Journal: Bioscience Reports

Article Title: Bafilomycin A1 activates HIF-dependent signalling in human colon cancer cells via mitochondrial uncoupling

doi: 10.1042/bsr20120085

Figure Lengend Snippet: Figure 1 Baf increases respiration in HCT116 cells through mitochondrial uncoupling (A) The O2 profiles in WT and SCO2 −/ −HCT116 cells treated with 0.25 μM Baf at 6 % hypoxia. Cells were loaded with an O2 sensing probe NanO2 and iO2 levels were monitored for 5 h. In control samples respiration was blocked with AA (10 μM). (B) Treatment with Baf partially depolarizes the mitochondrial membrane potential (m) in WT cells, as monitored by TMRM staining (20 nM in solution). (C) In SCO2 −/ −cells the m is reduced, as compared with WT. (D) A reduction in the m in SCO2 −/ −cells is not associated with a decrease in the mitochondrial size, as follows from the comparative analysis of MTG staining and VDAC1 protein levels. Bar represents 20 μm. Asterisks indicate statistical significance.

Article Snippet: Briefly, HCT116 cells were seeded on Ibidi chambers, grown and treated as described above, washed with PBS, fixed with 3.7 % PFA (paraformaldehyde) in PBS, quenched with 50 mM NH4Cl, permeabilized with 0.25 % TX100, blocked with 5 % BSA, incubated with anti-HIF-1α (Novus Biologicals) primary and fluorescent anti-mouse secondary antibodies conjugated with Alexa Fluor555 dye (Invitrogen), counterstained with DAPI, mounted with ProLong® Gold and air-dried.

Techniques: Control, Membrane, Staining

Journal: Bioscience Reports

Article Title: Bafilomycin A1 activates HIF-dependent signalling in human colon cancer cells via mitochondrial uncoupling

doi: 10.1042/bsr20120085

Figure Lengend Snippet: Figure 2 Stabilization of HIF-1α and HIF-2α in HCT116 cells upon Baf treatment (0.25 μM) is associated with mitochon- drial uncoupling (A) Western blot analysis demonstrates significant elevation in HIF-1α and HIF-2α levels in HCT116 WT cells treated with Baf for 5 h at 6 % O2. This effect is abolished by inhibiting mitochondrial respiration with AA. In the presence of DMOG, the level of HIF-1α and HIF-2α stabilization is similar in all samples, including those which are treated with Baf/AA. In the presence of CMA, HIF-1α and HIF-2α levels in WT cells are also increased significantly, yet to a lesser degree than upon Baf treatment. (B) Cellular ATP levels are reduced in WT cells treated with Baf/AA for 5 h at 6 % O2, regardless the presence of DMOG. Treatment with Baf in galactose ( + ) medium causes dramatic decrease in the ATP level. (C) The levels of PHD1, PHD3, VHL and ATP6V0C proteins are similar in WT and SCO2 −/ −cells, while the content of PHD2 in SCO2 −/ −cells is significantly lower than in WT control. Asterisks indicate statistically significant difference (∗P < 0.01 and ∗∗P < 0.001, Student’s t test) between the experimental samples and mock control (DMSO). Hash in (A) shows a significant difference between the effects of Baf and CMA.

Article Snippet: Briefly, HCT116 cells were seeded on Ibidi chambers, grown and treated as described above, washed with PBS, fixed with 3.7 % PFA (paraformaldehyde) in PBS, quenched with 50 mM NH4Cl, permeabilized with 0.25 % TX100, blocked with 5 % BSA, incubated with anti-HIF-1α (Novus Biologicals) primary and fluorescent anti-mouse secondary antibodies conjugated with Alexa Fluor555 dye (Invitrogen), counterstained with DAPI, mounted with ProLong® Gold and air-dried.

Techniques: Western Blot, Control

Journal: Bioscience Reports

Article Title: Bafilomycin A1 activates HIF-dependent signalling in human colon cancer cells via mitochondrial uncoupling

doi: 10.1042/bsr20120085

Figure Lengend Snippet: Figure 3 Nuclear localization and functional activity of HIF-1α in the HCT116 cells treated with Baf at 6 % O2 (A, B) Immunofluorescence analysis shows significant increase in nuclear HIF-1α staining in WT cells treated with Baf for 5 h. No effect of Baf or CMA on the nuclear HIF-1α accumulation is observed in SCO2 −/ −cells. Treatment with DMOG causes translocation of HIF-1 into the nuclei of both WT and SCO2 −/ −in an O2-independent manner. Nuclei are counterstained with DAPI. (C) Quantitative analysis of the data shown in (A, B) is performed by measuring HIF-1-specific fluorescence in the DAPI-positive regions. (D) DNA-binding analysis using TransAM® HIF-1 kit demonstrates an increase in HIF-1-HRE interaction in WT cells upon Baf treatment. (E) Treatment with Baf activates ‘hypoxia-specific’ gene expression in WT cells, as demonstrated using RT–PCR analysis. Data are presented as the ratio between mRNA levels in treated and non-treated cells. (F) Western blot analysis shows that PHD2 protein level is increased in WT cells treated with Baf and DMOG; however no increase in PHD2 levels in SCO2 −/ −cells is observed. Asterisks indicate statistically significant difference between the experimental samples and mock control (DMSO).

Article Snippet: Briefly, HCT116 cells were seeded on Ibidi chambers, grown and treated as described above, washed with PBS, fixed with 3.7 % PFA (paraformaldehyde) in PBS, quenched with 50 mM NH4Cl, permeabilized with 0.25 % TX100, blocked with 5 % BSA, incubated with anti-HIF-1α (Novus Biologicals) primary and fluorescent anti-mouse secondary antibodies conjugated with Alexa Fluor555 dye (Invitrogen), counterstained with DAPI, mounted with ProLong® Gold and air-dried.

Techniques: Functional Assay, Activity Assay, Staining, Translocation Assay, Binding Assay, Gene Expression, Reverse Transcription Polymerase Chain Reaction, Western Blot, Control

Journal: iScience

Article Title: Simulating cell-free chromatin using preclinical cancer models for liquid biopsy applications

doi: 10.1016/j.isci.2025.114113

Figure Lengend Snippet: MNase treatment of media from preclinical models reproducibly generates nucleosomal distributions from cfChromatin (A) HCT116-conditioned media without (left) or with (right) MNase treatment shows enrichment for mono- and oligo-nucleosome-sized cfDNA fragments. LM, lower marker; UM, upper marker. (B) MNase treatment generates significantly different proportions of mono-, di-, and tri-cell-free nucleosomes compared to no nuclease treatment, reproducible across various 2D and organoid culture models (CAMA-1 and MCF7 = breast adenocarcinoma, HCT116 = colorectal carcinoma, A549 = lung adenocarcinoma, SU-DHL-6 = diffuse large B cell lymphoma, BPTO.95 and DCBPTO.66 = breast cancer patient-derived tumor organoids, BXTO.64 = breast cancer patient-derived xenograft-derived organoid). Paired t test for fragments (<200 bp, p = 1.53 × 10 −7 ; 200–400 bp, p = 5.05 × 10 −4 ; 400–600 bp, p = 1.44 × 10 −3 ; >600 bp, p = 6.07 × 10 −8 ). Results from (B) are extracted from the BioAnalyzer traces shown in B. (C) HCT116 (top) and CAMA-1 (bottom) media samples were treated with a fixed concentration of MNase over a 0- to 30-min time course. The proportion of mononucleosome-sized fragments increases with digestion time, with a small fraction of mononucleosomes at earlier time points relative to later.

Article Snippet: Purified, pre-sheared, and double size-selected naked genomic DNA (gDNA) from SW48 cells and media-derived cfChromatin from HCT116 cells were subjected to nuclease digestion under varying conditions using DNase1L3 (Aviva Systems Biology, CAT# OPCA03343), DNase1 (New England Biolabs, CAT# M0303S), and MNase (New England Biolabs, CAT# M0247S).

Techniques: Marker, Derivative Assay, Concentration Assay

Journal: iScience

Article Title: Simulating cell-free chromatin using preclinical cancer models for liquid biopsy applications

doi: 10.1016/j.isci.2025.114113

Figure Lengend Snippet: Simulated cfChromatin reflects chromosomal variation and nucleosome profiles associated with gene expression and chromatin accessibility patterns (A) PCA of cfMNase-seq samples across various sample types and MNase digestion times ( n = 21) and samples from three external MNase-seq datasets for HCT116 ( n = 4) and MCF7 ( n = 8). The PCA was performed over 10,000 bp bins genome-wide. A visual representation of the 10,000 bp bin size is shown in A. Clusters were identified using k -means clustering. A silhouette score was calculated to validate clustering; cluster 1 (bottom right) had an average silhouette score of 0.92, cluster 2 (middle left) 0.94, and cluster 3 (top middle) 0.96. (B) Analysis of the association between cfMNase-seq coverage and gene expression around the TSS. Composite cfMNase-seq coverage profiles at TSSs falling within five FPKM levels shown for CAMA-1 (30-min digestion). Coverage is shown as average GC-corrected fragment midpoint coverage. The complementary analysis for all other models, information on the FPKM subsets, and coverage profiles across MNase digestion times are shown in . (C) Normalized central coverage metric from cfMNase-seq coverage profiles across different gene expression levels (Spearman correlation ρ = −0.85, p = 3.5 × 10 −16 ), shown for all samples with a 30-min digestion time. (D) Composite cfMNase-seq coverage profiles (mean ±95% confidence interval) at 6,260 sites with enriched chromatin accessibility for A549, shown for A549 and the average of BPTO.95, CAMA-1, DCBPTO.66, HCT116, MCF7, and SU-DHL-6 grouped together (all 30-min digestions). The complementary results for other model-specific enriched open chromatin sites are shown in D. (E) Normalized central coverage metric from cfMNase-seq coverage profiles across sites of enriched chromatin accessibility for each model (specific) compared to the average across the rest (others) (paired Wilcoxon signed-rank test p = 0.063).

Article Snippet: Purified, pre-sheared, and double size-selected naked genomic DNA (gDNA) from SW48 cells and media-derived cfChromatin from HCT116 cells were subjected to nuclease digestion under varying conditions using DNase1L3 (Aviva Systems Biology, CAT# OPCA03343), DNase1 (New England Biolabs, CAT# M0303S), and MNase (New England Biolabs, CAT# M0247S).

Techniques: Gene Expression, Genome Wide

Journal: iScience

Article Title: Simulating cell-free chromatin using preclinical cancer models for liquid biopsy applications

doi: 10.1016/j.isci.2025.114113

Figure Lengend Snippet: Simulated cfChromatin reflects cancer-specific chromatin features and overcomes signal dilution in low-burden patient plasma (A) Analysis of the association between CAMA-1 xenograft plasma WGS and gene expression around the TSS, shown for five binned FPKM levels. (B) Composite coverage profiles at 117,917 ATAC-seq open chromatin sites from independent breast cancer patient samples within TCGA after filtering out PBMC signal. Coverage profiles are shown for breast cancer patient plasma ( n = 54) across varying tumor fractions (breakdown shown in ), simulated cfChromatin from organoids (BXTO.64, BPTO.95, and DCBPTO.66), simulated cfChromatin from cell lines (30-min digestion CAMA-1 and MCF7), and CAMA-1 xenograft plasma WGS. (C) Composite coverage profiles shown at 56,484 ATAC-seq open chromatin sites from independent colon adenocarcinoma patient samples within TCGA after filtering out PBMC signal. Composite profiles are shown for colorectal cancer patient plasma WGS ( n = 27) and cell-line-simulated cfChromatin from HCT116 (30-min digestion). (D) Composite coverage profiles shown at 65,427 ATAC-seq open chromatin sites from independent lung adenocarcinoma patient samples within TCGA after filtering out PBMC signal. Composite profiles are shown for lung cancer patient plasma WGS ( n = 9) and cell-line-simulated cfChromatin from A549. Composite site analysis for all individual samples and the corresponding metrics are in . Within-figure abbreviations: BRCA, breast cancer; TF, tumor fraction; CRC, colorectal cancer; LUAD, lung adenocarcinoma.

Article Snippet: Purified, pre-sheared, and double size-selected naked genomic DNA (gDNA) from SW48 cells and media-derived cfChromatin from HCT116 cells were subjected to nuclease digestion under varying conditions using DNase1L3 (Aviva Systems Biology, CAT# OPCA03343), DNase1 (New England Biolabs, CAT# M0303S), and MNase (New England Biolabs, CAT# M0247S).

Techniques: Clinical Proteomics, Gene Expression

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: British Journal of Pharmacology

Article Title: N ‐Acylethanolamine acid amidase (NAAA) is dysregulated in colorectal cancer patients and its inhibition reduces experimental cancer growth

doi: 10.1111/bph.15737

Figure Lengend Snippet: The N ‐acylethanolamine acid amidase (NAAA) selective inhibitor AM9053 affects tumour cell proliferation. (a) Cell viability assay in HCT116 and (b) in healthy human colonic epithelial cells (HCEC) alone or in the presence of AM9053 (0.1–3 μM, 24 h) ( n = 5). Results are expressed as mean ± SD. (c) Cell proliferation rate of HCT116 cells incorporating bromodeoxyuridine (BrdU), alone or in the presence of AM9053 (0.1–3 μM, 24 h). Results are expressed as percentage of cell proliferation ( n = 5 independent experiments). Values are expressed as means ± SD; * P < 0.05 versus control, as assessed by one‐way ANOVA followed by Dunnett's multiple comparisons test. (d) Cell proliferation rate of HCEC incorporating BrdU, alone or in the presence of AM9053 (3 μM, 24 h). Results are expressed as percentage of cell proliferation ( n = 6). Values are expressed as means ± SD; n.s., not significant as assessed by unpaired Student's t ‐test. (e) The antiproliferative effect of AM9053 (3 μM) was also evaluated in the presence of GW6471 (3 μM, PPAR‐α antagonist), 5′‐iodoresiniferatoxin (I‐RTX 0.1 μM, TRPV1 antagonist), AM251 (1 μM, CB 1 antagonist) and AM630 (1 μM, CB 2 antagonist). All results are expressed as percentage of cell proliferation ( n = 5 independent experiments) and as means ± SD; * P < 0.05 versus control and/or AM9053 as assessed by one‐way ANOVA followed by Tukey's multiple comparisons test

Article Snippet: HCT116 cells (1.5 × 10 5 cells seeded in a six‐well plate) were transfected with siRNA for NAAA (Trilencer‐27 siRNA duplex, OriGene, Rockville, MD, USA) (10 nM) and/or a standard negative control (5 nM), resuspended in MegaTran 2.0 Transfection Reagent (OriGene, Rockville, MD, USA) and Opti‐MEM® (Thermo Fisher Scientific, Waltham, MA, USA) in a total volume of 100 μl and added drop by drop to cells for 4 h. The transfection was then stopped by adding DMEM supplemented with 20% FBS overnight and replaced with complete culture medium incubated at 37°C, 5% CO 2 for 48 h. Silencing efficiency was controlled by performing RT‐PCR, and the obtained siNAAA‐HCT116 was used as due.

Techniques: Viability Assay

Journal: British Journal of Pharmacology

Article Title: N ‐Acylethanolamine acid amidase (NAAA) is dysregulated in colorectal cancer patients and its inhibition reduces experimental cancer growth

doi: 10.1111/bph.15737

Figure Lengend Snippet: The inhibitor AM9053 blocks tumoural cell cycle. (a) Cell cycle analysis by flow cytometry of HCT116 cells treated or not with AM9053 (3 μM, 24 h). Results are expressed as fold change of the cells in each cell cycle phase ( n = 8 independent experiments) in order to uniform the % values of the cells in each phase of the cell cycle ( n = 1 outlier has been removed by ROUT test). Values are expressed as means ± SD; * P < 0.05 versus control, as assessed by unpaired Student's t test and/or Mann–Whitney test. (b) Representative density plots and cell percentage, indicating the G0/G1‐, G2/M‐ and S‐phase distribution of HCT116 cells treated (right figure) or not (left figure) with AM9053 (3 μM, 24 h). Gene expression, in HCT116 cells, alone or in the presence of AM9053 (3 μM, 24 h), of cyclin A2 (c), cyclin‐dependent kinase 2 (CDK2) (d), cyclin B1 (e) and cyclin‐dependent kinase 1 (CDK1) (f). Gene expression was measured by qRT‐PCR and calculated by using the 2 −ΔCt formula ( n = 6 independent experiments). Values are expressed as means ± SD; * P < 0.05 versus control and analysed by Student's t ‐test; n.s., not significant

Article Snippet: HCT116 cells (1.5 × 10 5 cells seeded in a six‐well plate) were transfected with siRNA for NAAA (Trilencer‐27 siRNA duplex, OriGene, Rockville, MD, USA) (10 nM) and/or a standard negative control (5 nM), resuspended in MegaTran 2.0 Transfection Reagent (OriGene, Rockville, MD, USA) and Opti‐MEM® (Thermo Fisher Scientific, Waltham, MA, USA) in a total volume of 100 μl and added drop by drop to cells for 4 h. The transfection was then stopped by adding DMEM supplemented with 20% FBS overnight and replaced with complete culture medium incubated at 37°C, 5% CO 2 for 48 h. Silencing efficiency was controlled by performing RT‐PCR, and the obtained siNAAA‐HCT116 was used as due.

Techniques: Cell Cycle Assay, Flow Cytometry, MANN-WHITNEY, Expressing, Quantitative RT-PCR

Journal: British Journal of Pharmacology

Article Title: N ‐Acylethanolamine acid amidase (NAAA) is dysregulated in colorectal cancer patients and its inhibition reduces experimental cancer growth

doi: 10.1111/bph.15737

Figure Lengend Snippet: Transient knock‐down of N ‐acylethanolamine acid amidase (NAAA) is associated with an antiproliferative effect. (a) NAAA gene expression in HCT116 cells alone (untransfected) or in the presence of scramble small interfering (si)RNA and/or NAAA siRNA ( n = 6 independent experiments). NAAA gene expression was measured by qRT‐PCR and calculated by using the 2 −ΔCt formula. Values are expressed as means ± SD; * P < 0.05 versus scramble siRNA and/or NAAA siRNA as assessed by one‐way ANOVA followed by Kruskal–Wallis comparisons test; n.s., not significant. (b) Cell proliferation rate of HCT116 cells incorporating BrdU in the following conditions: alone (untransfected), untransfected in the presence of AM9053 (3 μM, 24 h), scramble siRNA, NAAA siRNA and NAAA siRNA in the presence of AM9053 (3 μM, 24 h). Results are expressed as percentage of cell proliferation ( n = 5 independent experiments) and as means ± SD; * P < 0.05 versus untransfected and/or NAAA siRNA, as assessed by one‐way ANOVA followed by Tukey's multiple comparisons test; n.s., not significant. (c) Cell cycle analysis by flow cytometry of HCT116 cells in the presence of scramble siRNA and/or NAAA siRNA. Results are expressed as fold change of the cells in each cell cycle phase ( n = 6) in order to uniform the % values of the cells in each phase of the cell cycle. Values are expressed as means ± SD; * P < 0.05 versus scramble siRNA, as assessed by unpaired Student's t ‐test. (d) Representative density plots and cell percentage, indicating the G0/G1‐, G2/M‐ and S‐phase distribution of HCT116 cells in the presence of scramble siRNA (upper figure) and/or NAAA siRNA (lower figure). Gene expression, in HCT116 cells in the presence of scramble siRNA and/or NAAA siRNA, of cyclin A2 (e), CDK2 (f), cyclin B1 (g) and CDK1 (h). Gene expression was measured by qRT‐PCR and calculated by using the 2 −ΔCt formula ( n = 6) ( n = 1 outlier has been removed by ROUT test for cyclin B1 analysis). Values are expressed as means ± SD; * P < 0.05 versus scramble siRNA, as assessed by Student's t ‐test; n.s., not significant

Article Snippet: HCT116 cells (1.5 × 10 5 cells seeded in a six‐well plate) were transfected with siRNA for NAAA (Trilencer‐27 siRNA duplex, OriGene, Rockville, MD, USA) (10 nM) and/or a standard negative control (5 nM), resuspended in MegaTran 2.0 Transfection Reagent (OriGene, Rockville, MD, USA) and Opti‐MEM® (Thermo Fisher Scientific, Waltham, MA, USA) in a total volume of 100 μl and added drop by drop to cells for 4 h. The transfection was then stopped by adding DMEM supplemented with 20% FBS overnight and replaced with complete culture medium incubated at 37°C, 5% CO 2 for 48 h. Silencing efficiency was controlled by performing RT‐PCR, and the obtained siNAAA‐HCT116 was used as due.

Techniques: Expressing, Quantitative RT-PCR, Cell Cycle Assay, Flow Cytometry

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