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: iScience

Article Title: The TRIP12’s intrinsically disordered region induces chromatin condensates and interferes with nuclear processes

doi: 10.1016/j.isci.2025.114592

Figure Lengend Snippet: The IDR of TRIP12 is responsible for the formation of chromatin condensates (A) Prediction of TRIP12 3D-structure by AlphaFold model. Predicted local distance difference test (pLDDT) indicates the confidence level of the predicted structure. Dark blue, light blue, orange, and yellow represent very high, confident, low, and very low model confidence, respectively. (B) Graphical representation of TRIP12 different domains fused to the GFP reporter protein. Dark blue rectangles locate endogenous or artificial NLSs. IDR, intrinsically disordered region; ARM, armadillo domain; WWE, tryptophan-tryptophan-glutamate-rich domain; HECT, homologous to E6-AP carboxyl terminus; and GFP, green fluorescent protein. (C) Representative images of TRIP12-domains fused to GFP expressing HeLa S3 cells by immunofluorescence. Nuclei were counterstained with DAPI. Cells with three different GFP intensities are represented. Insertion of an artificial NLS in ARM-WWE-GFP, ΔIDR/ARM-WWE/HECT-GFP, HECT-GFP, and ΔIDR-GFP constructs allowed for nuclear localization with a faint presence in the cytoplasm. Scale bars represent 2 μm. (D) Determination of DNA granularity relative to GFP expression level in TRIP12-domains fused to GFP expressing HeLa S3 cells. For each cell, DAPI granularity and GFP expression were determined as described in “ ” on over 50 cells. Blue and green filled circles correspond to individual non-transfected and transfected cells, respectively. The linear regression curve is indicated in black. A Spearman r coefficient test and a two tailed-p value are indicated. (E) Representative images of chromatin condensates induced by TRIP12-IDR overexpression in HCT-116, U2OS, and hTert-RPE1 cell lines by immunofluorescence. Nuclei were counterstained with DAPI. Scale bars represent 2 μm. (F) Comparison of isoelectric point (pI) and capacity to form chromatin condensates (slope) of the different TRIP12-GFP constructs. The pI of the different TRIP12 fragments was determined using ProtParam on Expasy website. The capacity to form chromatin condensates is indicated by the slope values obtained in D, 2F, and C. A Spearman r coefficient test and a two tailed-p value are indicated. (G) Representative images of DNA organization in IDR-GFP deletion constructs in high expressing HeLa S3 cells (left). The cytoplasmic expression of 325-445-GFP constructs is explained by the loss of NLS sequences. Determination of DNA granularity relative to GFP expression level for the different IDR-GFP deletion constructs (right). The DAPI granularity and GFP expression were determined as described in “ ” on more than 40 cells. Blue and green filled circles correspond to individual non-transfected and transfected cells, respectively. The linear regression curve is indicated in black. Scale bars represent 2 μm. A Spearman r coefficient test and a two tailed-p value are indicated. (H) Comparison of isoelectric point (pI), capacity to form chromatin condensates (slope), and the length (in aa) of the different IDR-GFP deletion constructs. The pI and the length of the different TRIP12 fragments were determined using ProtParam on Expasy website. The capacity to form chromatin condensates was assessed from the slope values obtained in G. A Spearman r coefficient test and a two tailed-p value are indicated. (I) Representative image of cellular fractionation of HeLa S3 cells expressing 1-107-GFP, 108-207-GFP, 208-324-GFP, and 325-445-GFP constructs. GFP expression in soluble and chromatin-bound fractions was determined by western blot analysis. The level of HSP90 and PanH3 protein expression were used as loading and enrichment controls. The graph represents the percentage of GFP expression in the different fraction. Results are expressed as mean ± SEM of four different experiments. (J) Electric net charge of [1–107], [108–207], [208–324], and [325–445] fragments of TRIP12 IDR at pH 7.4 determined by Prot pi|Protein Tool (left graph). Percentage of basic, acidic, and uncharged residues in [1–107], [108–207], [208–324], and [325–445] fragments of TRIP12 IDR determined by Prot pi|Protein Tool (right graph). (K) Graphical representation of MED1-IDR fused to GFP protein. The dark blue rectangle indicates artificial NLSs. Representative images of MED1-IDR-GFP expressing HeLa S3 cells obtained by immunofluorescence. Nuclei were counterstained with DAPI. Cells with three different GFP intensities are represented. For each cell, DAPI granularity and GFP expression were determined as described in “ ” on more than 50 cells. Blue and green filled circles correspond to individual non-transfected and transfected cells, respectively. The linear regression curve is represented in black. Scale bars represent 2 μm. A Spearman r coefficient test and a two tailed-p value are indicated.

Article Snippet: HeLa S3 (CCL-2TM, female), HEK-293T (CRL-3216TM, female), U2OS (HTB-96 TM , female), hTERT RPE-1 (CRL-4000 TM , female) and HCT-116 (CCL-247 TM , male) cell lines were procured from ATCC ( https://www.atcc.org ).

Techniques: Expressing, Immunofluorescence, Construct, Transfection, Two Tailed Test, Over Expression, Comparison, Cell Fractionation, Western Blot

Journal: iScience

Article Title: FAM65A, as a potential predictor of prognosis, promotes colorectal cancer progression via activating Ras/ERK/RSK signaling

doi: 10.1016/j.isci.2026.114662

Figure Lengend Snippet: Ectopic expression of FAM65A promotes CRC cell proliferation, migration, and inhibits cell apoptosis in vitro (A) Western blot analysis the efficiency of FAM65A overexpression, accompanied by a quantitative assessment, n = 3, ∗∗ p < 0.01. (B) The findings from the CCK8 assay conducted on HCT116-MCS and HCT116-FAM65A cells are presented, n = 3, ∗∗∗ p < 0.001. (C) The outcomes of the colony formation assay for HCT116-MCS and HCT116-FAM65A cells are reported. (D) A quantitative analysis of the colony formation assay results is provided, n = 3, ∗∗∗ p < 0.001. (E) The results of the EdU assay in HCT116-MCS and HCT116-FAM65A cells are shown. Scale bars, 100 μm. (F) A quantitative analysis of the EdU assay results is included, n = 3, ∗∗∗ p < 0.001. (G) The findings from the apoptosis assay in HCT116-MCS and HCT116-FAM65A cells are presented. (H) A quantitative analysis of the apoptosis assay results is provided, n = 3, ∗∗∗ p < 0.001. (I) The results of the transwell migration assay for HCT116-MCS and HCT116-FAM65A cells are reported. Scale bars, 50 μm. (J) A quantitative analysis of the transwell migration assay results is included, n = 3, ∗∗∗ p < 0.001. (K) The outcomes of the wound healing assay in HCT116-MCS and HCT116-FAM65A cells are presented. Scale bars, 50 μm. (L) A quantitative analysis of the wound healing assay results is provided, n = 3, ∗∗ p < 0.01. (M) The western blot analysis illustrates the expression levels of Ki-67, cleaved caspase 3, Bcl-2, Bax, E-cadherin, N-cadherin, β-catenin, and vimentin in HCT116-MCS and HCT116-FAM65A cells. Data are presented as mean ± SEM of biologically independent experiments.

Article Snippet: HCT116 cell line , ATCC , CCL-247.

Techniques: Expressing, Migration, In Vitro, Western Blot, Over Expression, CCK-8 Assay, Colony Assay, EdU Assay, Apoptosis Assay, Transwell Migration Assay, Wound Healing Assay

Journal: iScience

Article Title: FAM65A, as a potential predictor of prognosis, promotes colorectal cancer progression via activating Ras/ERK/RSK signaling

doi: 10.1016/j.isci.2026.114662

Figure Lengend Snippet: FAM65A promotes CRC tumor progression is dependent on RSK phosphorylation (A) Results from protein kinase microarray analysis of HCT116-MCS and HCT116-FAM65A cells. (B) Quantitative assessment of the protein kinase microarray data, n = 2. (C) Western blot analysis demonstrating the expression levels of p -RSK and total RSK in HCT116-MCS and HCT116-FAM65A cells. (D) Western blot analysis of p -RSK and RSK expression in HCT116-FAM65A cells treated with 4 and 8 μM BRD7389 or without treatment. (E) Results from the CCK8 cell proliferation assay conducted on HCT116-FAM65A cells with and without the application of BRD7389, n = 3, ∗∗ p < 0.01, ∗∗∗ p < 0.001. (F) Outcomes of the colony formation assay performed on HCT116-FAM65A cells treated with BRD7389 or without treatment. (G) Quantitative analysis of the colony formation assay results, n = 3, ∗∗∗ p < 0.001. (H) Results from the EdU assay conducted on HCT116-FAM65A cells with and without the application of BRD7389. Scale bars, 100 μm. (I) Quantitative analysis of the EdU assay results, n = 3, ∗∗∗ p < 0.001. (J) Western blot analysis of Ki-67 expression in HCT116-FAM65A cells treated with BRD7389 or not. (K) Results from the apoptosis assay conducted on HCT116-FAM65A cells treated with BRD7389 or not. Scale bars, 50 μm. (L) Quantitative analysis of the apoptosis experiments, n = 3, ∗∗∗ p < 0.001. (M) Western blot analysis of the expression levels of cleaved Caspase 3, Bcl-2, and Bax in HCT116-FAM65A cells treated with BRD7389 or not. (N) Results from the Transwell migration assay conducted on HCT116-FAM65A cells with and without the application of BRD7389. Scale bars, 50 μm. (O) Quantitative analysis of the Transwell migration assay results, n = 3, ∗∗∗ p < 0.001. (P) Results from the wound healing assay performed on HCT116-FAM65A cells treated with BRD7389 or not. Scale bars, 50 μm. (Q) Quantitative analysis of the wound healing assay results, n = 3, ∗∗ p < 0.01, ∗∗∗ p < 0.001. (R) Western blot analysis the expression of EMT markers in HCT116-FAM65A cells treated with BRD7389 or not. Data are presented as mean ± SEM of biologically independent experiments.

Article Snippet: HCT116 cell line , ATCC , CCL-247.

Techniques: Phospho-proteomics, Microarray, Western Blot, Expressing, Proliferation Assay, Colony Assay, EdU Assay, Apoptosis Assay, Transwell Migration Assay, Wound Healing Assay

Journal: iScience

Article Title: FAM65A, as a potential predictor of prognosis, promotes colorectal cancer progression via activating Ras/ERK/RSK signaling

doi: 10.1016/j.isci.2026.114662

Figure Lengend Snippet: Ras/ERK signaling activation was indispensable for FAM65A-mediated RSK activation and CRC progression (A) Western blot analysis of Ras and p -ERK expression in HCT116-FAM65A cells treated with 10 μM Abd-7, or without treatment. (B) Results from the CCK8 cell proliferation assay conducted on HCT116-FAM65A cells with and without the application of Abd-7, n = 3, ∗∗∗ p < 0.001. (C) Colony formation assay performed on HCT116-FAM65A cells treated with Abd-7 or not. (D) Quantitative analysis of the colony formation assay results, n = 3, ∗∗∗ p < 0.001. (E) Results from the EdU assay conducted on HCT116-FAM65A cells with and without the application of Abd-7. Scale bars, 100 μm. (F) Quantitative analysis of the EdU assay results, n = 3, ∗∗∗ p < 0.001. (G) Western blot analysis of Ki-67, cleaved Caspase 3, Bcl-2, and Bax expression in HCT116-FAM65A cells treated with Abd-7 or not. (H) Results from the apoptosis assay conducted on HCT116-FAM65A cells treated with Abd-7 or not. Scale bars, 50 μm. (I) Quantitative analysis of the apoptosis experiments, n = 3, ∗∗∗ p < 0.001. (J) Results from the Transwell migration assay conducted on HCT116-FAM65A cells with and without the application of Abd-7. Scale bars, 50 μm. (K) Quantitative analysis of the Transwell migration assay results, n = 3, ∗∗∗ p < 0.001. (L) Results from the wound healing assay performed on HCT116-FAM65A cells treated with Abd-7 or not. Scale bars, 50 μm. (M) Quantitative analysis of the wound healing assay results, n = 3, ∗∗∗ p < 0.001. (N) Western blot analysis the expression of EMT markers in HCT116-FAM65A cells treated with Abd-7 or not. (O) Proposed model of FAM65A in CRC progression. Data are presented as mean ± SEM of biologically independent experiments.

Article Snippet: HCT116 cell line , ATCC , CCL-247.

Techniques: Activation Assay, Western Blot, Expressing, Proliferation Assay, Colony Assay, EdU Assay, Apoptosis Assay, Transwell Migration Assay, Wound Healing Assay

Journal: iScience

Article Title: A nuclear isoform of hydroxyacyl-COA dehydrogenase inhibits tumor progression in colorectal cancer

doi: 10.1016/j.isci.2025.114470

Figure Lengend Snippet: HADH-S is decreased in human colorectal cancer tissue (A) Immunofluorescence analysis of endogenous HADH expression using anti-HADH antibody in RKO and HCT116 cells. Scale bars, 10 μm. (B) Immunoblotting assessment of nuclear, cytosolic, and mitochondrial fractions of RKO and HCT116 cells. (C) The expression of HADH in different grade CRC tumors from the TCGA database. (D) Kaplan-Meier curves depict the overall survival rate in patients with colon cancer with high or low expression of HADH from the TCGA database. (E) Representative IHC staining of HADH in colorectal cancer tissues and para-cancer tissues. Scale bars, 50 μm. The red arrows indicate nuclear staining of HADH. (F) Comparison of HADH IRS in CRC tissues and paired para-cancer tissues (mean ± SD; two-tailed one-way analysis of variance). (G) Nuclear staining of HADH in CRC tissues and paired para-cancer tissues (mean ± SD; ∗ p < 0.05; two-tailed t test).

Article Snippet: HCT116 , ATCC , Cat#CCL-247.

Techniques: Immunofluorescence, Expressing, Western Blot, Immunohistochemistry, Staining, Comparison, Two Tailed Test

Journal: iScience

Article Title: A nuclear isoform of hydroxyacyl-COA dehydrogenase inhibits tumor progression in colorectal cancer

doi: 10.1016/j.isci.2025.114470

Figure Lengend Snippet: HADH-S inhibits the tumorigenesis of colorectal cancer (A) The proliferation assay of RKO cells stably expressing HADH, HADH-L, or HADH-S. Cell number at day 4 was statistically analyzed. (B) EdU incorporation assay in RKO expressing HADH, HADH-L, or HADH-S. Scale bars, 20 μm. The analysis of EdU incorporation data is shown in the right panel. (C) Immunoblotting analysis of FLAG-tagged HADH, HADH-L, and HADH-S in HCT116 cells. (D) The proliferation assay of HCT116 cells expressing HADH, HADH-L, or HADH-S. Cell number at day 4 was statistically analyzed. (E) EdU incorporation assay in HCT116 expressing HADH, HADH-L, or HADH-S. (F) Immunoblotting analysis of HADH knockdown in HCT116 cells. (G) The proliferation assay of HCT116 cells with or without HADH knockdown. Cell number at day 4 was statistically analyzed. (H) EdU incorporation assay in HCT116 with or without HADH knockdown. (I) Colony formation assay of HCT116 cells expressing HADH, HADH-L, or HADH-S. The analysis is shown in the right panel. (J) The volume of xenograft tumors from mice implanted with the indicated HCT116 cells. (K) The images of xenograft tumors from mice implanted with the indicated HCT116 cells. The analysis of tumor weight is shown in the right panel. All the data are presented as mean ± SD. For panels G and H, a two-tailed t test was used. For panels A, B, D, E, I, J, and K, one-way ANOVA was used to compare multiple groups.∗ p < 0.05, ∗∗ p < 0.01, ns not significant.

Article Snippet: HCT116 , ATCC , Cat#CCL-247.

Techniques: Proliferation Assay, Stable Transfection, Expressing, Western Blot, Knockdown, Colony Assay, Two Tailed Test

Journal: Nucleic Acids Research

Article Title: Novel intermolecular zinc fingers and redox-driven conformational changes dictate tumor suppressor ZMYND11’s role in cooperative recognition of diverse targets

doi: 10.1093/nar/gkag048

Figure Lengend Snippet: The WH-PHD domains of ZMYND11 are required for the inhibition of colorectal cancer cell in vitro proliferation. (A) Schematic representation of ZMYND11 domain organization. (B) ZMYND11 expression across cancer types analyzed using GEPIA2 . (C) 5-Ethynyl-2′-deoxyuridine (EdU) incorporation assay in HCT116 cells transfected with pcDNA3.1 (empty vector), pcDNA3.1-ZMYND11, or pcDNA3.1-ZMYND11-mut (ΔWP) (WH-PHD deletion mutant). Nuclei were counterstained with 4',6-diamidino-2-phenylindole (DAPI). Scale bar, 50 μm. (D) Quantification of EdU-positive cells. Comparisons were made relative to the ZMYND11 group: empty vector ( P = 2.64 × 10⁻ 7 ) and ZMYND11-mut (ΔWP) ( P = 0.0004). n = 14 independent samples. (E) CCK-8 cell proliferation assay of HCT116 cells transfected as in (C) . n = 8 independent samples. Data are presented as mean ± standard error of the mean (SEM). * P <.05; ** P <.01; *** P <.001.

Article Snippet: HCT116 cell lines were purchased from the American Type Culture Collection (ATCC) (Manassas, VA) and cultured in IMDM supplemented with 10% Fetal Bovine Serum.

Techniques: Inhibition, In Vitro, Expressing, Transfection, Plasmid Preparation, Mutagenesis, CCK-8 Assay, Proliferation Assay

Journal: Nucleic Acids Research

Article Title: Novel intermolecular zinc fingers and redox-driven conformational changes dictate tumor suppressor ZMYND11’s role in cooperative recognition of diverse targets

doi: 10.1093/nar/gkag048

Figure Lengend Snippet: The WH–PHD domains of ZMYND11 suppress colorectal cancer growth in vivo . (A) Representative xenograft tumors collected 6 weeks after subcutaneous transplantation of HCT116 cells transfected with pcDNA3.1 (empty vector), pcDNA3.1-ZMYND11, or pcDNA3.1-ZMYND11-mut (ΔWP). (B) Tumor weight quantification. Tumors from the ZMYND11 group were significantly smaller than those from the empty vector ( P = 0.0039) or ZMYND11-mut (ΔWP) ( P = 0.0061) groups. n = 5 mice per group. Data are shown as mean ± SEM. * P <.05; ** P <.01; *** P <.001.

Article Snippet: HCT116 cell lines were purchased from the American Type Culture Collection (ATCC) (Manassas, VA) and cultured in IMDM supplemented with 10% Fetal Bovine Serum.

Techniques: In Vivo, Transplantation Assay, Transfection, Plasmid Preparation

Journal: Nucleic Acids Research

Article Title: Novel intermolecular zinc fingers and redox-driven conformational changes dictate tumor suppressor ZMYND11’s role in cooperative recognition of diverse targets

doi: 10.1093/nar/gkag048

Figure Lengend Snippet: Diverse interactions mediated by the ZMYND11 PHD domain. (A) The surface potential of the ZMYND11 PHD dimer generated by the APBS electrostatics tool of PyMOL (unit: kT/e) was displayed as a color gradient ranging from red (negative) to blue (positive). (B) MBP pull-down assay demonstrating binding interactions between individual ZMYND11 domains and nucleosomes. (C) Co-IP revealing endogenous interaction between ALKBH6 and ZMYND11 in HCT116 cells using anti-HA and anti-Flag antibodies. (D) Immunohistochemical staining of colorectal cancer tissue microarrays showing nuclear and cytoplasmic co-localization of ALKBH6 (red) and ZMYND11 (green). Nuclei stained blue. n = 3 independent samples. Scale bar: 100 μm. (E) GST pull-down assay indicating that double mutations in PHD zinc finger sites weaken the interaction between ZMYND11 and ALKBH6. (F) GST pull-down assays showing direct interaction of individual ZMYND11 domains (PHD and CC-MYND) with ALKBH6 in vitro . H6: ALKBH6. (G) MST analysis of WPBP WT and WPBP C103A,C106A binding to dsDNA2. (H) MST analysis of WPBP WT and WPBP C103A,C106A binding to nucleosomes. All experiments were repeated at least three times with similar results. Data are presented as mean ± SEM.

Article Snippet: HCT116 cell lines were purchased from the American Type Culture Collection (ATCC) (Manassas, VA) and cultured in IMDM supplemented with 10% Fetal Bovine Serum.

Techniques: Generated, Pull Down Assay, Binding Assay, Co-Immunoprecipitation Assay, Immunohistochemical staining, Staining, In Vitro

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