Journal: JCI Insight

Article Title: The critical role of GRP78/BiP MARylation in ER stress of KRAS-mutant colorectal cancer

doi: 10.1172/jci.insight.182809

Figure Lengend Snippet: The analysis of overall survival ( A ), progression-free survival ( B ), disease-free survival ( C ), and disease-specific survival ( D ) analysis of patients with KRAS-WT and -mutant CRC based on the Cbioportal database. ( E ) The proportion of each mutation subtype in KRAS-mutant CRC. ( F ) The proportion of patients with KRAS-WT and -mutant patients in each subtype of CRC. ( G ) The mutation event frequency of various genes in KRAS-WT and -mutant CRC. ( H ) Differentially expressed genes in patients with KRAS-WT and -mutant CRC. ( I ) ART1 mRNA levels in KRAS-WT and -mutant CRC tissues. P < 0.05 by t test. ( J ) The positive intensity of ART1 in KRAS-WT and -mutant CRC tissues detected by immunohistochemistry. Original magnification, ×20 (top) and ×40 (bottom). ( K ) The expression of ART1 protein in KRAS-WT (SW48, Caco2, HT-29) and -mutant CRC cell lines (LoVo, HCT-116, SW480) observed by Western blot. Immunohistochemical detection of the expression of GRP78/BiP ( L ), HSC70 ( M ), and CHOP ( N ) in KRAS-WT and -mutant CRC tissues. Original magnification, ×200 (left) and ×400 (right). ( O ) The expression of GRP78/BiP, HSC70, and CHOP proteins in KRAS-WT and -mutant CRC cell lines detected by Western blot. ( P ) Transmission electron microscopy shows the morphology of the ER (arrows) in KRAS-WT and -mutant CRC cell lines. Original magnification, ×25,000.

Article Snippet: Human CRC cell lines SW48 (KRAS WT), HT-29 (KRAS WT), SW480 (G12V), and HCT116 (G13D) were obtained from American Type Culture Collection (ATCC).

Techniques: Mutagenesis, Immunohistochemistry, Expressing, Western Blot, Immunohistochemical staining, Transmission Assay, Electron Microscopy

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