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: PLoS ONE

Article Title: Use of Fluorescence Lifetime Imaging Microscopy (FLIM) as a Timer of Cell Cycle S Phase

doi: 10.1371/journal.pone.0167385

Figure Lengend Snippet: (a,b) Images of live synchronized HCT116 cells released from APH block without (a) and with (b) BrdU labeling (100 μM, 4 h), stained with HXT (1 μM, 30 min). Scale bar is 50 μm. (c) Representative examples of HXT fluorescence decays (data trace) for individual pixels in selected nuclei (indicated by circles on (b)) showing mono- and double-exponential fittings. (d) Average τ m (left) and intensity (right) signals for no BrdU (red, n = 12) and +BrdU (blue, n = 15) nuclei. Asterisks indicate significant difference between groups (p < 0.05): *—p < 0.001, **—p < 0.00001. Error bars show the standard deviation.

Article Snippet: Tumor spheroids were formed by seeding HCT116 cells on a Lipidure-coat TM plate (Amsbio, UK) at concentration of 200 cells/ well, and by growing them for 4 days.

Techniques: Blocking Assay, Labeling, Staining, Fluorescence, Standard Deviation

Journal: PLoS ONE

Article Title: Use of Fluorescence Lifetime Imaging Microscopy (FLIM) as a Timer of Cell Cycle S Phase

doi: 10.1371/journal.pone.0167385

Figure Lengend Snippet: ( a) Asynchronous and synchronized live HCT116 cells were incubated with BrdU (100 μM, 4 h) and stained with HXT (1 μM, 30 min). Immediately after FLIM cells were fixed with 4% paraformaldehyde and stained with anti-BrdU antibody. Scale bar is 50 μm. (b) Average (n = 5) distributions of τ m . Black arrow indicates threshold τ m , which differentiates between S phase and non S-phase cells. (c) Cell proliferation rates calculated by the different methods. Bar chart shows fractions of total cell numbers and standard deviation for +BrdU cells (S-phase). The mean values were calculated from five different images of the asynchronous and synchronized cell cultures.

Article Snippet: Tumor spheroids were formed by seeding HCT116 cells on a Lipidure-coat TM plate (Amsbio, UK) at concentration of 200 cells/ well, and by growing them for 4 days.

Techniques: Incubation, Staining, Standard Deviation

Journal: Non-coding RNA Research

Article Title: Telomerase RNA component lncRNA as potential diagnostic biomarker promotes CRC cellular migration and apoptosis evasion via modulation of β-catenin protein level

doi: 10.1016/j.ncrna.2023.03.004

Figure Lengend Snippet: LncRNAs profiling in CRC cell lines. Volcano plot of the pairwise comparison of lncRNAs expression in (A) HCT116 cell line and (B) SW620 cell line versus control cell line. Overexpressed lncRNAs are shown to the right of the plot (green) and were only selected if they passed the thresholds of false discovery rate (FDR) > 2 (horizontal blue line) and log2 fold change >1 (right vertical line). Accordingly, down-regulated lncRNAs are shown to the left of the plot (yellow) and were only selected if they passed the thresholds of FDR >2 (horizontal blue line) and log2 fold change <-1 (left vertical line). (C) Clustering graph of the lncRNA different expression level between the CRC cell lines. Significant from control at p < 0.05 and analyzed by two-way ANOVA followed by Tukey's multiple comparisons test.

Article Snippet: Flow cytometry HCT116 cells transfected with si-TERC and si-NC were harvested 48 h later for cell cycle analysis Phycoerythrin was used to stain cells with the PE-Texas Red™ Antibody Labeling Kit (Novus Biologicals™ 7670030).

Techniques: Comparison, Expressing, Control

Journal: Non-coding RNA Research

Article Title: Telomerase RNA component lncRNA as potential diagnostic biomarker promotes CRC cellular migration and apoptosis evasion via modulation of β-catenin protein level

doi: 10.1016/j.ncrna.2023.03.004

Figure Lengend Snippet: The wound healing was used for migration assay. (A) Images taken to HCT116 control cells, si-NC transfected cells, and si-TERC transfected cells at different time intervals. (B) The mean ± SD of wound width was significantly larger in si-TERC transfected cells than control and si-NC after 24 h. (C) The mean ± SD of migration rate was significantly decreased in si-TERC transfected cells than control and si-NC after 48 h. (D) The mean ± SD of wound area was significantly larger in si-TERC transfected cells than control and si-NC after 24 h. (E) The mean ± SD of wound closure was significantly decreased in si-TERC transfected cells than control and si-NC after 48 h *Significantly different in compare to control and si-NC at p < 0.05. This data was assessed by two-way ANOVA followed by Tukey's multiple comparisons test.

Article Snippet: Flow cytometry HCT116 cells transfected with si-TERC and si-NC were harvested 48 h later for cell cycle analysis Phycoerythrin was used to stain cells with the PE-Texas Red™ Antibody Labeling Kit (Novus Biologicals™ 7670030).

Techniques: Migration, Control, Transfection

Journal: Non-coding RNA Research

Article Title: Telomerase RNA component lncRNA as potential diagnostic biomarker promotes CRC cellular migration and apoptosis evasion via modulation of β-catenin protein level

doi: 10.1016/j.ncrna.2023.03.004

Figure Lengend Snippet: (A) Cell cycle analysis of HCT116 cell lines. (B) The mean ± SD of number of HCT116 cells were significantly increased in the G0/G1 phase after lncRNA TERC knockdown in comparing to control and si-N. In addition, the mean ± SD of number of HCT116 cells were significantly decreased in the S phase in comparing to control and si-NC. *Significantly different in compare to control and si-NC at p < 0.05. This data was assessed using two-way ANOVA followed by Tukey's multiple comparisons test.

Article Snippet: Flow cytometry HCT116 cells transfected with si-TERC and si-NC were harvested 48 h later for cell cycle analysis Phycoerythrin was used to stain cells with the PE-Texas Red™ Antibody Labeling Kit (Novus Biologicals™ 7670030).

Techniques: Cell Cycle Assay, Knockdown, Control

Journal: Non-coding RNA Research

Article Title: Telomerase RNA component lncRNA as potential diagnostic biomarker promotes CRC cellular migration and apoptosis evasion via modulation of β-catenin protein level

doi: 10.1016/j.ncrna.2023.03.004

Figure Lengend Snippet: (A) Flow cytometric analysis for cell apoptosis. (B) The mean ± SD of percentage of HCT116 apoptotic cells were significantly increased after lncRNA TERC knockdown in comparing to control and si-NC. *Significantly different in compare to control and si-NC at p < 0.001. This data was assessed using two-way ANOVA followed by Tukey's multiple comparisons test.

Article Snippet: Flow cytometry HCT116 cells transfected with si-TERC and si-NC were harvested 48 h later for cell cycle analysis Phycoerythrin was used to stain cells with the PE-Texas Red™ Antibody Labeling Kit (Novus Biologicals™ 7670030).

Techniques: Knockdown, Control

Journal: Non-coding RNA Research

Article Title: Telomerase RNA component lncRNA as potential diagnostic biomarker promotes CRC cellular migration and apoptosis evasion via modulation of β-catenin protein level

doi: 10.1016/j.ncrna.2023.03.004

Figure Lengend Snippet: β-catenin expression pattern in transfected HCT 116 cell line (A) The mean ± SD expression fold change of β-catenin mRNA in HCT 116 cell lines (B) Normalized level in TERC knockdown HCT116 cells and control cells. *Significantly different in compare to control and si-NC at p < 0.001. This data was assessed using two-way ANOVA followed by Tukey's multiple comparisons test.

Article Snippet: Flow cytometry HCT116 cells transfected with si-TERC and si-NC were harvested 48 h later for cell cycle analysis Phycoerythrin was used to stain cells with the PE-Texas Red™ Antibody Labeling Kit (Novus Biologicals™ 7670030).

Techniques: Expressing, Transfection, Knockdown, Control

Journal: Cancers

Article Title: Extracellular Vesicles in Colorectal Cancer: From Tumor Growth and Metastasis to Biomarkers and Nanomedications

doi: 10.3390/cancers15041107

Figure Lengend Snippet: Partial list of proteins identified in exosomes with evidence of biological effects and clinical implications in colorectal cancer.

Article Snippet: miR-221-3p , Colon cancer cells (HCT116 and Caco-2 cell lines) , Human endothelial cells (HUVECs) , Conditioned medium from HCT116 and Caco-2 cells; centrifugation; ExoQuickTM Exosome Precipitation Solution (System Biosciences) , Increased proliferation and motility of endothelial cells in vitro , Angiogenesis , Exhausts SOCS3, leading to STAT3 signaling pathway and upregulation of VEGFR2 , [ ] .

Techniques: Isolation, Centrifugation, Gradient Centrifugation, Ubiquitin Proteomics, Activation Assay, RNA Binding Assay, Expressing, Migration, Filtration, Mutagenesis

Journal: Cancers

Article Title: Extracellular Vesicles in Colorectal Cancer: From Tumor Growth and Metastasis to Biomarkers and Nanomedications

doi: 10.3390/cancers15041107

Figure Lengend Snippet: Partial list of ncRNAs (miRNAs and lnCRNAs) in exosomes with their biological effects and clinical implications in colorectal cancer.

Article Snippet: miR-221-3p , Colon cancer cells (HCT116 and Caco-2 cell lines) , Human endothelial cells (HUVECs) , Conditioned medium from HCT116 and Caco-2 cells; centrifugation; ExoQuickTM Exosome Precipitation Solution (System Biosciences) , Increased proliferation and motility of endothelial cells in vitro , Angiogenesis , Exhausts SOCS3, leading to STAT3 signaling pathway and upregulation of VEGFR2 , [ ] .

Techniques: Isolation, Transduction, Expressing, Plasmid Preparation, Centrifugation, Migration, In Vitro, Control, Biomarker Discovery, Activation Assay, Membrane, Injection, Filtration, Binding Assay, Permeability, In Vivo, Clinical Proteomics, Activity Assay, Inhibition, Protein-Protein interactions, Transfection, Extraction, Ubiquitin Proteomics, Mutagenesis, Electroporation, Stable Transfection, Gradient Centrifugation, Suspension, Derivative Assay, Purification, Alternative Splicing, RNA Binding Assay, Diagnostic Assay, Translocation Assay