human crc cell lines sw48  (ATCC)

Journal: Cancer Reports

Article Title: Transmembrane Protein TMEM59L Modulates 5‐ FU Resistance via PTPRN ‐Mediated DNA Damage Repair in Colorectal Cancer

doi: 10.1002/cnr2.70448

Figure Lengend Snippet: TMEM59L regulates colorectal cancer cells proliferation, migration, and invasion. (A) Western blotting confirmed expression of TMEM59L in different CRC cell lines. (B) Western blotting detects the knockdown of TMEM59L by shRNA and overexpression of TMEM59L by plasmid. (C) Downregulation of TMEM59L suppresses cell proliferation in HCT116 cells; overexpression of TMEM59L in SW480 promotes cell proliferation. (D) The function of TMEM59L on the migration and invasion ability of CRC cells was detected by Transwell assay. (E) E‐cadherin and Vimentin were evaluated through immunofluorescence staining in TMEM59L knockdown and overexpression CRC cells.

Article Snippet: Five human CRC cell lines (NCI‐H716, HCT116, COLO 320DM, SW48, SW480) were sourced from the American Type Culture Collection (ATCC, USA), while a 5‐fluorouracil‐resistant HCT116 subline (HCT116/FU, BNCC342640) was obtained from BNCC (China).

Techniques: Migration, Western Blot, Expressing, Knockdown, shRNA, Over Expression, Plasmid Preparation, Transwell Assay, Immunofluorescence, Staining

Journal: Cancer Reports

Article Title: Transmembrane Protein TMEM59L Modulates 5‐ FU Resistance via PTPRN ‐Mediated DNA Damage Repair in Colorectal Cancer

doi: 10.1002/cnr2.70448

Figure Lengend Snippet: TMEM59L was elevated in 5‐FU resistance CRC cell lines and reduced 5‐FU sensitivity. (A) The expression of TMEM59L in non‐responder and responder groups treated by 5‐FU ( n = 279 vs. 379, p = 0.0002), oxaliplatin ( n = 173 vs. 265, p = 0.012) and Capecitabine ( n = 62 vs. 47, p = 0.000019), respectively. (B) TMEM59L level in CRC cell lines and corresponding 5‐FU resistant cells was examined by western blot. (C, D) CCK‐8 assays of TMEM59L downregulation and upregulation on sensitivity of HCT116 and SW480 cells to 5‐FU; the half maximal inhibitory concentration (IC50) was calculated using GraphPad software. Data represent the mean ± SD ( n = 3), * p < 0.05 vs. HCT116 group, $ p < 0.05 vs. SW480 group.

Article Snippet: Five human CRC cell lines (NCI‐H716, HCT116, COLO 320DM, SW48, SW480) were sourced from the American Type Culture Collection (ATCC, USA), while a 5‐fluorouracil‐resistant HCT116 subline (HCT116/FU, BNCC342640) was obtained from BNCC (China).

Techniques: Expressing, Western Blot, CCK-8 Assay, Concentration Assay, Software

Journal: Cancer Reports

Article Title: Transmembrane Protein TMEM59L Modulates 5‐ FU Resistance via PTPRN ‐Mediated DNA Damage Repair in Colorectal Cancer

doi: 10.1002/cnr2.70448

Figure Lengend Snippet: Silencing of TMEM59L enhanced DNA damage and 5‐FU sensibility in colorectal cancer cells and drug‐resistant CRC cell lines. (A, B) γ‐H2AX foci formation in HCT116 and SW480 cells was detected by immunofluorescence 48 h after treatment with 5‐FU (25 μg/mL). (C) Intracellular ROS levels in HCT116 and SW480 cells treated with 5‐FU for 48 h were detected by reactive oxygen species detection kit. (D) Effect of TMEM59L on apoptosis in CRC cells induced by 5‐FU (25 μg/mL) treatment for 48 h was determined by flow cytometric analysis. (E) Downregulation of TMEM59L reduced colony formation of HCT116/FU and SW480/FU cells.

Article Snippet: Five human CRC cell lines (NCI‐H716, HCT116, COLO 320DM, SW48, SW480) were sourced from the American Type Culture Collection (ATCC, USA), while a 5‐fluorouracil‐resistant HCT116 subline (HCT116/FU, BNCC342640) was obtained from BNCC (China).

Techniques: Immunofluorescence

Journal: Cancer Reports

Article Title: Transmembrane Protein TMEM59L Modulates 5‐ FU Resistance via PTPRN ‐Mediated DNA Damage Repair in Colorectal Cancer

doi: 10.1002/cnr2.70448

Figure Lengend Snippet: TMEM59L regulated 5‐FU induced DNA damage and ROS through PTPRN. (A) Physical interactions with TMEM59L in GeneMANIA website. (B) Correlation analysis between TMEM59L and PTPRN in COAD from GEPIA database. (C) PTPRN expression in COAD and READ from the TCGA database analyzed by the GEPIA database. (D) Higher PTPRN expression was related to poorer OS in CRC patients from TCGA through Kaplan–Meier Plotter database. (E) PTPRN partially reversed the effect of TMEM59L on 5‐FU induced ROS of HCT116 and SW480 cells. (F) PTPRN partially reversed the effect of TMEM59L on 5‐FU induced DNA damage of HCT116 and SW480 cells.

Article Snippet: Five human CRC cell lines (NCI‐H716, HCT116, COLO 320DM, SW48, SW480) were sourced from the American Type Culture Collection (ATCC, USA), while a 5‐fluorouracil‐resistant HCT116 subline (HCT116/FU, BNCC342640) was obtained from BNCC (China).

Techniques: Expressing

Journal: iScience

Article Title: Single-molecule imaging quantifies oncogenic KRAS dynamics for enhanced accuracy of therapeutic efficacy assessment

doi: 10.1016/j.isci.2025.113374

Figure Lengend Snippet: Transient trapping of KRAS upon activation, as revealed by single-molecule imaging (A) Schematic diagram of the experimental setup for single-molecule imaging. (B) Western blot analysis of activated tdStayGold-KRAS (left) and endogenous KRAS (right) obtained by an RAS-GTP pull-down assay, as well as total RAS proteins (including both active and inactive forms) in whole SW48 cell lysates, before and after stimulation with 10 nM EGF. The proportion of activated RAS molecules was quantified based on the western blot data. Independent experimental results are presented, with bars representing the mean ± SEM. Data 2, 3, and 5 min after EGF stimulation were statistically compared with those before EGF stimulation using Welch’s t test. ∗ p < 0.05 and ∗∗∗ p < 0.001 (+Total includes all data 2, 3.5, and 5 min after stimulation). (C) Representative still image of single-molecule observation of tdStayGold-KRAS WT in SW48 cells (left), and trajectories of individual molecules acquired from 10-s movies recorded at video rate (33-ms resolution) 3.5 min after EGF stimulation (right, yellow lines: 1–100 frames, red lines: 101–200 frames, blue lines: 201–300 frames; see also ). (D) Representative 3-s trajectories of KRAS WT before (left) and 3.5 min after (right) EGF stimulation, showing transient trapping highlighted by red segments and arrowheads. (E) Image sequences (top) and trajectories (bottom) of individual KRAS molecules, exhibiting transient trapping, highlighted by red segments and arrowheads, alongside their respective trapping durations.

Article Snippet: The SW48 cell line (CCL-231) and the Caco-2 cell line (HTB-37) were obtained from the American Type Culture Collection (ATCC).

Techniques: Activation Assay, Imaging, Western Blot, Pull Down Assay

Journal: iScience

Article Title: Single-molecule imaging quantifies oncogenic KRAS dynamics for enhanced accuracy of therapeutic efficacy assessment

doi: 10.1016/j.isci.2025.113374

Figure Lengend Snippet: Quantitative analysis of transient trapping of single KRAS WT and mutant molecules reveals activation with greater sensitivity than biochemical assays (A and B) Western blot analysis of activated tdStayGold-KRAS mutant proteins obtained via the RAS-GTP pull-down assay and total tdStayGold-KRAS mutant proteins (including both active and inactive forms) in whole SW48 cell lysates before and after EGF stimulation (top panel in A). The proportion of activated KRAS mutant proteins was quantified based on the western blot data. Results from independent experiments are displayed, with bars representing the mean ± SEM. Data at 2, 3.5, and 5 min after EGF stimulation were statistically compared with those before EGF stimulation (bottom panel in A; +Total includes all data 2, 3.5, and 5 min after stimulation) using Welch’s t test. ∗ p < 0.05, ∗∗ p < 0.01, and ∗∗∗ p < 0.001 throughout this study. Data of KRAS WT and mutants 2–5 min after stimulation (+Total in A) were compared as well (B). (C) Representative 3-s trajectories of oncogenic KRAS mutants after EGF stimulation, showing alternating diffusion and transient trapping (highlighted by red segments and arrowheads, see also ). (D and E) Temporal fractions (D) and frequencies (E) of transient trapping of KRAS WT and mutant proteins observed 2−5 min after EGF stimulation in SW48 cells. (F and G) Time course of temporal fractions (F) and frequencies (G) of KRAS trapping 2, 3.5, and 5 min after EGF stimulation in SW48 cells. The temporal fraction and frequency of trapped molecules are presented as box-and-whisker plots, showing the minimum, maximum, sample median, sample mean (circle), first and third quartiles, and whiskers extending to a maximum of 1.5× interquartile range beyond the box. Data after EGF stimulation were compared either among mutants (D and E) or with those before EGF stimulation (F and G) using Welch’s t test.

Article Snippet: The SW48 cell line (CCL-231) and the Caco-2 cell line (HTB-37) were obtained from the American Type Culture Collection (ATCC).

Techniques: Mutagenesis, Activation Assay, Western Blot, Pull Down Assay, Diffusion-based Assay, Whisker Assay

Journal: iScience

Article Title: Single-molecule imaging quantifies oncogenic KRAS dynamics for enhanced accuracy of therapeutic efficacy assessment

doi: 10.1016/j.isci.2025.113374

Figure Lengend Snippet: Quantitative analysis of the duration and zone size of KRAS trappings revealed that prolonged KRAS trappings in smaller membrane zones increase upon EGF stimulation (A–C) Distribution of trapping durations (A) and trapping zone sizes (B) of KRAS WT and oncogenic mutants before and after EGF stimulation in SW48 cells. The zone sizes of individual KRAS trapping events are plotted against trapping duration in (C), along with Spearman’s rank correlation coefficient (ρ). (D) Comparison of the trapping zone sizes among KRAS WT and oncogenic mutants after EGF stimulation. The size distribution of individual trapping zones is presented using both violin plots and box-and-whisker plots, showing the sample median, sample mean (circle), first and third quartiles, and whiskers extending to a maximum of 1.5× interquartile range beyond the box. Statistical analysis was performed using Welch’s t test. ∗∗∗ p < 0.001.

Article Snippet: The SW48 cell line (CCL-231) and the Caco-2 cell line (HTB-37) were obtained from the American Type Culture Collection (ATCC).

Techniques: Membrane, Comparison, Whisker Assay

Journal: iScience

Article Title: Single-molecule imaging quantifies oncogenic KRAS dynamics for enhanced accuracy of therapeutic efficacy assessment

doi: 10.1016/j.isci.2025.113374

Figure Lengend Snippet: PS depletion attenuates the transient trapping of KRAS WT and oncogenic mutant molecules (A; left) Schematic representation of single-molecule imaging of a PS probe (evectin2 [2xPH]) in SW48 cells, with (bottom) or without (top) PS depletion via PSD expression. (Right) Schematic representation of single-molecule imaging of KRAS in SW48 cells with (bottom) or without (top) PS depletion. Activated KRAS (KRAS-GTP) forms nanoclusters facilitated by PS and/or CRD of BRAF, which associates with PS in the membrane. (B) Fluorescence images of SF650B-Halo7-evectin2 (2xPH) and mCherry-PSD in the presence (bottom) or absence (top) of PSD expression. Images were acquired using oblique angle illumination and TIRFM. Single fluorescent spots of evectin2 (2xPH) recruited to the PM are indicated by yellow arrowheads. (C) Quantification of evectin2 (2xPH) fluorescent spots recruited to the PM with or without PSD expression. Values were normalized to both total probe expression (measured via whole-cell fluorescence under oblique-angle illumination) and the observation area. (D–F) Temporal fractions of transient trapping (D), distributions of trapping durations (E), and trapping zone sizes (F) for KRAS WT, G13D, and G12V, with or without PSD expression, measured 2−5 min after EGF stimulation. The normalized number of recruited PS probe spots and the temporal fraction of trapped molecules are presented as box-and-whisker plots, displaying the minimum, maximum, sample median, sample mean (circle), first and third quartiles, and whiskers extending to a maximum of 1.5× interquartile range beyond the box. The size distribution of individual trapping zones is presented using both violin plots and box-and-whisker plots, illustrating the sample median, sample mean (circle), first and third quartiles, and whiskers extending to a maximum of 1.5× interquartile range beyond the box. Statistical analysis was performed using Welch’s t test.

Article Snippet: The SW48 cell line (CCL-231) and the Caco-2 cell line (HTB-37) were obtained from the American Type Culture Collection (ATCC).

Techniques: Mutagenesis, Imaging, Expressing, Membrane, Fluorescence, Whisker Assay

Journal: iScience

Article Title: Single-molecule imaging quantifies oncogenic KRAS dynamics for enhanced accuracy of therapeutic efficacy assessment

doi: 10.1016/j.isci.2025.113374

Figure Lengend Snippet: Quantitative analysis of transient trapping of individual SOS1 and BRAF molecules reveals that the association of KRAS with SOS1 and BRAF induces transient trapping (A) Representative images of single-molecule observations of tdStayGold-SOS1 or tdStayGold-BRAF recruited to the PM before and 3 min (SOS1) or 3.5 min (BRAF) after EGF stimulation in SW48 cells (left and middle panels) and trajectories of individual molecules acquired from 10-s movies recorded at video rate (33-ms resolution) after EGF stimulation (right panels, yellow lines: 1–100 frames; red lines: 101–200 frames; blue lines: 201–300 frames; see also and ). (B) Time course of the recruitment ratio of SOS1 or BRAF molecules to the PM. Data were normalized to values observed 1 min after EGF stimulation and are represented as mean ± SEM. (C) Representative 3-s trajectories of individual SOS1 and BRAF molecules 3 min (SOS1) or 3.5 min (BRAF) after EGF stimulation, exhibiting transient trapping highlighted by red segments and arrowheads. (D) Temporal fraction of transient trapping for single KRAS S17N, SOS1, and BRAF molecules in SW48 cells 1–3 min (SOS1) or 3.5 min (KRAS S17N and BRAF) after EGF stimulation. Data are presented as box-and-whisker plots, displaying the minimum, maximum, sample median, sample mean (circle), first and third quartiles, and whiskers extending to a maximum of 1.5× interquartile range beyond the box. (E–G) Distribution of trapping durations (E) and zone sizes (F) for KRAS S17N, SOS1, and BRAF molecules after EGF stimulation in SW48 cells. The size distribution is presented using both violin plots and box-and-whisker plots, illustrating the sample median, sample mean (circle), first and third quartiles, and whiskers extending to a maximum of 1.5× interquartile range beyond the box. The zone sizes of individual trapping events are plotted against trapping duration in (G) with Spearman’s rank correlation coefficient (ρ). Statistical analyses were performed using Welch’s t test. ∗∗∗ p < 0.001.

Article Snippet: The SW48 cell line (CCL-231) and the Caco-2 cell line (HTB-37) were obtained from the American Type Culture Collection (ATCC).

Techniques: Whisker Assay

Journal: iScience

Article Title: Single-molecule imaging quantifies oncogenic KRAS dynamics for enhanced accuracy of therapeutic efficacy assessment

doi: 10.1016/j.isci.2025.113374

Figure Lengend Snippet: Dual-color single-molecule imaging reveals the association of KRAS with SOS1 and BRAF within trapping zones (A–F) Simultaneous observation of single tdStayGold-KRAS molecules (green) and TMR-Halo7-SOS1 (A–C) or BRAF (D–F) molecules (magenta) after EGF stimulation in SW48 cells. Representative wide-field dual-color images (A and D), trajectories (B and E), and enlarged image sequences (C and F) highlight transient trapping events occurring exclusively during colocalization, as indicated by yellow circles and arrowheads, along with trapping durations (see also and ). (G) Schematic representation illustrating KRAS, which undergoes transient trapping due to association with GEF and BRAF. (H) Schematic diagram showing how prolonged bulk signaling may arise from the integration of short, pulse-like activation events of individual KRAS molecules.

Article Snippet: The SW48 cell line (CCL-231) and the Caco-2 cell line (HTB-37) were obtained from the American Type Culture Collection (ATCC).

Techniques: Imaging, Activation Assay

Journal: iScience

Article Title: Single-molecule imaging quantifies oncogenic KRAS dynamics for enhanced accuracy of therapeutic efficacy assessment

doi: 10.1016/j.isci.2025.113374

Figure Lengend Snippet: Cetuximab treatment suppresses the transient trapping of KRAS molecules in a mutant-dependent manner (A) Time course of temporal trapping fractions for KRAS WT and oncogenic mutants 2, 3.5, and 5 min after EGF stimulation under cetuximab treatment in SW48 cells (+Total includes all data 2, 3.5, and 5 min after stimulation). (B and C) Distributions of trapping zone sizes (B) and trapping durations (C) for KRAS WT and oncogenic mutants, with and without cetuximab treatment, 2–5 min after EGF stimulation in SW48 cells. (D–F) The temporal trapping fractions (D), trapping zone sizes (E), and their activation levels (quantified from western blot analysis; see B and S5C) (F) of KRAS WT and oncogenic mutants in the presence of cetuximab 2–5 min (D and E) and 3.5 min (F) after EGF stimulation. The temporal fraction of trapped molecules is presented using box-and-whisker plots, showing the minimum, maximum, sample median, sample mean (circle), first and third quartiles, and whiskers extending to a maximum of 1.5× interquartile range beyond the box. The size distribution of individual trapping zones is presented using both violin plots and box-and-whisker plots, illustrating the sample median, sample mean (circle), first and third quartiles, and whiskers extending to a maximum of 1.5× interquartile range beyond the box. The percentage of activated KRAS molecules is shown as bar graphs representing the mean ± SEM. Statistical analyses were performed using Welch’s t test. ∗ p < 0.05, ∗∗ p < 0.01, and ∗∗∗ p < 0.001.

Article Snippet: The SW48 cell line (CCL-231) and the Caco-2 cell line (HTB-37) were obtained from the American Type Culture Collection (ATCC).

Techniques: Mutagenesis, Activation Assay, Western Blot, Whisker Assay

Journal: iScience

Article Title: Single-molecule imaging quantifies oncogenic KRAS dynamics for enhanced accuracy of therapeutic efficacy assessment

doi: 10.1016/j.isci.2025.113374

Figure Lengend Snippet: Transient trapping of oncogenic KRAS mutants is disrupted by KRAS inhibitors, and combination therapies targeting upstream regulators of KRAS exhibit additive effects Comparison of the effect of cetuximab, KRAS inhibitor, or combination therapies involving a KRAS inhibitor and cetuximab, SOS1 inhibitor, or SHP2 inhibitor on the transient trapping of KRAS G12D and G12C after EGF stimulation in SW48 cells. (A) The temporal trapping fractions of KRAS 2–5 min after stimulation. Data are presented as box-and-whisker plots, displaying the minimum, maximum, sample median, sample mean (circle), first and third quartiles, and whiskers extending to a maximum of 1.5× interquartile range beyond the box. (B) The trapping zone sizes of KRAS 2–5 min after stimulation. Data are presented as both violin plots and box-and-whisker plots, illustrating the sample median, sample mean (circle), first and third quartiles, and whiskers extending to a maximum of 1.5× interquartile range beyond the box. (C) The trapping durations of KRAS 2–5 min after stimulation. Statistical analyses were performed using Welch’s t test. Significance levels of p values comparing KRAS inhibitor monotherapy with each combination therapy, or between combination therapies, were adjusted using Bonferroni correction as follows: ∗ p < 0.017, ∗∗ p < 0.003, and ∗∗∗ p < 0.0003.

Article Snippet: The SW48 cell line (CCL-231) and the Caco-2 cell line (HTB-37) were obtained from the American Type Culture Collection (ATCC).

Techniques: Comparison, Whisker Assay