human hct116 cells  (ATCC)

Journal: Nucleic Acids Research

Article Title: Cytoplasmic poly-adenosine binding proteins modulate susceptibility of mRNAs to Pumilio-mediated decay

doi: 10.1093/nar/gkag075

Figure Lengend Snippet: The poly(A) tail is necessary for PUM-mediated repression. ( A ) PUM1&2 repression activity was measured in HCT116 cells using Nano-luciferase reporters with three PREs within a minimal 3′ UTR with cleavage and poly-adenylation signals (Nluc 3×PRE), calculated relative to a version wherein the PRE sequences were mutated (indicated in red text) to prevent PUM binding (Nluc 3×PREmt). PUM repression of the poly-adenylated reporter was compared to a Nluc reporter that has a 3′ end generated by the MALAT1 non-coding RNA (Nluc 3×PRE MALAT), which is processed by RNase P mediated cleavage to form a triple helix structure (PBD: 4PLX). Derivatives of the Nluc 3×PRE were constructed with internal poly(A) tracts of either 20 (A20) or 60 (A60) adenosines, inserted between the PREs and the MALAT1 triple helix. Firefly luciferase (Fluc) served as an internal control to normalize transfection efficiency. ( B ) PRE dependent repression by endogenous PUM1 and PUM2 was measured as log(2) fold change of each Nluc 3×PRE reporter relative to its corresponding 3×PREmt reporter. Mean fold change is plotted along with individual replicate data points. n = 9; three experiments, each with three biological replicates; ± standard deviation (SD). For significance calling, * P < .05, ** P < .01, *** P < .001, and **** P < .0001, based on ordinary one-way ANOVA and Tukey test for multiple comparisons. Asterisks above the axis denote significance relative to the 3×PREmt version of each reporter type, whereas below the bars are calculated relative to the poly-adenylated Nluc reporters. ( C ) Western blot confirming the depletion of PAN2 and CNOT1 proteins by RNAi in HCT116 cells. GAPDH served as a loading control. n = 3 experimental replicates. ( D ) The effect of CCR4-NOT or PAN2 and PAN3 knockdown on PUM repression of the Nluc 3×PRE reporter, relative to the mutant Nluc 3×PREmt, was measured in HCT116 cells, in comparison to cells transfected with NTC siRNAs. n = 9; three experiments, each with three biological replicates; ± SD.

Article Snippet: Human HCT116 cells (ATCC, CCL-247) were cultured at 37°C under 5% CO 2 in McCoy’s 5A modified medium (Gibco, Fisher 16600082) with 10% (v/v) fetal bovine serum (Genesee 25–514) and antibiotics (100 U/ml penicillin and 100 μg/ml streptomycin, Thermo Fisher Scientific).

Techniques: Activity Assay, Luciferase, Binding Assay, Generated, Construct, Control, Transfection, Standard Deviation, Western Blot, Knockdown, Mutagenesis, Comparison

Journal: Nucleic Acids Research

Article Title: Cytoplasmic poly-adenosine binding proteins modulate susceptibility of mRNAs to Pumilio-mediated decay

doi: 10.1093/nar/gkag075

Figure Lengend Snippet: PUM1 interacts with endogenous PABPCs through the RRM domain independent of RNA. ( A ) PABPC1 and PABPC4 co-immunoprecipitate with PUM1 from HCT116 cell extracts treated with RNases A and One. PUM1, eIF4E, eIF4G, PABPC1, PABPC4, and GAPDH were detected by western blot. ( B ) Denaturing formaldehyde agarose gel analysis confirmed depletion of RNA in the HCT116 cell extracts before (−) or after treatment with RNase A and RNase One (+). The 18S and 28S rRNA bands, detected by ethidium bromide, are indicated on the right. ( C ) Human PABPC1 domain architecture showing the N-terminal RRM domains with the critical eIF4G binding site residues and the proline-rich linker and C-terminal region PABC domain containing the MLLE motif residues important binding by PAM2-domain containing proteins. ( D ) Western blot of the co-immunoprecipitation analysis of endogenous PUM1 with either HaloTag (HT), as a negative control, or HT-PABPC1 full-length (aa1–636) or HT-RRMs 1–4 (aa 1–370). All input samples were treated with RNase A and One. IgG beads and GAPDH served as negative controls. Dashed vertical lines in the panels indicate that the images were cropped to show relevant lanes. ( E ) Western blot of PUM1 immunoprecipitates to detect association with either wild-type full-length HT-PABPC1, or mutant versions wherein the eIF4G binding site is mutated (M161A and D165A) or the MLLE motif is mutated to (MLLEmt: M584G, L585A, L586A, and E587R), or the HT-MLLE domain (aa 542–636). All input samples were treated with RNase A and One. ( F ) Western blot of the co-immunoprecipitation analysis of endogenous PUM1 to detect association with either wild-type full-length HT-PABPC1 or the RNA-binding mutant version (RBmt). All input samples were treated with RNase A and One. HT and H3 served as negative controls.

Article Snippet: Human HCT116 cells (ATCC, CCL-247) were cultured at 37°C under 5% CO 2 in McCoy’s 5A modified medium (Gibco, Fisher 16600082) with 10% (v/v) fetal bovine serum (Genesee 25–514) and antibiotics (100 U/ml penicillin and 100 μg/ml streptomycin, Thermo Fisher Scientific).

Techniques: Western Blot, Agarose Gel Electrophoresis, Binding Assay, Immunoprecipitation, Negative Control, Mutagenesis, RNA Binding Assay

Journal: Nucleic Acids Research

Article Title: Cytoplasmic poly-adenosine binding proteins modulate susceptibility of mRNAs to Pumilio-mediated decay

doi: 10.1093/nar/gkag075

Figure Lengend Snippet: Poly(A)-binding proteins PABPC1 and PABPC4 are necessary for PUM-mediated repression. ( A ) Western blot analysis confirms auxin induced depletion of degron-tagged PABPC1-AID and RNAi depletion of PABPC4 either individually or in combination in HCT116 cells. RNAi was performed by transfecting cells with either PABPC4 or NTC siRNAs. PABPC1-AID protein was depleted upon treatment with indole-3-acetic acid (+IAA), in comparison to vehicle only control (−IAA). GAPDH served as a loading control. ( B )PUM-mediated repression of Nluc 3×PRE reporter expression was measured in HCT116 cells, relative to the mutant PRE version. Individual and combined effects of PABPC4 RNAi and IAA-induced depletion of PABPC1-AID on PUM repression were tested. n = 9; three experiments, each with three biological replicates; ± SD. For significance calling, * P < .05, ** P < .01, *** P < .001, **** P < .001, based on ordinary one-way ANOVA and Tukey test for multiple comparisons. ( C ) Western blot analysis of samples from a representative experimental replicate for experiments shown in panels ( D ) and ( E ). GAPDH served as a loading control and PUM1 and PUM2 protein levels were detected as controls. The effect of combined PABPC4 RNAi and PABPC1-AID depletion on PUM repression of Nluc reporters containing the 3′ UTRs of the natural, PRE-containing, PUM target mRNAs FZD8 in panel (D) and CDKN1B in panel (E) were measured, relative to matched PRE mutant versions. n = 9; three experiments, each with three biological replicates; ± SD. ( F ) Live cell numbers were measured in control or PABPC1-AID and PABPC4 RNAi depletion conditions from reporter assays shown in panel (B). n = 12; three experiments, each with two biological replicates, two technical replicates; ± SD. ( G ) Cell viability was measured in control or PABPC1-AID and PABPC4 RNAi depletion conditions from reporter assays shown in panel (B). n = 12; three experiments, each with two biological replicates, two technical replicates; ± SD.

Article Snippet: Human HCT116 cells (ATCC, CCL-247) were cultured at 37°C under 5% CO 2 in McCoy’s 5A modified medium (Gibco, Fisher 16600082) with 10% (v/v) fetal bovine serum (Genesee 25–514) and antibiotics (100 U/ml penicillin and 100 μg/ml streptomycin, Thermo Fisher Scientific).

Techniques: Binding Assay, Western Blot, Comparison, Control, Expressing, Mutagenesis

Journal: Nucleic Acids Research

Article Title: Cytoplasmic poly-adenosine binding proteins modulate susceptibility of mRNAs to Pumilio-mediated decay

doi: 10.1093/nar/gkag075

Figure Lengend Snippet: PUMs do not disrupt PABPC1 RNA binding. ( A ) RNA co-immunoprecipitation (RIP) of endogenous PABPC1 with Nluc 3×PRE or 3×PREmt reporter RNAs was performed from HCT116 cell extracts. The top panel shows western blot detection of endogenous PABPC1 protein in the 1% input cell extract and 25% of the immunoprecipitate PABPC1 or negative control IgG RIP samples. The bottom panel shows detection of Nluc mRNA mRNAs by northern blotting. 1% of input and 75% of the RIP purified RNA samples were loaded on the denaturing formaldehyde-MOPS agarose gel, respectively. Three biological replicates were analyzed for each condition. ( B ) Enrichment of Nluc 3×PRE or 3×PREmt reporter RNA in PABPC1 RIP samples was measured as fold increase in the mRNA relative to the IgG control. Nluc RNA level in each RIP sample was normalized to its level in the respective input sample. n = 3 biological replicates; ± SD. No significant (ns) difference in binding of each mRNA to PABPC1 was detected based on an unpaired student’s t -test.

Article Snippet: Human HCT116 cells (ATCC, CCL-247) were cultured at 37°C under 5% CO 2 in McCoy’s 5A modified medium (Gibco, Fisher 16600082) with 10% (v/v) fetal bovine serum (Genesee 25–514) and antibiotics (100 U/ml penicillin and 100 μg/ml streptomycin, Thermo Fisher Scientific).

Techniques: RNA Binding Assay, Immunoprecipitation, Western Blot, Negative Control, Northern Blot, Purification, Agarose Gel Electrophoresis, Control, Binding Assay

Journal: Nucleic Acids Research

Article Title: Cytoplasmic poly-adenosine binding proteins modulate susceptibility of mRNAs to Pumilio-mediated decay

doi: 10.1093/nar/gkag075

Figure Lengend Snippet: PABPC1&4 depletion accelerates mRNA degradation independent of PUM activity. ( A ) Experimental strategy for measuring mRNA decay rates in response to PUM repression and depletion of PABPC1-AID and PABPC4. After RNAi knockdown of PABPC4 (48 h total) and auxin (+IAA) induced degradation of PABPC1-AID (24 h total), the tet-off regulated expression of Nluc 6xPRE or Nluc 6xPREmt produced a pulse of nascent Nluc mRNA in HCT116 cells. Time course shows the procedure for RNAi induced PABPC4 depletion and auxin (+IAA) induced PABPC1-AID depletion. The timeline for transfection of the Tet-off Nluc reporters is shown, along with the suppression of the reporters by doxycycline (+Dox) and transcriptional pulse caused by its removal (-Dox) for 2 h. RNA samples were collected before the pulse (−2 h) and at 0, 1, 2, 4, 6, and 8 h post-pulse to measure decay rates by northern blot. ( B ) Tet-off transcription shut-off was performed to compare the half-lives of the Nluc 6xPRE and 6xPREmt reporter mRNAs under NTC or PABPC1&C4 depletion. A representative northern blot of Nluc reporters and the 18S ribosomal rRNA internal control is shown. Replicate blots are shown in . ( C ) Decay rates of Nluc 6xPRE and Nluc 6xPREmt in response to depletion of PABPC1 and PABPC4 (left). The fraction of each Nluc mRNA remaining, normalized to internal control 18S rRNA, is plotted relative to time (hours) after inhibition of transcription. First order exponential decay trend lines, calculated by non-linear regression analysis, are plotted for each experimental condition from three experimental replicates. n = 3; ± SD. On the right, mean Nluc mRNA half-lives from the experimental replicates are compared. n = 3; ±SD. For significance calling, * P < .05, ** P < .01, *** P < .001, and **** P < .0001, based on ordinary one-way ANOVA and Tukey test for multiple comparisons. ( D ) A representative western blot confirming depletion of PABPC1 and PABPC4 by AID and RNAi, respectively.

Article Snippet: Human HCT116 cells (ATCC, CCL-247) were cultured at 37°C under 5% CO 2 in McCoy’s 5A modified medium (Gibco, Fisher 16600082) with 10% (v/v) fetal bovine serum (Genesee 25–514) and antibiotics (100 U/ml penicillin and 100 μg/ml streptomycin, Thermo Fisher Scientific).

Techniques: Activity Assay, Knockdown, Expressing, Produced, Transfection, Northern Blot, Control, Inhibition, Western Blot

Journal: Nucleic Acids Research

Article Title: Cytoplasmic poly-adenosine binding proteins modulate susceptibility of mRNAs to Pumilio-mediated decay

doi: 10.1093/nar/gkag075

Figure Lengend Snippet: PUMs and PABPCs have opposing effects on mRNA stability. ( A ) Expression levels of the PRE-containing PUM target mRNAs ITGA2 and SMPDL3A were measured by RT-qPCR in RNA samples purified from HCT116 cells wherein PUM1 and PUM2 were depleted by RNAi. Changes in mRNA levels were determined relative to cells treated with the NTC siRNAs. The non-targeted GAPDH mRNA and the non-adenylated MALAT1 non-coding RNA served as controls. ( B ) Expression levels of the PRE-containing PUM target mRNAs ITGA2 and SMPDL3A were measured by RT-qPCR in RNA samples purified from HCT116 cells wherein PABPC1-AID and PABPC4 were depleted by auxin treatment (+IAA) and RNAi, respectively. Changes in mRNA levels were determined relative to cells treated with vehicle only (−IAA) and the NTC siRNA. The non-targeted GAPDH mRNA and the non-adenylated MALAT1 non-coding RNA served as controls. In both panels (A) and (B), transcript level was measured by RT-qPCR, normalized to 18S rRNA, and plotted as fold change relative to the non-depleted control condition. n = 3 biological replicates, ± SD. For significance calling, * P < .05, ** P < .01, *** P < .001, and **** P < .0001, ns = not significant based on unpaired two-tailed t -tests. ( C ) Western blot analysis confirmed RNAi depletion of PUM1 and PUM2 in biological replicates in panel A. GAPDH served as a loading control. ( D ) Western blot analysis of PABPC1-AID and PABPC4 confirmed their depletion by auxin induced degradation and RNAi, respectively, in samples from panel (B). Histone H3 served as a loading control.

Article Snippet: Human HCT116 cells (ATCC, CCL-247) were cultured at 37°C under 5% CO 2 in McCoy’s 5A modified medium (Gibco, Fisher 16600082) with 10% (v/v) fetal bovine serum (Genesee 25–514) and antibiotics (100 U/ml penicillin and 100 μg/ml streptomycin, Thermo Fisher Scientific).

Techniques: Expressing, Quantitative RT-PCR, Purification, Control, Two Tailed Test, Western Blot

Journal: Nucleic Acids Research

Article Title: Cytoplasmic poly-adenosine binding proteins modulate susceptibility of mRNAs to Pumilio-mediated decay

doi: 10.1093/nar/gkag075

Figure Lengend Snippet: PABPC overexpression alleviates PUM repression and requires RNA binding. ( A ) Western blot analysis of halotag (HT) and HT-PABPC1 titration for samples from a representative experimental replicate of panel (B). The amount of transfected plasmid for each effector is shown at the top. GAPDH served as a loading control. PUM1 and PUM2 were detected as an additional control. ( B ) Reporter assay showing the effect of HT-PABPC1 overexpression on PUM repression of the Nluc 3×PRE reporter in wild-type HCT116 cells, calculated relative to the Nluc 3×PREmt reporter. Halotag served as a negative control. n = 9; three experiments, each with three biological replicates; ± SD. For significance calling, * P < .05, ** P < .01, *** P < .001, and *** P < .0001, based on ordinary one-way ANOVA and Tukey test for multiple comparisons. Significance indicated above the X-axis indicates relative to 3×PREmt reporter, whereas significance calling shown below is indicated by the respective brackets. ( C ) Graph of the fold change in HT-PABPC1 exogenous expression over endogenous PABPC1 levels calculated from three experimental replicates including the western blot shown in panel (A). n = 3; ± SD. ( D ) Western blot analysis of halotag (HT), HT-PABPC1 full-length, HT-PABPC1 full-length RNA-binding mutant (RBmt), HT-RRM1-4 domains, and HT-RRM1-4 RBmt samples taken from a representative experimental replicate of panel (E). GAPDH served as a loading control. PUM1 and PUM2 were detected as controls. ( E ) Reporter assay showing effect of HT-PABPC1 full-length, HT-RRM1-4 domains, and RNA-binding mutants versions when overexpressed on PUM repression of the Nluc 3×PRE reporter in HCT116 cells. n = 9; three experiments, each with three biological replicates; ± SD. Halotag served as a negative control.( F ) Western blot analysis of overexpressed HT-PABPC1 on PUM repression and the effect of the mutation of the eIF4G binding site mutant (M161A, D165A), or the MLLE motif (MLLEmt), or the RNA-binding defective mutant (RBmt) from a representative experimental replicate of panel (G). H3 served as a loading control. PUM1 and PUM2 were detected as additional controls. ( G ) Reporter assay showing effect of HT-PABPC1 full-length mutants on PUM repression in wild-type HCT116 cells. n = 9; three experiments, each with three biological replicates; ± SD. Halotag served as a negative control.

Article Snippet: Human HCT116 cells (ATCC, CCL-247) were cultured at 37°C under 5% CO 2 in McCoy’s 5A modified medium (Gibco, Fisher 16600082) with 10% (v/v) fetal bovine serum (Genesee 25–514) and antibiotics (100 U/ml penicillin and 100 μg/ml streptomycin, Thermo Fisher Scientific).

Techniques: Over Expression, RNA Binding Assay, Western Blot, Titration, Transfection, Plasmid Preparation, Control, Reporter Assay, Negative Control, Expressing, Mutagenesis, Binding Assay

Journal: Nucleic Acids Research

Article Title: Cytoplasmic poly-adenosine binding proteins modulate susceptibility of mRNAs to Pumilio-mediated decay

doi: 10.1093/nar/gkag075

Figure Lengend Snippet: PABPC1 does not disrupt PUM binding to Nluc 3×PRE mRNA. ( A ) Co-immunoprecipitation analysis of PUM1 binding to the Nluc 3×PRE reporter in the presence of overexpressed halotag (HT) or HT-PABPC1. The top two panels show western blot detection of PUM1 protein and HT-PABPC1 in the 2% of the input HCT116 cell extracts and 25% of the PUM1 RNA co-immunoprecipitates (RIP) from three biological replicate samples per condition. The bottom panel shows detection of Nluc mRNA by northern blotting. For northern blots, 1% of input samples and 75% of the RIP samples were loaded on the formaldehyde-MOPS agarose gel, respectively. Co-immunoprecipitation with IgG beads served as a negative control. ( B ) Fold increase of the Nluc 3×PRE mRNA in the input samples from cells expressing HT-PABPC1 are plotted relative to the HT control, based on data in panel (A). n = 3 biological replicates, ± SD. For significance calling, *** P < .001 based on an unpaired student’s t -test. ( C ) Fold enrichment of the Nluc 3×PRE mRNA in PUM1 RIP samples was measured relative to the IgG control. Importantly, the mRNA levels in each RIP sample was normalized to that present in the respective input samples. n = 3 biological replicates, ± SD. For significance calling, the difference between HT and HT-PABPC1 was not significant ( P > 0.05 = ns) based on an unpaired student’s t -test.

Article Snippet: Human HCT116 cells (ATCC, CCL-247) were cultured at 37°C under 5% CO 2 in McCoy’s 5A modified medium (Gibco, Fisher 16600082) with 10% (v/v) fetal bovine serum (Genesee 25–514) and antibiotics (100 U/ml penicillin and 100 μg/ml streptomycin, Thermo Fisher Scientific).

Techniques: Binding Assay, Immunoprecipitation, Western Blot, Northern Blot, Agarose Gel Electrophoresis, Negative Control, Expressing, Control

Journal: Nucleic Acids Research

Article Title: Cytoplasmic poly-adenosine binding proteins modulate susceptibility of mRNAs to Pumilio-mediated decay

doi: 10.1093/nar/gkag075

Figure Lengend Snippet: PABPC1 overexpression stabilizes mRNAs, blocking PUM-mediated mRNA degradation. ( A ) Northern blot analysis of Nluc reporter mRNA containing either six wild-type PREs (6×PRE) or mutant PREs (6×PREmt) in HCT116 cells transfected with halotag (HT) or HT-PABPC1 expression plasmids. 18S rRNA served as a loading control, and ethidium bromide staining confirmed RNA integrity and loading. Three biological replicate samples are shown for each condition. As a marker for the Nluc mRNA with the poly(A) tail removed (A0), one RNA sample from the HT control was treated with oligo-dT15 and RNase H (RH + dT). Poly-adenylated (pA) and deadenylated (A0) Nluc species are indicated on the right. ( B ) Western blot analysis of effector protein expression. PABPC1 antibody detected both endogenous PABPC1 and overexpressed HT-PABPC1, while HT antibody confirmed the expression of HT. ( C ) Fold changes of either Nluc 6×PRE or Nluc 6×PREmt mRNA levels from panel (A) in response to overexpressed HT-PABPC1 were calculated relative to their respective HT negative controls. Data represent mean values ± SD, n = 3 biological replicates. ( D ) Fold changes of either Nluc 6×PRE mRNA levels from panel (A) in response to overexpressed HT-PABPC1 or the negative control HT were calculated relative to their respective or Nluc 6×PREmt negative controls. Data represent mean values ± SD, n = 3 biological replicates. For significance calling, * P < .05, ** P < .01, based on ordinary one-way ANOVA and Tukey test for multiple comparisons. ( E ) Tet-off transcription shut-off was performed to compare the half-lives of the Nluc 6×PRE and 6×PREmt reporter mRNAs in response to overexpressed HT-PABPC1 or negative control halotag (HT). A representative northern blot of Nluc reporters and the 18S ribosomal rRNA internal control is shown. Replicate blots are shown in . ( F ) Decay rates of Nluc 6×PRE and Nluc 6×PREmt in response to overexpression of HT-PABPC1, in comparison to HT. The fraction of each Nluc mRNA remaining, normalized to 18S rRNA, is plotted relative to time (hours) after inhibition of transcription. First-order exponential decay trend lines, calculated by non-linear regression analysis, are plotted for each experimental condition from three experimental replicates. n = 3; ± SD. On the right, mean Nluc mRNA half-lives from the experimental replicates are compared. n = 3; ± SD. For significance calling, * P < .05, ** P < .01, *** P < .001, based on ordinary one-way ANOVA and Tukey test for multiple comparisons. ( G ) A representative western blot confirming expression of HT-PABPC1 relative to endogenous PABPC1 using anti-PABPC1 antibody in each condition used for mRNA decay analysis. Western blot also confirmed expression of the HT protein. Histone H3 served as a loading control.

Article Snippet: Human HCT116 cells (ATCC, CCL-247) were cultured at 37°C under 5% CO 2 in McCoy’s 5A modified medium (Gibco, Fisher 16600082) with 10% (v/v) fetal bovine serum (Genesee 25–514) and antibiotics (100 U/ml penicillin and 100 μg/ml streptomycin, Thermo Fisher Scientific).

Techniques: Over Expression, Blocking Assay, Northern Blot, Mutagenesis, Transfection, Expressing, Control, Staining, Marker, Western Blot, Negative Control, Comparison, Inhibition

Journal: Medical Oncology (Northwood, London, England)

Article Title: Investigating the effects of platelets, platelet releasate and aspirin on colorectal cancer cell proliferation, migration and invasion

doi: 10.1007/s12032-026-03264-z

Figure Lengend Snippet: Platelet activation and binding to colorectal cancer cells. Whole blood obtained from healthy individuals ( n = 5) was treated with DPBS or 1mM aspirin before incubation with different concentrations of HCT15 or HCT116 colorectal cancer cells. Platelet activation was measured using flow cytometry for the platelet activation markers ( a ) P-selectin, ( b ) LAMP-3 and ( c ) activated GPIIb/IIIa. Representative images of platelets binding to d) HCT15 and e) HCT116 cells. Platelets were stained using an anti-CD41 antibody (green) and CRC cells were stained using an anti-EpCAM antibody (red) and DAPI (blue) and imaged using immunofluorescence microscopy. Data presented as box whisker plots for n = 5 experiments. * P < 0.05, ** P < 0.01

Article Snippet: HCT15 and HCT116 human colorectal cancer cells (ATCC, Manassas, USA), RPMI 1640 + L-glutamine, fetal bovine serum, penicillin/streptomycin and Dulbecco’s phosphate buffer saline (Gibco, Waltham, USA); Vacuette tube 9mL ACD-B (Greiner Bio-One, Kremsmünster, Austria); prostaglandin I 2 (PGI 2 ) sodium salt (Cayman Chemicals, Ann Arbor, USA); thrombin receptor activating peptide (Roche, Basel Switzerland); CD41 antibody (Abcam, Cambridge, UK); EpCAM (D4K8R) XP antibody, anti-mouse IgG (H + L), F(ab’)2 fragment Alexa Fluor 488, anti-rabbit IgG (H + L), F(ab’) 2 fragment Alexa Fluor 555 and ProLong Gold Antifade Reagent with DAPI (Cell Signalling Technology, Danvers, USA); Vacutainer Citrate 2.7mL tubes, TruCount tubes, CD42b PE, CD62p APC, CD63 PE-Cy7 and PAC-1 FITC antibodies (BD Biosciences, Franklin Lakes, USA); paraformaldehyde 16% solution, EM grade (Electron Microscopy Sciences, Hatfield, USA); sodium chloride 0.9% for irrigation (Baxter, Deerfield, USA); CellTrace Carboxyfluorescein succinimidyl ester (CFSE) Cell Proliferation Kit (Thermo Fisher Scientific, Waltham, USA); cell culture plates, Transwell 6.5 mm 8.0 μm polycarbonate insert, Matrigel and 5mL polystyrene round-bottom Tube 12 × 75 mm (Corning, Corning, USA), crystal violet solution, aspirin and phenazine methosulfate (PMS) (Sigma-Aldrich, St. Louis, USA).

Techniques: Activation Assay, Binding Assay, Incubation, Flow Cytometry, Staining, Immunofluorescence, Microscopy, Whisker Assay

Journal: Medical Oncology (Northwood, London, England)

Article Title: Investigating the effects of platelets, platelet releasate and aspirin on colorectal cancer cell proliferation, migration and invasion

doi: 10.1007/s12032-026-03264-z

Figure Lengend Snippet: The effect of platelets and platelet releasate on colorectal cancer cell migration and invasion. Inserts in transwell assays were ( a ) uncoated to measure migration or ( b ) Matrigel coated to measure invasion of HCT15 and HCT116 cells co-incubated with platelets or platelet-releasate from healthy individuals ( n = 5) for 24 h. Platelets were pretreated with DPBS or 1mM aspirin and platelet releasate was generated from platelets pretreated with DPBS or aspirin. Data is presented box whisker plots for n = 5 experiments. * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001

Article Snippet: HCT15 and HCT116 human colorectal cancer cells (ATCC, Manassas, USA), RPMI 1640 + L-glutamine, fetal bovine serum, penicillin/streptomycin and Dulbecco’s phosphate buffer saline (Gibco, Waltham, USA); Vacuette tube 9mL ACD-B (Greiner Bio-One, Kremsmünster, Austria); prostaglandin I 2 (PGI 2 ) sodium salt (Cayman Chemicals, Ann Arbor, USA); thrombin receptor activating peptide (Roche, Basel Switzerland); CD41 antibody (Abcam, Cambridge, UK); EpCAM (D4K8R) XP antibody, anti-mouse IgG (H + L), F(ab’)2 fragment Alexa Fluor 488, anti-rabbit IgG (H + L), F(ab’) 2 fragment Alexa Fluor 555 and ProLong Gold Antifade Reagent with DAPI (Cell Signalling Technology, Danvers, USA); Vacutainer Citrate 2.7mL tubes, TruCount tubes, CD42b PE, CD62p APC, CD63 PE-Cy7 and PAC-1 FITC antibodies (BD Biosciences, Franklin Lakes, USA); paraformaldehyde 16% solution, EM grade (Electron Microscopy Sciences, Hatfield, USA); sodium chloride 0.9% for irrigation (Baxter, Deerfield, USA); CellTrace Carboxyfluorescein succinimidyl ester (CFSE) Cell Proliferation Kit (Thermo Fisher Scientific, Waltham, USA); cell culture plates, Transwell 6.5 mm 8.0 μm polycarbonate insert, Matrigel and 5mL polystyrene round-bottom Tube 12 × 75 mm (Corning, Corning, USA), crystal violet solution, aspirin and phenazine methosulfate (PMS) (Sigma-Aldrich, St. Louis, USA).

Techniques: Migration, Incubation, Generated, Whisker Assay

Journal: Medical Oncology (Northwood, London, England)

Article Title: Investigating the effects of platelets, platelet releasate and aspirin on colorectal cancer cell proliferation, migration and invasion

doi: 10.1007/s12032-026-03264-z

Figure Lengend Snippet: The effect of platelets and platelet releasate on colorectal cancer cell proliferation. a ) HCT15 and b ) HCT116 cells stained with CFSE were co-incubated with platelets or platelet releasate from healthy individuals ( n = 6) for 48 h. Platelets were pretreated with DPBS or 1mM aspirin and platelet releasate was generated from platelets pretreated with DPBS or aspirin. The cells were analysed using flow cytometry and ModFit LT software which calculated a proliferation index. Data is presented box whisker plots for n = 6 experiments. ( c ) HCT15 and ( d ) HCT116 cells were co-incubated for 48 h with platelets from healthy individuals ( n = 5) pretreated with DPBS or 1mM aspirin, with proliferation determined using MTS. Data is presented as the mean with error bars representing the standard error of the mean for n = 5 experiments. ( e ) HCT15 and ( g ) HCT116 cells were incubated with 1mM aspirin for 48 h ( n = 10) and the effect it had on proliferation was determined using MTS proliferation assays. Data is presented box whisker plots for n = 10 experiments. ** P < 0.01

Article Snippet: HCT15 and HCT116 human colorectal cancer cells (ATCC, Manassas, USA), RPMI 1640 + L-glutamine, fetal bovine serum, penicillin/streptomycin and Dulbecco’s phosphate buffer saline (Gibco, Waltham, USA); Vacuette tube 9mL ACD-B (Greiner Bio-One, Kremsmünster, Austria); prostaglandin I 2 (PGI 2 ) sodium salt (Cayman Chemicals, Ann Arbor, USA); thrombin receptor activating peptide (Roche, Basel Switzerland); CD41 antibody (Abcam, Cambridge, UK); EpCAM (D4K8R) XP antibody, anti-mouse IgG (H + L), F(ab’)2 fragment Alexa Fluor 488, anti-rabbit IgG (H + L), F(ab’) 2 fragment Alexa Fluor 555 and ProLong Gold Antifade Reagent with DAPI (Cell Signalling Technology, Danvers, USA); Vacutainer Citrate 2.7mL tubes, TruCount tubes, CD42b PE, CD62p APC, CD63 PE-Cy7 and PAC-1 FITC antibodies (BD Biosciences, Franklin Lakes, USA); paraformaldehyde 16% solution, EM grade (Electron Microscopy Sciences, Hatfield, USA); sodium chloride 0.9% for irrigation (Baxter, Deerfield, USA); CellTrace Carboxyfluorescein succinimidyl ester (CFSE) Cell Proliferation Kit (Thermo Fisher Scientific, Waltham, USA); cell culture plates, Transwell 6.5 mm 8.0 μm polycarbonate insert, Matrigel and 5mL polystyrene round-bottom Tube 12 × 75 mm (Corning, Corning, USA), crystal violet solution, aspirin and phenazine methosulfate (PMS) (Sigma-Aldrich, St. Louis, USA).

Techniques: Staining, Incubation, Generated, Flow Cytometry, Software, Whisker Assay

Journal: Oncology Letters

Article Title: CRISPR/Cas9-mediated claudin-2 knockout in HCT116 cells reveals its key role in colorectal cancer progression

doi: 10.3892/ol.2025.15407

Figure Lengend Snippet: ICE analysis of CRISPR/Cas9-mediated CLDN2 -KO efficiency in HCT116 cells. ICE software analysis confirmed successful editing of the CLDN2 locus with an efficiency of ~91%. The editing score reflects the proportion of indels detected in the cell population. ICE, Inference of CRISPR Edits; CLDN2 , claudin-2; Wt, wild-type; KO, knockout; indel, insertion-deletion.

Article Snippet: The human CRC cell line HCT116 (cat. no. CCL-247; American Type Culture Collection) was obtained from Synthego.

Techniques: CRISPR, Software, Knock-Out

Journal: Oncology Letters

Article Title: CRISPR/Cas9-mediated claudin-2 knockout in HCT116 cells reveals its key role in colorectal cancer progression

doi: 10.3892/ol.2025.15407

Figure Lengend Snippet: Wound healing assay assessing cell migration in Wt and CLDN2 -KO HCT116 cells. (A) Representative images of wound closure at 0 and 24 h post-scratch (scale bar, 100 µm). (B) Quantification of wound closure percentage. Wt cells achieved ~96% closure, while CLDN2 -KO cells demonstrated 41% closure after 24 h (P=0.0027; unpaired two-tailed t-test; n=3). Data are presented as the mean ± SEM. **P<0.01. Wt, wild-type; CLDN2 -KO, claudin-2 knock out.

Article Snippet: The human CRC cell line HCT116 (cat. no. CCL-247; American Type Culture Collection) was obtained from Synthego.

Techniques: Wound Healing Assay, Migration, Two Tailed Test, Knock-Out

Journal: Oncology Letters

Article Title: CRISPR/Cas9-mediated claudin-2 knockout in HCT116 cells reveals its key role in colorectal cancer progression

doi: 10.3892/ol.2025.15407

Figure Lengend Snippet: Gene expression analysis of invasion- and metastasis-related genes in CLDN2 -KO vs. Wt HCT116 cells. Reverse transcription-quantitative PCR exhibited significant downregulation of multiple target genes, including ZONAB, NDRG1, CLDN14, CLDN23, Bcl-2, p53 and Bcl-6. Gene expression levels were normalized to GAPDH. Data are represented as mean ± SEM (n=3). Statistical comparisons were made using unpaired two-tailed t-tests. *P<0.05. ns, not significant; Wt, wild-type; CLDN2 -KO, claudin-2 knock out; ZO-1, zonula occludens-1; VDR , vitamin D receptor; ZONAB, ZO-1 -associated nucleic acid binding protein; NDRG1, N-Myc downstream-regulated gene 1; APC, adenomatous polyposis coli; AF-6/AFDN , Afadin; TJP1, tight junction protein 1; YBX3, Y-box binding protein 3; PTMS, parathymosin; TCN-1, transcobalamin 1.

Article Snippet: The human CRC cell line HCT116 (cat. no. CCL-247; American Type Culture Collection) was obtained from Synthego.

Techniques: Gene Expression, Reverse Transcription, Real-time Polymerase Chain Reaction, Two Tailed Test, Knock-Out, Binding Assay

Journal: Oncology Letters

Article Title: CRISPR/Cas9-mediated claudin-2 knockout in HCT116 cells reveals its key role in colorectal cancer progression

doi: 10.3892/ol.2025.15407

Figure Lengend Snippet: Relative expression levels of metastasis-associated genes in Wt and CLDN2 -KO HCT116 cells. Gene expression was quantified using reverse transcription-quantitative PCR. Each bar represents the mean ± SEM of three independent experiments. Values were calculated using the 2 −ΔΔCq method and normalized to GAPDH. Expression in Wt cells was set to 1.0 and knock out values were expressed relative to this baseline. Each bar corresponds to a specific gene and the height of the bars indicates the magnitude of the fold-change observed in the CLDN2 -KO samples compared with the Wt samples. The highest bars on the figure represent the gene with the lowest degree of expression variation. IL-6 exhibited the lowest degree of downregulation (fold-change, 0.718), whereas AF-6 demonstrated the most pronounced reduction (fold-change, 0.008). Wt, wild type; ZO-1 , zonula occludens-1; VDR , vitamin D receptor; ZONAB , ZO-1-associated nucleic acid binding protein; NDRG1, N-Myc downstream-regulated gene 1; APC, adenomatous polyposis coli; AF-6 , Afadin; PTMS, parathymosin; TCN-1, transcobalamin 1; CLDN2 -KO, claudin-2 knock out; AU, arbitrary Units.

Article Snippet: The human CRC cell line HCT116 (cat. no. CCL-247; American Type Culture Collection) was obtained from Synthego.

Techniques: Expressing, Gene Expression, Reverse Transcription, Real-time Polymerase Chain Reaction, Knock-Out, Binding Assay

Journal: Materials Today Bio

Article Title: Primary tissue metabolic fingerprinting for efficient diagnosis of lymph node metastasis and metabolic reprogramming mechanisms in colorectal cancer

doi: 10.1016/j.mtbio.2025.102712

Figure Lengend Snippet: Biological function validation of metabolic biomarkers in CRC lymph node metastasis. Relative abundance of (A) γ-glutamylcysteine (γ-Glu-Cys) and (B) glutathione (GSH) in LNM-versus LNM + CRC tissues. (C) Representative images of immunohistochemical (IHC) staining of GCLC, GCLM and GSS in CRC tissues (n = 10). Scale bar: 200 μm. (D) Quantitative IHC score analysis for GCLC, GCLM and GSS, showing significant upregulation of GCLC in LNM-positive samples (mean ± SEM, Mann-Whitney U test). (E) Glutamate-cysteine ligase (GCL) activity in CRC tissues (n = 6, mean ± SEM, Mann-Whitney U test)). (F) GCLC protein level was determined through western blot analysis following the transfection of GCLC and control siRNAs. (G) Cell migration ability was estimated using a scratch wound healing assay (n = 3, mean ± SEM, Mann-Whitney U test). (H) Transwell migration ability of HCT116 cells after GCLC and control siRNAs transfection (n = 3, mean ± SEM, Mann-Whitney U test). (I) Protein expression analysis of GCLC in CRC samples from The Cancer Genome Atlas (TCGA) database, confirming upregulation in LNM + cases (Mann-Whitney U test, p < 0.001). (J) Schematic illustration depicting the metabolic reprogramming mechanism in primary CRC leading to LNM, highlighting cysteine depletion, GCLC-driven GSH accumulation, and enhanced tumor migration.

Article Snippet: The HCT116 human CRC cell line was obtained from the American Type Culture Collection (ATCC).

Techniques: Biomarker Discovery, Immunohistochemical staining, Immunohistochemistry, MANN-WHITNEY, Activity Assay, Western Blot, Transfection, Control, Migration, Wound Healing Assay, Expressing

Journal: Nucleic Acids Research

Article Title: NSMF modulates replication stress to facilitate colorectal cancer progression

doi: 10.1093/nar/gkaf1521

Figure Lengend Snippet: NSMF knockdown inhibits CRC cell proliferation and cell-cycle progression ( A ) Correlation analysis between NSMF expression and replication stress score in CRC cell line from CCLE dataset. ( B ) Cell growth curves of HCT116 and SW480 cells following transfection with control or NSMF siRNAs over 5 days, measured by direct cell counting ( n = 3). Data represent the mean ± SD. **** P < .0001, two-way ANOVA with repeated measures, followed by Tukey’s multiple comparisons test. ( C ) Colony formation assay showing the effect of NSMF knockdown in HCT116 and SW480 cells. Representative images (upper panel) and quantification of colonies area (lower panel) are shown. Data represent mean ± SD. *** P < .001, unpaired two-tailed Student’s t -test (HCT116, n = 2; SW480, n = 3). ( D ) Relative cell number of five CRC cell lines (HCT116, SW480, SW620, SNU-407, and RKO) measured 3 days after NSMF knockdown compared to control. Data represent the mean ± SEM. ( n = 3) *** P < .001, **** P < .0001, unpaired two-tailed Student’s t -test. ( E ) Cell-cycle analysis by flow cytometry in HCT116 cells transfected with siCtrl or siNSMF#1. Representative flow cytometry plots (left) and quantification of cell cycle distribution in G1, S, and G2/M phases with percentages indicated in bar graph (right). Data are presented as the mean ± SD ( n = 3). All experiments were independently performed at least three times, and representative results are shown.

Article Snippet: The normal colon-derived cell line CCD-18Co, human CRC cell lines HCT116, and the human lung fibroblast cell line IMR-90 were obtained from the American Type Culture Collection (ATCC, Manassas, VA).

Techniques: Knockdown, Expressing, Transfection, Control, Cell Counting, Colony Assay, Two Tailed Test, Cell Cycle Assay, Flow Cytometry

Journal: Nucleic Acids Research

Article Title: NSMF modulates replication stress to facilitate colorectal cancer progression

doi: 10.1093/nar/gkaf1521

Figure Lengend Snippet: NSMF depletion affects DNA replication dynamics and induces replication stress. (A, B) DNA fiber analysis examining the effect of NSMF on replication dynamics in HCT116 cells. For normal conditions, cells were sequentially labeled with CldU and IdU for 30 min each ( A ). For replication stress conditions, cells were treated with 2 mM hydroxyurea (HU) for 2 h between CldU and IdU pulses ( B ). Representative DNA fiber images (upper) and quantification of IdU tract lengths (lower). The median value is indicated, derived from the analysis of 200 or more IdU and CldU tracts per experimental condition. **** P < .0001, two-tailed Mann–Whitney test. ( C, D ) Quantification of newly fired origins ( C ), IdU-only fibers) and stalled forks ( D ), CldU-only fibers) in HCT116 cells transfected with siCtrl or siNSMF#1. A total of 300–350 fibers from 7 to 18 randomly selected non-overlapping images per condition were analyzed. Data represent mean ± SEM. * P < .05, **** P < .0001. Statistical significance was assessed using a Mann–Whitney test. Results are representative of three independent experiments. ( E ) BrdU pulse-chase analysis of cell-cycle kinetics in HCT116 cells transfected with siCtrl or siNSMF#1 following release from a HU block (2 mM, 12 h). Representative results from three independent experiments are shown. ( F ) Immunofluorescence analysis of phospho-RPA2 foci in control (shCtrl) and NSMF-depleted (shNSMF#1 and #2) HCT116 cells under normal (NT) or HU treatment (2 mM, 16 h) conditions. The number of phospho-RPA2 foci per cell was quantified from at least 54 cells across two independent experiments. Scale bar, 10 μm. Data are presented as median. * P < .05, **** P < .0001, n.s., not significant, Kruskal–Wallis test followed by Dunn’s multiple comparisons test. ( G ) γH2AX staining for control (shCtrl) or NSMF-depleted (shNSMF#2) HCT116 cells treated with HU treatment (2 mM, 16 h), and directly fixed or allowed to grow in the complete medium for 5 h in the absence of HU. The number of γH2AX foci per cell was quantified from at least 106 cells across three independent experiments. Scale bar, 10 μm. Data are presented as median. **** P < .0001, n.s., not significant, Kruskal–Wallis test followed by Dunn’s multiple comparisons test.

Article Snippet: The normal colon-derived cell line CCD-18Co, human CRC cell lines HCT116, and the human lung fibroblast cell line IMR-90 were obtained from the American Type Culture Collection (ATCC, Manassas, VA).

Techniques: Labeling, Derivative Assay, Two Tailed Test, MANN-WHITNEY, Transfection, Pulse Chase, Blocking Assay, Immunofluorescence, Control, Staining

Journal: Nucleic Acids Research

Article Title: NSMF modulates replication stress to facilitate colorectal cancer progression

doi: 10.1093/nar/gkaf1521

Figure Lengend Snippet: NSMF mitigates replication stress and prevents oncogene-induced senescence. ( A ) SA-β-galactosidase (SA-β-Gal) staining in stable NSMF knockdown (shNSMF #1 and #2) or control (shCtrl) HCT116 cells. Representative images (upper) and quantification of SA-β-gal positive cells (lower). Scale bar, 50 μm. Data are presented as mean ± SEM from 150 cells across seven images obtained from three independent experiments. ** P < .01, *** P < .001, one-way ANOVA followed by Dunnett’s multiple comparisons test. ( B ) GSEA plot showing enrichment of cellular senescence-related genes in RNA-seq data from Nsmf +/+ ; Apc Min/+ and Nsmf −/− ; Apc Min/+ intestinal tumor (upper). Heatmap visualization of differentially expressed senescence- and SASP-related genes between genotypes (lower). ( C ) qRT-PCR analysis of senescence-associated genes in intestinal tumors from Nsmf +/+ ; Apc Min/+ ( n = 4) and Nsmf −/− ; Apc Min/+ ( n = 3) mice. Data represent the mean ± SEM. * P < .05, ** P < .01, *** P < .001, unpaired two-tailed t -test with Holm–Sidak correction for multiple comparisons. ( D ) Western blot analysis of p16INK4A and p21CIP1 in intestinal tumor tissues from Nsmf +/+ ; Apc Min/+ and Nsmf −/− ; Apc Min/+ mice. GAPDH served as a loading control. ( E ) Schematic representation of the experimental design for the oncogene-induced senescence model. IMR-90 cells were transduced with lentiviruses encoding either GFP-vector or GFP-NSMF. Following selection, senescence was induced by expression of oncogenic Ras G12V . ( F ) Western blot analysis of the indicated proteins on day 4 after induction of oncogenic Ras G12V expression. α-Tubulin was used as a loading control. ( G ) SA-β-Gal staining in IMR-90 cells 8 days post-transduction. Representative images (left) and quantification of SA-β-Gal positive cells (right). Scale bar, 20um. Data are presented as mean ± SEM ( n = 4–6 independent images per sample). *** P < .001, n.s., not significant, one-way ANOVA followed by Tukey’s HSD test. ( H ) Immunofluorescence analysis of γH2AX in GFP-vector or GFP-NSMF expressing IMR-90 cells with or without Ras G12V . Quantification of γH2AX foci per GFP-positive cell was performed in at least 42 cells per group. Scale bar, 20 μm. Data are presented as median. ** P < .01, **** P < .0001, n.s., not significant, Kruskal–Wallis test followed by Dunn’s multiple comparisons test. All experiments were independently performed at least three times, and representative results are shown. ( I ) Correlation of NSMF expression with genomic instability in pan-cancer analysis. Genomic instability was assessed across 4315 pan-cancer samples from TCGA using multiple genomic instability features, including frequency of LOH, HRD-related LOH frequency, telomeric allelic imbalance, large-scale transitions, mutation burden per sample, and weighted genome integrity index. Tumors were categorized based on genomic instability scores as low (<25%, below first quartile), medium (25%–75%, between first and third quartile), or high (>75%, above third quartile). Statistical comparisons of NSMF expression levels across groups were performed using Wilcoxon rank-sum test. ( J ) Hypothetical model illustrating the role of NSMF in regulating replication stress, highlighting its critical function in alleviating excessive replication stress and preventing cytotoxic DNA damage. This regulatory activity supports a controlled level of genomic instability, thereby promoting CRC progression. Figure was created using BioRender.com.

Article Snippet: The normal colon-derived cell line CCD-18Co, human CRC cell lines HCT116, and the human lung fibroblast cell line IMR-90 were obtained from the American Type Culture Collection (ATCC, Manassas, VA).

Techniques: Staining, Knockdown, Control, RNA Sequencing, Quantitative RT-PCR, Two Tailed Test, Western Blot, Transduction, Plasmid Preparation, Selection, Expressing, Immunofluorescence, Mutagenesis, Activity Assay