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: Genes

Article Title: Isoform-Specific Transcriptomic Effects of miR-133A1 , miR-133A2 , and miR-133B in a Colorectal Cancer Cell Line

doi: 10.3390/genes16111322

Figure Lengend Snippet: Functional categorization of differentially expressed genes regulated by miR-133 isoforms. ( A ) Schematic representation of mature miR-133 isoform sequences. miR-133A1 and miR-133A2 are identical, while miR-133B contains a G→A substitution at position 22. The seed region (positions 2–8, dashed purple box) is identical across all isoforms. ( B ) qRT-PCR validation of miR-133 overexpression in SW48 cells. Data represent mean ± SD (n = 3). (** p < 0.01, *** p < 0.001 vs. vector control). DOX(−): non-induced; DOX(+): doxycycline-induced. (C-E) Functional categorization of differentially expressed genes in miR-133A1 ( C ), miR-133A2 ( D ), and miR-133B ( E ) stable cell lines. Pie charts show proportional distribution across biological processes; bar graphs show directional regulation (red: upregulated; blue: downregulated).

Article Snippet: Human colorectal cancer SW48 cells were obtained from the American Type Culture Collection (ATCC, Manassas, VA, USA; Cat. No. CCL-231) and maintained in RPMI-1640 medium (Gibco) supplemented with 10% fetal bovine serum (FBS; HyClone, Logan, UT, USA), 100 U/mL penicillin, and 100 μg/mL streptomycin at 37 °C in a humidified atmosphere with 5% CO 2 .

Techniques: Functional Assay, Quantitative RT-PCR, Biomarker Discovery, Over Expression, Plasmid Preparation, Control, Stable Transfection

Journal: Genes

Article Title: Isoform-Specific Transcriptomic Effects of miR-133A1 , miR-133A2 , and miR-133B in a Colorectal Cancer Cell Line

doi: 10.3390/genes16111322

Figure Lengend Snippet: Comparative analysis of differentially expressed genes regulated by miR-133 isoforms. ( A ) Venn diagram showing the overlap of differentially expressed genes among SW48-KI133A1, SW48-KI133A2, and SW48-KI133B cells compared with parental SW48 controls. Numbers indicate isoform-specific and shared upregulated (red) and downregulated (blue) genes. ( B – D ) Scatter plots depicting log2-normalized expression changes in SW48-KI133A1 ( B ), SW48-KI133A2 ( C ), and SW48-KI133B ( D ) relative to SW48 control cells. Red dots represent significantly upregulated genes, while blue dots indicate significantly downregulated genes. The results highlight both common and isoform-specific transcriptional signatures regulated by miR-133 isoforms.

Article Snippet: Human colorectal cancer SW48 cells were obtained from the American Type Culture Collection (ATCC, Manassas, VA, USA; Cat. No. CCL-231) and maintained in RPMI-1640 medium (Gibco) supplemented with 10% fetal bovine serum (FBS; HyClone, Logan, UT, USA), 100 U/mL penicillin, and 100 μg/mL streptomycin at 37 °C in a humidified atmosphere with 5% CO 2 .

Techniques: Expressing, Control

Journal: Genes

Article Title: Isoform-Specific Transcriptomic Effects of miR-133A1 , miR-133A2 , and miR-133B in a Colorectal Cancer Cell Line

doi: 10.3390/genes16111322

Figure Lengend Snippet: Hierarchical clustering and correlation analysis of differentially expressed genes in miR-133 isoform-expressing SW48 cells. ( A ) Heatmaps showing hierarchical clustering of differentially expressed genes at three-fold change thresholds (>1.5, >2.0, and >3.0) for SW48-KI 133A1 , SW48-KI 133A2 , and SW48-KI 133B cells compared with parental SW48 controls. Rows represent individual genes, and columns represent two independent biological replicates. Red indicates upregulated genes, and blue indicates downregulated genes. At higher thresholds, core isoform-specific genes (labeled on the right) are highlighted. KI 133A1 and KI 133A2 show similar expression patterns, whereas KI 133B displays a distinct transcriptional profile. ( B ) Pearson correlation matrix (left) and scatter plots (right) of all biological replicates across samples. Strong correlations (r = 0.95–0.99) confirm high reproducibility between replicates and support the robustness of differential expression analysis. These results indicate that the observed transcriptomic differences among isoforms are biologically reproducible rather than experimental noise. Asterisks indicate statistical significance (*** p < 0.001).

Article Snippet: Human colorectal cancer SW48 cells were obtained from the American Type Culture Collection (ATCC, Manassas, VA, USA; Cat. No. CCL-231) and maintained in RPMI-1640 medium (Gibco) supplemented with 10% fetal bovine serum (FBS; HyClone, Logan, UT, USA), 100 U/mL penicillin, and 100 μg/mL streptomycin at 37 °C in a humidified atmosphere with 5% CO 2 .

Techniques: Expressing, Labeling, Quantitative Proteomics

Journal: Genes

Article Title: Isoform-Specific Transcriptomic Effects of miR-133A1 , miR-133A2 , and miR-133B in a Colorectal Cancer Cell Line

doi: 10.3390/genes16111322

Figure Lengend Snippet: Comparative GO and KEGG enrichment analysis of differentially expressed genes across miR-133 isoforms. The upper panel presents a merged visualization of enriched GO categories (BP: Biological Process, CC: Cellular Component, MF: Molecular Function) combining data from all three miR-133 isoforms in a single integrated bar plot. The lower panel displays KEGG pathway enrichment analysis. Bar plots show enrichment in SW48-KI 133A1 (red/pink bars), SW48-KI 133A2 (blue/light blue bars), and SW48-KI 133B (dark blue/yellow bars) compared with parental SW48 controls. For each isoform, darker-colored bars represent downregulated gene sets and lighter-colored bars represent upregulated gene sets. Bars are grouped by functional category to facilitate direct comparison across the three isoforms. The side-by-side presentation reveals highly convergent enrichment patterns across all three miR-133 isoforms, with consistent regulation of cell proliferation, metal ion homeostasis, extracellular components, protein binding functions, and cancer-associated signaling pathways.

Article Snippet: Human colorectal cancer SW48 cells were obtained from the American Type Culture Collection (ATCC, Manassas, VA, USA; Cat. No. CCL-231) and maintained in RPMI-1640 medium (Gibco) supplemented with 10% fetal bovine serum (FBS; HyClone, Logan, UT, USA), 100 U/mL penicillin, and 100 μg/mL streptomycin at 37 °C in a humidified atmosphere with 5% CO 2 .

Techniques: Functional Assay, Comparison, Protein Binding, Protein-Protein interactions

Journal: Genes

Article Title: Isoform-Specific Transcriptomic Effects of miR-133A1 , miR-133A2 , and miR-133B in a Colorectal Cancer Cell Line

doi: 10.3390/genes16111322

Figure Lengend Snippet: Heatmap representation of fold change values for representative genes regulated by miR-133 isoforms. Heatmaps show the relative expression (fold change) of selected genes in SW48-KI 133-A1 , SW48-KI 133-A2 , and SW48-KI 133-B compared with parental SW48 controls. Red indicates upregulation (fold change > 1.5), and blue indicates downregulation (fold change < 0.67). ( A ) Apoptosis-related genes. ( B ) Cell cycle genes. ( C ) Cell migration genes. ( D ) Immune response genes. ( E ) Cell differentiation genes. ( F ) Angiogenesis-related gene. ( G ) Inflammatory response genes. Note that some panels share common genes (e.g., PPIA) as these genes are functionally involved in multiple biological processes.

Article Snippet: Human colorectal cancer SW48 cells were obtained from the American Type Culture Collection (ATCC, Manassas, VA, USA; Cat. No. CCL-231) and maintained in RPMI-1640 medium (Gibco) supplemented with 10% fetal bovine serum (FBS; HyClone, Logan, UT, USA), 100 U/mL penicillin, and 100 μg/mL streptomycin at 37 °C in a humidified atmosphere with 5% CO 2 .

Techniques: Expressing, Migration, Cell Differentiation

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