Journal: PLOS Genetics

Article Title: Co-depletion of NIPBL and WAPL balance cohesin activity to correct gene misexpression

doi: 10.1371/journal.pgen.1010528

Figure Lengend Snippet: (A) Cartoon depicting the roles of the two opposing cohesin regulators; NIPBL loads cohesin onto chromatin and is required for loop extrusion whereas WAPL opens the ring and removes it. (B) Fluorescent western blot to NIPBL (top band, see arrow) and WAPL in nuclear (nuc) and chromatin-bound (chr) subcellular protein fractionations of RNAi control, NIPBL, or WAPL depleted HCT116 cells. All bands are from the same blot. (C) Mean fold change (%) of NIPBL and WAPL bound to chromatin in each respective knockdown. Each symbol represents a biological replicate, error bars represent standard deviation. (D) Fluorescent western blot to RAD21 in nuclear (nuc) and chromatin-bound (chr) subcellular protein fractionations of RNAi control, NIPBL, or WAPL depleted HCT116 cells. All bands are from the same blot. (E) Mean fold change (%) of RAD21 bound to chromatin in each respective knockdown. Each symbol represents a biological replicate, error bars represent standard deviation. (F) Cell growth measured in 24-hour increments following RNAi or auxin treatment. Each bar represents the mean of 3 biological replicates and error bars represent the standard deviation. (G) Representative immunofluorescence images of mitotic cells stained for α-tubulin (cyan) and phospho-Histone H3 (PH3; red). Top row are HCT116 cells following 72 hour treatment with RNAi against control, NIPBL, or WAPL. Bottom row are HCT116-RAD21-AID cells -/+ auxin for 6 or 24 hours. Scale bar, 5μm. (H) Average percentage of abnormal mitotic cells in RNAi control, NIPBL, or WAPL depleted HCT116 cells and HCT116-RAD21-AID cells -/+ auxin for 6 or 24 hours. Each symbol represents a biological replicate, error bars represent standard deviation. (I) Oligopaint design for three neighboring domains at chr2:217-222Mb. (J) Representative FISH images for three domains at chr2:217-222Mb in RNAi control, NIPBL, and WAPL depleted HCT116 cells. Dashed line represents nuclear edge, scale bar, 5μm (above) or 1μm (below). (K) Cumulative frequency distribution of overlap between the neighboring domains D1 and D2 on chr2 in RNAi control (n = 1,170 chromosomes), NIPBL (n = 1,177 chromosomes), or WAPL (n = 1,136 chromosomes) depleted HCT116 cells. Two-tailed Mann-Whitney test, **** p < 0.0001. (L) Cumulative frequency distribution of overlap between the neighboring domains D2 and D3 on chr2 in RNAi control (n = 1,202 chromosomes), NIPBL (n = 1,284 chromosomes), or WAPL (n = 1,149 chromosomes) depleted HCT116 cells. Two-tailed Mann-Whitney test, **** p < 0.0001. (M) Change in contact frequency across 18 domain pairs in NIPBL, or WAPL depleted HCT116 cells and HCT116-RAD21-AID cells treated with auxin for 6 hours. Each dot represents the median of ≥ 4 biological replicates at each locus.

Article Snippet: For experiments in the HCT116-RAD21-AID cell line, 7.5x10 4 cells in supplemented McCoy’s 5A media -/+ 500 μM auxin were seeded in 384-well plates (Perkin Elmer 6057300) and incubated at 37°C for 6 h. For RNAi experiments in the HCT116 cell line, plates were seeded with siRNA (see RNAi section for details) diluted in Opti-MEM reduced serum medium to a final concentration of 25nM per well.

Techniques: Western Blot, Control, Knockdown, Standard Deviation, Immunofluorescence, Staining, Two Tailed Test, MANN-WHITNEY

Journal: PLOS Genetics

Article Title: Co-depletion of NIPBL and WAPL balance cohesin activity to correct gene misexpression

doi: 10.1371/journal.pgen.1010528

Figure Lengend Snippet: (A) Fluorescent western blot to NIPBL (top band, see arrow) and WAPL in nuclear (nuc) and chromatin-bound (chr) subcellular protein fractionations of RNAi control, NIPBL, WAPL, and double knockdown (dKD) depleted HCT116 cells. All bands are from the same blot with different exposures to optimize band detection. (B) Mean fold change (%) of NIPBL and WAPL bound to chromatin in the double knockdown condition. Each symbol represents a biological replicate, error bars represent standard deviation. (C) Fluorescent western blot to RAD21 in nuclear (nuc) and chromatin-bound (chr) subcellular protein fractionations of RNAi control and NIPBL and WAPL double knockdown (dKD) depleted HCT116 cells. All bands from the same blot. (D) Mean fold change (%) of RAD21 bound to chromatin in RNAi control, NIPBL, WAPL, and double knockdown (dKD) depleted HCT116 cells. Each symbol represents a biological replicate, error bars represent standard deviation. (E) Representative FISH images for three domains at chr2:217-222Mb in RNAi control, NIPBL, WAPL, and NIPBL and WAPL co-depleted HCT116 cells. Dashed line represents nuclear edge, scale bar, 5μm (above) or 1μm (below). (F) Cumulative frequency distribution of overlap between the neighboring domains D1 and D2 on chr2 in RNAi control (n = 2,172 chromosomes), NIPBL (n = 1,514 chromosomes), WAPL (n = 1,704 chromosomes), or dKD (n = 1,620 chromosomes) depleted HCT116 cells. Two-tailed Mann-Whitney test, **** p < 0.0001, ns = not significant (p = 0.79). (G) Cumulative frequency distribution of overlap between the neighboring domains D2 and D3 on chr2 in RNAi control (n = 2,188 chromosomes), NIPBL (n = 1,571 chromosomes), WAPL (n = 1,719 chromosomes), or dKD (n = 1,661 chromosomes) depleted HCT116 cells. Two-tailed Mann-Whitney test, **** p < 0.0001, ** p = 0.0014. (H) Change in contact frequency across 18 domain pairs in HCT116 cells depleted for NIPBL, WAPL, or both. Each dot represents the median of ≥ 4 biological replicates at each locus.

Article Snippet: For experiments in the HCT116-RAD21-AID cell line, 7.5x10 4 cells in supplemented McCoy’s 5A media -/+ 500 μM auxin were seeded in 384-well plates (Perkin Elmer 6057300) and incubated at 37°C for 6 h. For RNAi experiments in the HCT116 cell line, plates were seeded with siRNA (see RNAi section for details) diluted in Opti-MEM reduced serum medium to a final concentration of 25nM per well.

Techniques: Western Blot, Control, Knockdown, Standard Deviation, Two Tailed Test, MANN-WHITNEY

Journal: Biomedicines

Article Title: Small Molecule 20S Proteasome Enhancer Regulates MYC Protein Stability and Exhibits Antitumor Activity in Multiple Myeloma

doi: 10.3390/biomedicines10050938

Figure Lengend Snippet: MYC-luciferase reporter assay: ( A ) MYC-luciferase reporter assay in stable transfected HCT-116 cells treated with various concentrations of TCH-165 (EC 50 2.57 μM; 95% CI 2.46–2.95); ( B ) MYC-luciferase reporter assay in stably transfected HCT-116 cells treated with vehicle, TCH-165 (3 μM), bortezomib (BTZ, 10 nM) and bortezomib (2 h pre-incubation, 10 nM) followed by TCH-165 (3 μM). *** p < 0.001.

Article Snippet: The MYC Reporter (Luc)-HCT-116 Cell line, Catalog #60520, was bought from BPS Bioscience.

Techniques: Luciferase, Reporter Assay, Transfection, Stable Transfection, Incubation

Journal: bioRxiv

Article Title: A scalable tumor-vasculature-on-chip for CAR T cell trafficking and efficacy studies

doi: 10.64898/2026.02.05.703975

Figure Lengend Snippet: (A) Schematic representation of the concept of immune checkpoint inhibition. Interaction between PD-1 and PD-L1 is blocked by Nivolumab which restores release of cytotoxic molecules by the CAR T cell (red) (B) Heatmap showing an overview of changes in tumor area and release of Granzyme B, IL-6, sPD-L1 and TNFα in HT-29-GFP and HCT116-GFP co-cultures in response to both single and combination treatments with Nivolumab, Ipilimumab and Temozolomide. Data used to generate the heatmap can be found in supplementary figures 7 and 8. (C,D) PCA plots showing response of HT-29-GFP and HCT116-GFP co-cultures to EpCAM-CD28 CAR T cells and treatments. (E) PCA plot showing the response of HT-29-GFP and HCT116-GFP co-cultures to combination treatments. Datapoints are scaled for the initial tumor cell density at the start of the co-culture. Pink circle indicates a small cluster of replicates consisting of HCT116-GFP cultures exposed to Nivolumab, Ipilimumab and Temozolomide. (F) Contribution and directionality of the different features of the principal components. (G) Heatmap showing morphometric parameters measured in the endothelial vessels of HT-29-GFP and HCT116-GFP co-cultures exposed to EpCAM-CD28 CART cells and treatments. Shown are fold changes against untreated controls. (H) Correlation distance matrix showing similarity between treatment responses based on patterns in relative change in tumor area, cytokine release and endothelial response. Data are presented as unsquared correlation distances, with values <1 indicating similarity in response while values >1 indicate distinct responses. A value of 1 means no relation between observed responses and a value of 0 indicates an identical response.

Article Snippet: Human colorectal cancer cell line HCT116-GFP (GeneCopoeia, SL024) was cultured in RPMI-1640 (Gibco, 11875093) supplemented with 10% Fetal Bovine Serum and 1 % Penicillin-Streptomycin.

Techniques: Inhibition, Co-Culture Assay

Journal: bioRxiv

Article Title: A scalable tumor-vasculature-on-chip for CAR T cell trafficking and efficacy studies

doi: 10.64898/2026.02.05.703975

Figure Lengend Snippet: (A) HCT116-GFP tumor cells proliferate after introduction in the lumenised ECM and cause regression of the endothelial vessel over time. The fluorescent signal could be used to create a binary mask that was used to track growth of HCT116-GFP tumor cells from day 5 until day 8. Fluorescent area at each timepoint was normalized against the area at t=1 for each individual replicate (n=15). (B) Effect of treatments on HT-29-GFP tumor area. Data was normalized against the untreated control at each timepoint (n=8-16). (C) Effect of treatments on HCT116-GFP tumor area. Data was normalized against the untreated control at each timepoint (n=8-14). Data included in the graphs are presented as mean ± SD.

Article Snippet: Human colorectal cancer cell line HCT116-GFP (GeneCopoeia, SL024) was cultured in RPMI-1640 (Gibco, 11875093) supplemented with 10% Fetal Bovine Serum and 1 % Penicillin-Streptomycin.

Techniques: Control

Journal: bioRxiv

Article Title: A scalable tumor-vasculature-on-chip for CAR T cell trafficking and efficacy studies

doi: 10.64898/2026.02.05.703975

Figure Lengend Snippet: (A) Analysis of cytokine levels (pg/mL) in media samples after 72 hours of co-culture of HT-29-GFP and HCT116-GFP tumor cells with EpCAM-CD28 CAR T cells. Co-cultures were untreated or exposed to Nivolumab, Ipilimumab and/or Temozolomide (n=8-14). (B) Cytokine levels in panel A were normalized against untreated controls per experiment resulting in fold changes that were used for principal component analysis (n=8-14). Data included in the graphs are presented as mean ± SD.

Article Snippet: Human colorectal cancer cell line HCT116-GFP (GeneCopoeia, SL024) was cultured in RPMI-1640 (Gibco, 11875093) supplemented with 10% Fetal Bovine Serum and 1 % Penicillin-Streptomycin.

Techniques: Co-Culture Assay

Journal: bioRxiv

Article Title: A scalable tumor-vasculature-on-chip for CAR T cell trafficking and efficacy studies

doi: 10.64898/2026.02.05.703975

Figure Lengend Snippet: (A) Endothelial vessels were co-cultured with either HT-29-GFP or HCT116-GFP tumor cells and stained with VE-cadherin after the 72-hour co-culture. Co-cultures were either untreated or exposed to single or combination treatments with Nivolumab, Ipilimumab and Temozolomide in the presence of EpCAM-CD28 CAR T cells (E:T = 1:1). The endothelial response was assessed by analysis of VE-cadherin objects using a range of different morphological and spatial descriptors using IN Carta® (n=7-14) and normalization against respective untreated controls containing CAR T cells for each parameter. Data are presented as mean ± SD. (B) Heatmap showing change in morphometric parameters measured in endothelial vessels, co-cultured with either HT-29-GFP or HCT116-GFP tumor cells, upon addition of EpCAM-CD28 CAR T cells (E:T = 1:1). Data is presented as fold changes against respective controls without CAR T cells.

Article Snippet: Human colorectal cancer cell line HCT116-GFP (GeneCopoeia, SL024) was cultured in RPMI-1640 (Gibco, 11875093) supplemented with 10% Fetal Bovine Serum and 1 % Penicillin-Streptomycin.

Techniques: Cell Culture, Staining, Co-Culture Assay

Journal: Oncotarget

Article Title: Humanized anti-hepatocyte growth factor (HGF) antibody suppresses innate irinotecan (CPT-11) resistance induced by fibroblast-derived HGF

doi:

Figure Lengend Snippet: A. Human lung and colorectal cancer cells were plated into 96-well plates at a density of 3 × 10 3 cells/well on day 0. Under serum free culture media (black bar), cells were incubated with CCD-18co colonic fibroblast CM (white bar) and/or CPT-11 for 48 h. Inhibition of cell proliferation was determined by MTT assay. Significant differences were evaluated using an unpaired two-tailed Student's t -test. (Error bars denote the standard deviation [SD]) (* p < 0.05, ** p < 0.01 and *** p < 0.001). B. Induction of apoptosis in colorectal cancer cells. Cells were stained with PI and Annexin V-FITC, followed by flow cytometry analysis. Cells were treated with CPT-11 under serum free media or CCD-18co colonic fibroblast CM. The apoptotic cells were determined by Annexin V-FITC positive staining.

Article Snippet: The human colorectal cancer cell lines, HCT116, HT29 and DLD-1 and lung cancer cell line 226Br were cultured in RPMI1640 (Welgene, Seoul, Korea) containing 10% fetal bovine serum (FBS; Welgene) and antibiotics (100 mg/L penicillin and 100 mg/L streptomycin; GIBCO-BRL Life Technologies; Gaithersburg, MD, USA).

Techniques: Incubation, Inhibition, MTT Assay, Two Tailed Test, Standard Deviation, Staining, Flow Cytometry

Journal: Oncotarget

Article Title: Humanized anti-hepatocyte growth factor (HGF) antibody suppresses innate irinotecan (CPT-11) resistance induced by fibroblast-derived HGF

doi:

Figure Lengend Snippet: A. HGF secreted by cancer cells (HCT-116 and DLD-1) and colonic fibroblasts (CCD-18co) were measured. Cells were cultured with serum free medium for 24 h and HGF concentrations were determined by ELISA. B. CM from fibroblast activates c-MET receptors. HCT-116 and DLD-1 cells were cultured with serum free media or CCD-18co CM for 1 h. Cells were collected, and the indicated proteins were detected by western blotting. C. Colonic fibroblast cells promote CPT-11 resistance of colorectal cancer cells (HCT-116 and DLD-1). Cancer cells were cultured with (white bar) or without (black bar) CCD-18co cells, in the presence or absence of CPT-11 (1.25-20 μM) for 48 h, and inhibition of cell proliferation was determined by MTT assay. Significant differences were evaluated using an unpaired two-tailed Student's t -test. (Error bars denote the standard deviation [SD]) (* p < 0.05 and *** p < 0.001). D. Co-culture with colonic fibroblast CCD-18co cells increases c-MET receptor activation in colorectal cancer cells. HCT-116 and DLD-1 cells were co-cultured with CCD-18co cells for 24 h. Lysates were analyzed for c-MET activation by western blotting. E. Inhibition of HGF production from fibroblast by transfection with HGF siRNA. Colonic fibroblast cells were transfected with 10 nM HGF siRNA or scramble siRNA. After transfection, cells were collected and lysates were submitted to Western blotting to quantify HGF. F. HCT-116 and DLD-1 cells were cultured with CM from HGF siRNA transfected fibroblast for 48 h in the presence or absent of CPT-11. Cell viability was determined by MTT assay. Significant differences were evaluated using an unpaired two-tailed Student's t -test. (Error bars denote the standard deviation [SD]) (* p < 0.05, ** p < 0.01 and *** p < 0.001).

Article Snippet: The human colorectal cancer cell lines, HCT116, HT29 and DLD-1 and lung cancer cell line 226Br were cultured in RPMI1640 (Welgene, Seoul, Korea) containing 10% fetal bovine serum (FBS; Welgene) and antibiotics (100 mg/L penicillin and 100 mg/L streptomycin; GIBCO-BRL Life Technologies; Gaithersburg, MD, USA).

Techniques: Cell Culture, Enzyme-linked Immunosorbent Assay, Western Blot, Inhibition, MTT Assay, Two Tailed Test, Standard Deviation, Co-Culture Assay, Activation Assay, Transfection