (n = 2). ( k ) Uric acid levels normalized to PCV in SW620 WT cells treated for 24 h with 1 μM TH9619 or TH9975 and 50 μM hypoxanthine (cultured in RPMI-dFBS), means ± SD (n = 3).

Article Snippet: SW620 MTHFD2 −/− and SW620 SHTM1 −/− cells were purchased from Synthego using single-guide RNA sequences targeting exon 2 (5′-CGCCAACCAGGAUCACACUC-3′ for MTHFD2 −/− ) or exon 4 (5′-UCAGGUGGCCCCCAUCCGGA-3′ for SHMT1 −/− ).

Techniques: Cell Culture

Journal: Nature Metabolism

Article Title: Formate overflow drives toxic folate trapping in MTHFD1 inhibited cancer cells

doi: 10.1038/s42255-023-00771-5

Figure Lengend Snippet: ( a ) Top: Reductive methylation reaction described by Schittmayer et al . to stabilize the THF intermediates, followed by the cleavage of the glutamate tails by glutamate carboxypeptidase . Bottom: Suggested mechanism for the tandem mass spectrometry fragmentation of 10-CHO-THF. ( b , c ) Protein expression of MTHFD2 was analyzed by Western Blot in MDA-MB-468 WT, MDA-MB-468 MTHFD2 -/- , MDA-MB-231 and SW620 cells to validate MTHFD2 KO in MDA-MB-468 cells and different expression levels in WT cells. β-actin was used as loading control. Images are representative of 3 independent repeats. Signal intensity was quantified and normalized to loading control and global mean and is shown in Fig. . ( d ) Absolute cell count was measured as cell number/uL by flow cytometry in MDA-MB-468 WT and MDA-MB-468 MTHFD2 -/- in response to 96 h treatment with 1 μM TH9619 and 50 μM hypoxanthine and 1 mM formate (cultured in RPMI-dFBS), means ± SD (n = 2 for WT and n = 4 for MTHFD2 -/- ); one-way ANOVA with Tukey’s multiple comparisons test was performed for MTHFD2 -/- cells. Representative images for Fig. of MDA-MB-468 MTHFD2 -/- cell density and morphology are shown at 0 h and 72 h after treatment with 1 μM TH9619, 50 μM hypoxanthine and 1 mM formate (cultured in RPMI-dFBS). ( e , f ) Cell death analysis measured by AnnexinV/PI staining of MDA-MB-231, MDA-MB-468 WT and MTHFD2 -/- cells in response to 96 h treatment with 1 μM TH9619, 50 μM hypoxanthine (H), 50 μM thymidine (T) and 1 mM formate (F, cultured in RPMI-dFBS), means ± SD (n = 2-6).

Article Snippet: SW620 MTHFD2 −/− and SW620 SHTM1 −/− cells were purchased from Synthego using single-guide RNA sequences targeting exon 2 (5′-CGCCAACCAGGAUCACACUC-3′ for MTHFD2 −/− ) or exon 4 (5′-UCAGGUGGCCCCCAUCCGGA-3′ for SHMT1 −/− ).

Techniques: Methylation, Mass Spectrometry, Expressing, Western Blot, Control, Cell Counting, Flow Cytometry, Cell Culture, Staining

Journal: Nature Metabolism

Article Title: Formate overflow drives toxic folate trapping in MTHFD1 inhibited cancer cells

doi: 10.1038/s42255-023-00771-5

Figure Lengend Snippet: a , TH9619 blocks purine synthesis in MTHFD2 −/− cells. b , Exogenous supply of sodium formate and hypoxanthine can induce the TH9619 folate trapping mechanism and block thymidylate synthesis in MTHFD2 −/− cells. c – g , Cell viability dose–response curves of SW620 MTHFD2 −/− cells treated for 96 h with TH9619 ( c ), TH9975 ( d ), MTX ( e ), DS18561882 ( f ) or SHIN1 ( g ) and 50 μM hypoxanthine, 1 mM sodium formate, 50 μM thymidine or vehicle (cultured in RPMI-dFBS), two representative experiments displayed as means ± s.d. ( n = 4 independent cell cultures). h , Hypoxanthine and TH9619 cause folate trapping in WT cells. Treatment with AICAr bypasses the block in de novo purine synthesis and releases the folate trap by AICAR Tfase consumption of 10-CHO-THF. i – k , Cell viability dose–response curves of SW620 WT cells treated for 96 h with TH9619 ( i ), TH9975 ( j ) or MTX ( k ) and vehicle or increasing AICAr concentrations (cultured in RPMI-dFBS), two representative experiments are shown as means ± s.d. ( n = 4 independent cell cultures for TH9619 and TH9965, n = 2 for MTX). l , [U- 13 C]serine-derived formate release rate of SW620 WT cells treated for 24 h with 1 μM TH9619 or TH9975, 50 μM hypoxanthine and 50 μM thymidine (cultured in RPMI-dFBS), means ± s.d. ( n = 3); one-way ANOVA with Dunnett’s multiple comparisons test. P values indicate comparisons to the DMSO control.

Article Snippet: SW620 MTHFD2 −/− and SW620 SHTM1 −/− cells were purchased from Synthego using single-guide RNA sequences targeting exon 2 (5′-CGCCAACCAGGAUCACACUC-3′ for MTHFD2 −/− ) or exon 4 (5′-UCAGGUGGCCCCCAUCCGGA-3′ for SHMT1 −/− ).

Techniques: Blocking Assay, Cell Culture, Derivative Assay, Control

Journal: Nature Metabolism

Article Title: Formate overflow drives toxic folate trapping in MTHFD1 inhibited cancer cells

doi: 10.1038/s42255-023-00771-5

Figure Lengend Snippet: a – c , Proliferation of ( a , b ) SW620 WT and ( c ) MTHFD2 −/− cells measured as fold change of cell confluence after ( a ) 1 μM TH9619 or TH9975 ( b , c ) with or without 50 μM hypoxanthine and 50 μM thymidine (cultured in RPMI-FBS or RPMI-dFBS). AUC of proliferation curves is shown as means ± standard error (( a ) n = 3 for WT, n = 4 for MTHFD2 −/− ( b , c ) n = 4); one-way ANOVA with ( a ) Šídák’s multiple comparisons test or ( b , c ) Tukey’s multiple comparisons test. d , e , Cell cycle analysis of SW620 WT and MTHFD2 −/− cells treated with TH9619, 50 μM thymidine (T), 250 μM hypoxanthine (H) or vehicle (cultured in ( d ) RPMI-FBS or ( e ) RPMI-dFBS), means ± s.d. ( n = 3); one-way ANOVA with Tukey’s multiple comparisons test for S-phase. f – h , Cell death analysis by annexinV/PI staining of SW620 WT and MTHFD2 −/− cells after 96 h treatment with 1 μM TH9619 or TH9975 and ( f , g ) 50 μM hypoxanthine (H) and 50 μM thymidine (T) cultured in ( f ) RPMI-FBS or ( g ) RPMI-dFBS, or ( h ) treated with T and 1 mM formate (F) cultured in RPMI-dFBS, means ± s.d. ( n = 3); one-way ANOVA analysis with Tukey’s multiple comparisons test for pooled early and late apoptotic populations. i , MTHFD2 protein levels in SW620, MDA-MB468 and MDA-MB-231 cells normalized to β-actin, means ± s.d. ( n = 3); one-way ANOVA analysis with Tukey’s multiple comparisons test. j , Formate release rates of SW620, MDA-MB-468 and MDA-MB-231 cells, means ± s.d. ( n = 7 for SW620, n = 12 for MDA-MB-468 and n = 3 for MDA-MB-231). k , Proliferation of MDA-MB-468 WT and MTHFD2 −/− cells and MDA-MB-231 cells measured as fold change of cell confluence in response to 1 μM TH9619 and 50 μM hypoxanthine and 1 mM sodium formate (cultured in RPMI-dFBS). AUC of curves is shown as means ± s.e. ( n = 3 for MDA-MB-468 and n = 4 for MDA-MB-231); one-way ANOVA with Tukey’s multiple comparisons test. l , Cell death analysis of MDA-MB-468 WT and MTHFD2 −/− cells after 96 h treatment with 1 μM TH9619 and 50 μM hypoxanthine (H) and 1 mM formate (F) (cultured in RPMI-dFBS), means ± s.d. ( n = 4); one-way ANOVA analysis with Tukey’s multiple comparisons test for pooled early and late apoptotic populations.

Article Snippet: SW620 MTHFD2 −/− and SW620 SHTM1 −/− cells were purchased from Synthego using single-guide RNA sequences targeting exon 2 (5′-CGCCAACCAGGAUCACACUC-3′ for MTHFD2 −/− ) or exon 4 (5′-UCAGGUGGCCCCCAUCCGGA-3′ for SHMT1 −/− ).

Techniques: Cell Culture, Cell Cycle Assay, Staining