sw620 human colorectal cancer cell line  (ATCC)

Journal: Journal of Genetic Engineering & Biotechnology

Article Title: Potential role of Adansonia digitata nanoparticles on colorectal cancer induced by colorectal cancer cells (SW620) in nude mice

doi: 10.1016/j.jgeb.2026.100659

Figure Lengend Snippet: Oxidative stress markers in nude mice colon treated with SW620, AD, ADNPs1, and ADNPs2. A significant change at p < 0.05 in MDA and a non-significant change at p > 0.05 in SOD and catalase activity. Values presented as mean ± S.E., a, b different superscripts within columns are significantly different ( P < 0.05).

Article Snippet: The SW620 human colorectal cancer cell line was obtained from the American Type Culture Collection (ATCC, Rockville, MD, USA).

Techniques: Activity Assay

Journal: Journal of Genetic Engineering & Biotechnology

Article Title: Potential role of Adansonia digitata nanoparticles on colorectal cancer induced by colorectal cancer cells (SW620) in nude mice

doi: 10.1016/j.jgeb.2026.100659

Figure Lengend Snippet: Anti-apoptotic/pro-apoptotic protein level markers in nude mice colon treated with SW620, AD, ADNPs1, and ADNPs2. A significant change at p < 0.05 compared to the SW620 group in cytochrome c and a non-significant change at p > 0.05 in caspase3, BAX, and BCL-2. Values presented as mean ± S.E., a, b different superscripts within columns are significantly different ( P < 0.05).

Article Snippet: The SW620 human colorectal cancer cell line was obtained from the American Type Culture Collection (ATCC, Rockville, MD, USA).

Techniques:

Journal: Journal of Genetic Engineering & Biotechnology

Article Title: Potential role of Adansonia digitata nanoparticles on colorectal cancer induced by colorectal cancer cells (SW620) in nude mice

doi: 10.1016/j.jgeb.2026.100659

Figure Lengend Snippet: TGF-β, TNF-α, INOS, and IL-1β mRNA expression in nude mice colon treated with SW620, AD, ADNPs1, and ADNPs2. A significant change at p < 0.05 compared to the SW620 group in TGF-β, and a non-significant change at p > 0.05 in TNF-α, IL-1β, and INOS. Values presented as mean ± S.E., a, b, c different superscripts within columns are significantly different ( P < 0.05).

Article Snippet: The SW620 human colorectal cancer cell line was obtained from the American Type Culture Collection (ATCC, Rockville, MD, USA).

Techniques: Expressing

Journal: bioRxiv

Article Title: Molidustat Targets a Synthetic Lethal Vulnerability in APC-Mutant Colorectal Cancer through GSTP1 and PHD2 Co-Inhibition

doi: 10.64898/2026.01.31.702998

Figure Lengend Snippet: A. Percentage of Cleaved Caspase 3/7 positive HT29 (top), and RKO (bottom) cells. Cells were treated with Molidustat for 48 hours at indicated concentrations, 10uM Staurosporine was used as a positive control (100% cell death). Mean + SEM is assessed by unpaired two tailed Student’s t-test, **p<0.01, (ns) non-significant. B. Representative images of Cleaved Caspase-3/7 signal in DMSO, Molidustat (90 μM), and Staurosporine treated cells. Scale bar: 300 μm. C. Representative Western Blot of PHD2 levels in HT29 cells. D. Percentage confluency of HT29 cells post-transfection with the indicated guide RNAs. E. Cleaved Caspase-3/7 signal in HT29 cells post-transfection with the indicated crRNAs. Mean + SEM is assessed by two-way ANOVA, *p<0.05. N = 3 biologically independent experiments.

Article Snippet: Human colorectal cancer cell lines HT29 and RKO were obtained from the American Type Culture Collection (ATCC) and maintained in Dulbecco’s Modified Eagle Medium (DMEM; Sigma-Aldrich, D6429) supplemented with 10% (v/v) fetal bovine serum (FBS; Gibco, 16000044), 1% (v/v) penicillin-streptomycin (Gibco, 15140122), and 2 mM L-glutamine (Sigma-Aldrich, G7513).

Techniques: Positive Control, Two Tailed Test, Western Blot, Transfection

Journal: The Journal of Biological Chemistry

Article Title: Mitotic chromatin compaction tethers extrachromosomal DNA to chromosomes and prevents their mis-segregation into micronuclei

doi: 10.1016/j.jbc.2025.111081

Figure Lengend Snippet: The prometaphase spread technique untethers ecDNA and chromosomes by decompacting mitotic chromatin. A , schematic of an ecDNA-containing cell undergoing mitosis (from left to right : prophase, metaphase, anaphase, telophase). Acentric ecDNA ( red ) have been observed to tether to or “hitchhike” on chromosomes ( blue ) during mitosis to ensure their proper segregation and inheritance by daughter cells. B , prometaphase spreads performed on colcemid-arrested COLO320DM cells with the conventional hypotonic solution (75 mM KCl) and higher osmolarity solutions (100 mM and 125 mM KCl), producing varying amounts of chromosome (DAPI, blue ) individualization and ecDNA ( MYC FISH, red ) untethering. C , quantification of panel B . Left : boxplots quantifying chromosome individualization in prometaphase spreads performed with varying solution osmolarity; from left to right , n = 3, 3, 3 biological replicates and 95, 92, 105 cells; one-way ANOVA, F = 100.7, p < 0.001; ∗∗ p < 0.01 by Tukey’s honestly significant difference (HSD), ns = not significant. Right : quantification of ecDNA untethering; one-way ANOVA, F = 48.3, p < 0.001. Chromosome individualization is represented by the number of connected components identified as chromosomes by ecSeg. ecDNA untethering is represented by the number of ecDNA unattached to chromosomes divided by the total number of ecDNA not completely surrounded by chromosomes. D , prometaphase spreads performed with incubation in 1× PBS using COLO320DM cells pretreated for 8 h with vehicle (0.1% DMSO) or varying concentrations of Trichostatin A (TSA). E , quantification of panel D . Left : quantification of chromosome individualization in prometaphase spreads performed with 1× PBS on cells pretreated with TSA; n = 3, 3, 3, 3, 3, 3 biological replicates and 88, 113, 96, 80, 76, 84 cells; one-way ANOVA, F = 38.5, p < 0.001; ∗∗ p < 0.01 by Tukey’s HSD, ns = not significant. Right : quantification of ecDNA untethering; one-way ANOVA, F = 166.5, p < 0.001. F , linear regression analysis of median chromosome individualization v er s us ecDNA untethering of prometaphase spread conditions in panels B – E (n = 9). G , schematic: electrostatic/hydrophobic mitotic compaction forces at the level of nucleosomes tether ecDNA ( red ) to mitotic chromosomes ( blue ). In panels B–E , scale bar = 10 μm and each data point in graphs represents one cell.

Article Snippet: Human colorectal cancer cell lines COLO320DM and COLO320HSR were purchased from ATCC.

Techniques: Incubation

Journal: The Journal of Biological Chemistry

Article Title: Mitotic chromatin compaction tethers extrachromosomal DNA to chromosomes and prevents their mis-segregation into micronuclei

doi: 10.1016/j.jbc.2025.111081

Figure Lengend Snippet: Hypotonic conditions and HDAC inhibition untether ecDNA. A , fixed metaphase COLO320DM cells cultured on glass coverslips treated for 15 min with 1× media, 0.75× media (not shown), 0.5× media, 1:1 mix of 1× PBS with 1× media (1× PBS-media, not shown), and 1:1 mix of 1.5× PBS with 1× media (1.25× PBS-media); dashed outlines indicate metaphase plate chromosomes (DAPI, blue ), arrowheads indicate untethered ecDNA (identified by MYC FISH signal, red ). B , boxplots quantifying untethered ecDNA per cell from panel A , represented by % of MYC FISH signal in a cell unattached to chromosomes aligned at the metaphase plate (non-overlapping pixels); from left to right, n = 4, 6, 6, 7, 4 biological replicates and 427, 672, 691, 532, 686 cells; one-way ANOVA, F = 882.3, p < 0.001; ∗ p < 0.05, ∗∗ p < 0.01 by Tukey’s HSD. C , live imaging of COLO320DM cells expressing H2B-emiRFP670 (chromatin, blue ) and tetR-mNeonGreen which binds to a tetO-96mer repeat sequence inserted near MYC loci ( MYC , red ). Cells were arrested at metaphase with 10 μM MG132 and placed in hypotonic 0.5× media at t = 0 min to decompact chromatin; arrows indicate untethered ecDNA; after image acquisition at t = 15 min, cells were placed in relatively normotonic 1× PBS-media to recompact chromatin; arrows indicate ecDNA-ecDNA tethering. Representative images of n = 5 biological replicates and >50 cells. D , fixed metaphase COLO320DM cells cultured on glass coverslips treated for 8 h with vehicle (0.1% DMSO) or indicated concentrations of TSA; dashed outlines indicate metaphase plate chromosomes (DAPI, blue ), arrowheads indicate untethered ecDNA (identified by MYC FISH signal, red ). E , boxplots quantifying untethered ecDNA per cell from panel D ; from left to right , n = 3, 3, 3, 3 biological replicates and 98, 144, 161, 246 cells; one-way ANOVA, F = 34.9, p < 0.001; ∗∗ p < 0.01 by Tukey’s HSD. F , modified volcano plot summarizing the results of the targeted screen for drugs with the ability to untether ecDNA and chromosomes, quantified by the area of the convex hull of the prometaphase spread produced without hypotonic solution incubation. TSA (0.5 μM) was included as positive control. padj = adjusted two-tailed Student’s t test p value using Bonferroni multiple test correction (44 total comparisons were made). Each data point represents the average of all cells for each condition. G , metaphase COLO320DM cells cultured on glass coverslips treated for 24 h with vehicle (0.1% DMSO) or indicated concentrations of LMK235; dashed outlines indicate metaphase plate chromosomes, arrowheads indicate untethered ecDNA. H , quantification of untethered ecDNA per cell from panel G; n = 11, 3, 7, 4, 3, 3 biological replicates and 1278, 501, 875, 472, 571, 388 cells; one-way ANOVA, F = 735.1, p < 0.001; ∗∗ p < 0.01 by Tukey’s HSD, ns = not significant. In all panels, scale bar = 10 μm. Except in panel F , each data point in graphs represents one cell.

Article Snippet: Human colorectal cancer cell lines COLO320DM and COLO320HSR were purchased from ATCC.

Techniques: Inhibition, Cell Culture, Imaging, Expressing, Sequencing, Modification, Produced, Incubation, Positive Control, Two Tailed Test

Journal: The Journal of Biological Chemistry

Article Title: Mitotic chromatin compaction tethers extrachromosomal DNA to chromosomes and prevents their mis-segregation into micronuclei

doi: 10.1016/j.jbc.2025.111081

Figure Lengend Snippet: Hypotonic conditions and HDAC inhibition lead to mis-segregation of ecDNA into micronuclei. A , live cell imaging of COLO320DM cells undergoing mitosis (chromatin labeled by H2B-emiRFP670, blue ) and ( MYC loci labeled by tetR-mNeonGreen, red ). Mitotic cells were placed in 0.5× media at t = 0 min; dashed outlines indicate chromosomes/primary nuclei, arrowheads indicate untethered ecDNA, arrows indicate micronuclei. Representative image of n = 5 biological replicates and >50 cells. B , fixed newly-divided daughter COLO320DM cells, as identified by the presence of Aurora B staining, treated for 6 h with 1× media, 0.75× media (not shown), 0.5× media, 1× PBS-media (not shown), and 1.25× PBS-media; dashed outlines indicate primary nuclei, arrowheads indicate micronuclei. C , quantification of panel B . Left : quantification of the percentage of daughter cell pairs with micronuclei; chi-squared, ∗ p < 0.05, ∗∗ p < 0.01. Right : quantification of the percentage of all MYC FISH signal per daughter cell pair inside micronuclei as opposed to the primary nuclei (symlog scale, linear ≤2, log >2); one-way ANOVA, F = 151.4, p < 0.001, ∗ p < 0.05, ∗∗ p < 0.01; n = 3, 3, 3, 6, 4 biological replicates and 601, 233, 148, 881, 836 daughter cell pairs. D and E , fixed newly-divided daughter COLO320HSR cells, treated similarly as in panels B and C . E , left : chi-squared, ∗∗ p < 0.01, ns = not significant. Right : one-way ANOVA, F = 0.9, p = 0.49; n = 3, 3, 3, 5, 3 biological replicates and 680, 256, 179, 578, 487 daughter cell pairs. F , quantification of pan-Histone H3 acetylation (pan-H3ac) IF in cytospin preparations of COLO320DM prometaphase spreads with chromosome and ecDNA segmentation by ecSeg (see C ); cells were treated in vehicle or LMK235 for 24 h; n = 2, 2, 2, 2 biological replicates and 52, 44, 75, 84 cells; one-way ANOVA, chromosomes: F = 67.6, p < 0.001, ecDNA: F = 81.3, p < 0.001, ∗ p < 0.05, ∗∗ p < 0.01 by Tukey’s HSD. G , newly-divided daughter COLO320DM cells, as identified by the presence of Aurora B staining (not shown), treated for 24 h with vehicle (0.1% DMSO) or indicated concentrations of LMK235; dashed outlines indicate primary nuclei, arrowheads indicate micronuclei. H , quantification of panel G . Left : quantification of the percentage of daughter cell pairs with micronuclei; chi-squared, ∗∗ p < 0.01. Right : quantification of the percentage of all MYC FISH signal per daughter cell pair inside micronuclei (symlog scale, linear ≤2, log >2); one-way ANOVA, F = 151.4, p < 0.001, ∗ p < 0.05, ∗∗ p < 0.01; n = 5, 5, 4 biological replicates and 713, 683, 420 daughter cell pairs. I , same as panel F , for COLO320HSR cells (chromosomes only; see E ); n = 2, 2, 2, 2 biological replicates and 85, 85, 63, 99 cells; one-way ANOVA, F = 204.1, p < 0.001, ∗∗ p < 0.01 by Tukey’s HSD. J and K , newly-divided daughter COLO320HSR cells, treated similarly as in panels G and H. K , left : chi-squared. Right : one-way ANOVA, F = 0.1, p = 0.88; n = 4, 3, 3 biological replicates and 486, 383, 335 daughter cell pairs. In all panels, scale bar = 10 μm and each data point in graphs represents one cell or daughter cell pair.

Article Snippet: Human colorectal cancer cell lines COLO320DM and COLO320HSR were purchased from ATCC.

Techniques: Inhibition, Live Cell Imaging, Labeling, Staining

Journal: The Journal of Biological Chemistry

Article Title: Mitotic chromatin compaction tethers extrachromosomal DNA to chromosomes and prevents their mis-segregation into micronuclei

doi: 10.1016/j.jbc.2025.111081

Figure Lengend Snippet: Ki67 gain-of-function untethers ecDNA from chromosomes. A , fixed COLO320DM cells cultured on coverslip and incubated in 0.5× media for 15 min to visualize and colocalize individual ecDNA ( MYC FISH) with Ki67 IF ( arrowheads ). B , line profile of MYC FISH and Ki67 IF intensity along the line indicated in panel A; polynomial curves were fitted to the line profiles. C , metaphase COLO320DM cells cultured on glass coverslips 2 to 4 days post mCherry-only or Ki67-mCherry expression plasmid transfection; cells were categorized based on mCherry fluorescence: - indicates lack of mCherry expression, ++ indicates top 20% tile mCherry expression by fluorescence intensity, + indicates the remaining cells that express mCherry; dashed outlines indicate chromosomes aligned at the metaphase plate, arrowheads indicate untethered ecDNA. D , boxplots quantifying ecDNA untethering in plasmid transfected cells from panel C ; mCh = mCherry expression category, Plas = plasmid transfected; from left to right ; n = 4, 4, 4, 4, 4, 4 biological replicates and 20, 63, 22, 116, 221, 86 total cells; two-way ANOVA, mCherry expression category (- v er s us + v er s us ++): F = 123.9, p < 0.001; plasmid transfected (mCherry-only v er s us Ki67-mCherry): F = 40.5, p < 0.001; interaction: F = 33.8, p < 0.001; ∗∗ p < 0.01 by Tukey’s HSD, ns = not significant. E , newly-divided daughter COLO320DM cells, as identified by the presence of Aurora B staining (not shown), 2 to 4 days post mCherry-only or Ki67-mCherry expression plasmid transfection (mCherry ++ cells are shown); dashed outlines indicate primary nuclei, arrowheads indicate micronuclei. F , quantification of panel E . Left : quantification of the percentage of daughter cell pairs with micronuclei; chi-squared, ∗∗ p < 0.01. Right: quantification of the percentage of all MYC FISH signal per daughter cell pair inside micronuclei (symlog scale, linear ≤2, log >2); two-way ANOVA, mCherry expression category (- v er s us + v er s us ++): F = 7.6, p < 0.001; plasmid transfected (mCherry-only v er s us Ki67-mCherry): F = 7.6, p = 0.0058; interaction: F = 4.9, p = 0.0073; ∗∗ p < 0.01; n = 7, 7, 7, 7, 7, 7 biological replicates and 93, 312, 104, 179, 400, 147 daughter cell pairs. G and H , newly-divided daughter COLO320HSR cells, transfected similarly as in panels E and F (mCherry ++ cells are shown). H , left : chi-squared, ∗ p < 0.05. Right : two-way ANOVA, mCherry expression category (- v er s us + v er s us ++): F = 0.7, p = 0.49; plasmid transfected (mCherry-only v er s us Ki67-mCherry): F = 0.1, p = 0.77; interaction: F = 0.9, p = 0.39; n = 4, 4, 4, 4, 4, 4 biological replicates and 168, 245, 107, 202, 227, 111 daughter cell pairs. In all panels, scale bar = 10 μm. Except in panel B , each data point in graphs represents one cell or daughter cell pair.

Article Snippet: Human colorectal cancer cell lines COLO320DM and COLO320HSR were purchased from ATCC.

Techniques: Cell Culture, Incubation, Expressing, Plasmid Preparation, Transfection, Fluorescence, Staining

Journal: The Journal of Biological Chemistry

Article Title: Mitotic chromatin compaction tethers extrachromosomal DNA to chromosomes and prevents their mis-segregation into micronuclei

doi: 10.1016/j.jbc.2025.111081

Figure Lengend Snippet: Ki67 loss-of-function decreases ecDNA untethering from chromosomes. A , metaphase COLO320DM wildtype (NT) and MKI67 knockout cells (gRNA #1 and #2), incubated for 15 min in 1× media (not shown), 0.75× media, and 0.5× media (not shown); dashed outlines indicate chromosomes aligned at the metaphase plate, arrowheads indicate untethered ecDNA. B , quantification of ecDNA untethering of COLO320DM wildtype and MKI67 knockout clones from panel A (each data point represents the weighted average of at least 95 cells from each clone) after 15 min incubation in 1× media ( left ; n = 4, 6, 5 clones; one-way ANOVA, F = 1.7, p = 0.23), 0.75× media ( middle ; n = 4, 6, 5 clones; one-way ANOVA, F = 12.4, p = 0.001), and 0.5× media ( right ; n = 4, 6, 5 clones; one-way ANOVA, F = 0.1, p = 0.99); ∗∗ p < 0.01 by Tukey’s HSD, ns = not significant; error bars = mean ± standard deviation. C , metaphase COLO320DM wildtype (NT) and MKI67 knockout cells (gRNA #1 and #2), treated for 24 h with vehicle (not shown), 0.2 μM LMK235, and 0.5 μM LMK235 (not shown); dashed outlines indicate chromosomes aligned at the metaphase plate, arrowheads indicate untethered ecDNA. D , quantification of ecDNA untethering of COLO320DM wildtype and MKI67 knockout clones from panel C (each data point represents the weighted average of at least 60 cells from each clone) after 24 h treatment with vehicle (0.1% DMSO, left ; n = 4, 3, 3 clones; one-way ANOVA, F = 3.9, p = 0.073), 0.2 μM LMK235 ( middle ; n = 4, 3, 3 clones; one-way ANOVA, F = 100.8, p < 0.001, ∗∗ p < 0.01 by Tukey’s HSD), and 0.5 μM LMK235 ( right ; n = 4, 3, 3 clones; one-way ANOVA, F = 0.8, p = 0.51); error bars = mean ± standard deviation; MKI67 knockout clones are gRNA #1 clones 1, 4, and 5, and gRNA #2 clones 1, 4, and 5 (see , A – C ). E , schematic: the biological surfactant Ki67 ( green ) coats the surface of mitotic ecDNA ( red ) and chromosomes ( blue ), helping to prevent tethering by electrostatic repulsion and steric hindrance. In all panels, scale bar = 10 μm.

Article Snippet: Human colorectal cancer cell lines COLO320DM and COLO320HSR were purchased from ATCC.

Techniques: Knock-Out, Incubation, Clone Assay, Standard Deviation

Journal: The Journal of Biological Chemistry

Article Title: Mitotic chromatin compaction tethers extrachromosomal DNA to chromosomes and prevents their mis-segregation into micronuclei

doi: 10.1016/j.jbc.2025.111081

Figure Lengend Snippet: HDAC inhibition leads to a loss of oncogene copy number in COLO320DM cells, but not in COLO320HSR cells. A and B , quantification of MYC DNA expression in COLO320DM ( A ) and COLO320HSR ( B ) cells treated with LMK235; qPCR, 2 -ΔΔCT analysis (normalized to LINE1 copy number and vehicle control); x-axis = symlog scale, linear ≤2, log >2. Error bars = mean ± 95% confidence interval. Drug-containing media was replaced every 2 to 3 days. A , treatment with vehicle (0.1% DMSO), 0.2 μM, 0.3 μM, or 1 μM LMK235 for 1 day (one-way ANOVA, F = 4.1, p = 0.036, n = 4, 4, 4, 3), 2 days (F = 31.3, p < 0.001, n = 4, 4, 4, 3), 10 days (F = 5.4, p = 0.017, n = 6, 6, 6, no 1 μM), 30 days (F = 19.8, p = 0.0023, n = 3, 3, 3, no 1 μM); 0 days: pretreatment (one-way ANOVA, F = 0.4, p = 0.77, n = 3, 3, 3, 3); # p < 0.05, ## p < 0.01 (0.2 μM LMK235 v er s us vehicle); ∗ p < 0.05, ∗∗ p < 0.01 (0.3 μM LMK235 v er s us vehicle); ˆˆ p < 0.01 (1 μM LMK235 v er s us vehicle) by Tukey’s HSD; B , treatment with vehicle, 0.3 μM LMK235, or 1 μM LMK235 for 2 days (one-way ANOVA, F = 2.2, p = 0.17, n = 4, 4, 4), 5 days (F = 0.5, p = 0.62, n = 4, 4, 4), 10 days (F = 10.1, p = 0.019, n = 4, 4), and 30 days (F = 2.7, p = 0.15, n = 4, 4); 0 days: pretreatment (one-way ANOVA, F = 0.8, p = 0.48); ∗ p < 0.05 (0.3 μM LMK235 v er s us vehicle). C , schematic, top : under normal mitotic conditions, ecDNA tether to chromosomes throughout mitosis to ensure their segregation into the primary nuclei of divided daughter cells; bottom : under conditions that decompact chromatin (hypotonic conditions and HDAC inhibition) or prevent ecDNA-chromosome interaction at their surfaces (Ki67 overexpression), ecDNA untether from chromosomes, leading some to be mis-segregated into micronuclei. D and E , schematics of a colloidal and surface chemistry-based framework for approaching ecDNA and chromosome compaction and tethering during mitosis. Arrow 1 represents biophysical changes to the colloidal particles (nucleosomes and chromatin molecules) or the solution/suspension medium (cytosol), such as alterations to the intracellular ionic strength or to the acetylation state of chromatin. Arrow 2 represents changes in the surfactant (Ki67) concentration of the system. Both sets of changes affect particle-particle and particle-solution interactions.

Article Snippet: Human colorectal cancer cell lines COLO320DM and COLO320HSR were purchased from ATCC.

Techniques: Inhibition, Expressing, Control, Over Expression, Suspension, Concentration Assay

Journal: Clinical and Translational Medicine

Article Title: RNF39 promotes colorectal cancer progression by driving RINT1 degradation and suppressing ER stress‐induced apoptosis

doi: 10.1002/ctm2.70577

Figure Lengend Snippet: RNF39 regulates proliferation and invasion in COAD cells. (A) RNF39 mRNA expression in HCT116 and SW480 cells transfected with two independent shRNAs targeting RNF39 (shRNF39‐1 and shRNF39‐2) or a negative control (sh‐NC), assessed by qRT‐PCR. (B) Representative images of colony formation assays in RNF39‐knockdown and control cells. (C, D) Quantification of colony numbers in HCT116 (C) and SW480 (D) cells following RNF39 knockdown. (E) Representative images of transwell invasion assays in RNF39‐silenced and control cells. (F, G) Quantification of invaded cells in HCT116 (F) and SW480 (G) after RNF39 knockdown. (H, I) Western blot (H) and qRT‐PCR (I) validation of RNF39 overexpression in HCT116 and SW480 cells transfected with Flag‐RNF39 or empty vector (EV). (J) Representative images of EdU staining in control and RNF39‐overexpressing cells; Hoechst was used to stain nuclei. (K) Quantification of EdU‐positive cells in HCT116 and SW480 cells. (L) Representative images of colony formation assays following RNF39 overexpression. (M, N) Quantification of colony numbers in HCT116 (M) and SW480 (N) cells with or without RNF39 overexpression. (O) Representative images of transwell invasion assays in RNF39‐overexpressing and control cells. (P, Q) Quantification of invaded cells in HCT116 (P) and SW480 (Q) with or without RNF39 overexpression.

Article Snippet: HCT116 and SW480 human colorectal cancer cell lines were obtained from the American Type Culture Collection and verified by short tandem repeat profiling.

Techniques: Expressing, Transfection, Negative Control, Quantitative RT-PCR, Knockdown, Control, Western Blot, Biomarker Discovery, Over Expression, Plasmid Preparation, Staining

Journal: Clinical and Translational Medicine

Article Title: RNF39 promotes colorectal cancer progression by driving RINT1 degradation and suppressing ER stress‐induced apoptosis

doi: 10.1002/ctm2.70577

Figure Lengend Snippet: RNF39 E3 ligase activity is required for COAD cell oncogenicity and tumour growth in vivo. (A) Representative images of colonies formed by RNF39⁺/⁺ and RNF39 − / − HCT116 and SW480 cells in a colony formation assay. (B) Quantification of the number of colonies in A. (C) Representative images of invaded RNF39⁺/⁺ and RNF39 − / − HCT116 and SW480 cells in a Matrigel‐coated transwell assay. (D) Quantification of invaded cells in C. (E) Representative images of wound closure in RNF39⁺/⁺ and RNF39 − / − HCT116 cells at 0, 24 and 48 h in a wound healing assay. (F) Quantification of the relative migration rate in E. (G) Representative images of wound closure in RNF39⁺/⁺ and RNF39 − / − SW480 cells in a wound healing assay. (H) Quantification of the relative migration rate in G. (I) Immunoblot validation of RNF39 expression in wild‐type (RNF39⁺/⁺) and knockout (RNF39 − / − ) HCT116 and SW480 cells reconstituted with empty vector (EV), wild‐type RNF39 (WT) or catalytically inactive mutant RNF39^C108S. (J) Representative colony formation assay images from the indicated HCT116 and SW480 cell groups. (K, L) Quantification of relative colony numbers in HCT116 (K) and SW480 (L) RNF39 − / − cells reconstituted with EV, WT or C108S. (M) Representative images of transwell invasion assays in the indicated cell groups. (N, O) Quantification of invaded cells in HCT116 (N) and SW480 (O) cells from transwell assays. (P) Tumour growth curves showing volume measurements over time in nude mice subcutaneously injected with RNF39⁺/⁺ or RNF39 − / − HCT116 cells ( n = 5 per group). (Q) Representative image of excised tumours from both groups at endpoint. (R) Quantification of tumour weights at harvest from RNF39⁺/⁺ and RNF39 − / − xenografts.

Article Snippet: HCT116 and SW480 human colorectal cancer cell lines were obtained from the American Type Culture Collection and verified by short tandem repeat profiling.

Techniques: Activity Assay, In Vivo, Colony Assay, Transwell Assay, Wound Healing Assay, Migration, Western Blot, Biomarker Discovery, Expressing, Knock-Out, Plasmid Preparation, Mutagenesis, Injection

Journal: Clinical and Translational Medicine

Article Title: RNF39 promotes colorectal cancer progression by driving RINT1 degradation and suppressing ER stress‐induced apoptosis

doi: 10.1002/ctm2.70577

Figure Lengend Snippet: RNF39 promotes ubiquitin‐dependent degradation of RINT1 in colorectal adenocarcinoma cells. (A, B) Western blot analysis of RINT1 protein levels in HCT116 (A) and SW480 (B) cells following RNF39 overexpression. (C, D) Western blot showing increased RINT1 protein levels upon RNF39 knockdown (shRNF39) in HCT116 (C) and SW480 (D) cells. (E–H) qRT‐PCR analysis of RINT1 mRNA levels in RNF39‐overexpressing (E, F) or RNF39‐depleted (G, H) cells, indicating no significant changes. (I) Time‐course western blot analysis of RINT1 protein stability following cycloheximide (CHX) treatment (0–10 h) in SW480 cells with or without RNF39 knockdown. (J) Quantification of RINT1 protein levels over time from (I), normalized to time 0 and plotted as relative intensity. (K) TUBE2 pulldown assay of ubiquitinated RINT1 in control and RNF39‐depleted SW480 cells. (L) Ubiquitination of RINT1 in HEK293T cells. Cells were co‐transfected with HA‐RINT1, Flag‐RNF39 (or vector) and His‐tagged K48‐only ubiquitin (His‐K48‐Ub). HA‐RINT1 was immunoprecipitated, and K48‐linked polyubiquitinated RINT1 was detected by anti‐His immunoblotting. (M) In vitro ubiquitination of RINT1. Lysates from HEK293T cells expressing HA‐RINT1 and/or Flag‐RNF39 were incubated with recombinant E1, E2, ATP and wild‐type ubiquitin. After HA immunoprecipitation, RINT1–ubiquitin conjugates were detected by anti‐HA immunoblotting and were only evident when both RNF39 and E1/E2 were present.

Article Snippet: HCT116 and SW480 human colorectal cancer cell lines were obtained from the American Type Culture Collection and verified by short tandem repeat profiling.

Techniques: Ubiquitin Proteomics, Western Blot, Over Expression, Knockdown, Quantitative RT-PCR, Control, Transfection, Plasmid Preparation, Immunoprecipitation, In Vitro, Expressing, Incubation, Recombinant

Journal: Clinical and Translational Medicine

Article Title: RNF39 promotes colorectal cancer progression by driving RINT1 degradation and suppressing ER stress‐induced apoptosis

doi: 10.1002/ctm2.70577

Figure Lengend Snippet: RNF39 modulates cellular sensitivity to ER stress and UPR‐mediated apoptosis in colorectal cancer cells. (A, B) qRT‐PCR analysis of CHOP mRNA levels in HCT116 and SW480 cells following RNF39 overexpression (A) or knockdown (B). (C) Representative images of colony formation assays in RNF39⁺/⁺ and RNF39 − / − HCT116 and SW480 cells treated with thapsigargin (TG) for the indicated time points (0,.5, 1.5, 3 h). (D, E) Quantification of relative colony numbers in HCT116 (D) and SW480 (E) cells after TG exposure. (F) Representative images of transwell invasion assays performed on RNF39⁺/⁺ and RNF39 − / − cells treated with TG for 0–3 h. (G, H) Quantification of invaded cells in HCT116 (G) and SW480 (H) cells under the indicated TG treatment durations. (I) Caspase‐3/7 activity assay in RNF39⁺/⁺ and RNF39 − / − cells treated with TG for 3 h. Data represent mean ± SD of triplicates.

Article Snippet: HCT116 and SW480 human colorectal cancer cell lines were obtained from the American Type Culture Collection and verified by short tandem repeat profiling.

Techniques: Quantitative RT-PCR, Over Expression, Knockdown, Activity Assay

Journal: Clinical and Translational Medicine

Article Title: RNF39 promotes colorectal cancer progression by driving RINT1 degradation and suppressing ER stress‐induced apoptosis

doi: 10.1002/ctm2.70577

Figure Lengend Snippet: RNF39 modulates colorectal cancer progression and ER stress responses through RINT1 degradation. (A) Immunoblot validation of RNF39 and RINT1 knockdown in HCT116 and SW480 cells transduced with sh‐NC, shRNF39, shRINT1 or shRNF39+shRINT1. (B) Representative images of colony formation assays across the four groups. (C, D) Quantification of colony‐forming ability in HCT116 (C) and SW480 (D) cells. (E) Representative images from transwell invasion assays in each group. (F, G) Quantification of invaded cells in HCT116 (F) and SW480 (G) cells. (H) Caspase‐3/7 activity assay in SW480 cells, comparing apoptotic response across groups. (I) Tumour growth curves from xenograft experiments using stably transduced HCT116 cells implanted into BALB/c nude mice ( n = 5 per group). (J) Representative images of excised tumours at day 28. (K) Tumour weights at endpoint across the four experimental conditions. Data are presented as mean ± SD; p ‐values were determined by one‐way ANOVA with post‐hoc testing.

Article Snippet: HCT116 and SW480 human colorectal cancer cell lines were obtained from the American Type Culture Collection and verified by short tandem repeat profiling.

Techniques: Western Blot, Biomarker Discovery, Knockdown, Transduction, Activity Assay, Stable Transfection