Journal: Redox Biology

Article Title: Targeting HIF-1α promotes ferroptosis and boosts antitumor immunity in MSS colorectal cancer

doi: 10.1016/j.redox.2026.104151

Figure Lengend Snippet: A hypoxia-characteristic cluster identified by Microwell-seq exhibited pronounced ferroptosis resistance in MSS CRC cells. (A and B) t-distributed stochastic neighbor embedding (t-SNE) plot of Microwell-seq analysis based on gene expressions of SW480 and WiDr. (C and D) Comparative gene set enrichment analysis (GSEA) of signaling pathways in different clusters. The red color represents up-regulation and blue represents down-regulation, calculated with the formula: ± Log2|NES/p.adjust|. Grey color represents no enrichment in the indicated pathway. NES: normalized enrichment score. (E and F) GSEA analysis showed the indicated pathway activity between the hypoxia cluster and other clusters. (G and H) Correlation analysis of hypoxia scores, glycolysis scores, and ferroptosis suppressor scores in WiDr and SW480 cells was performed using Pearson's method. (I and J) The ferroptosis suppressor score of SW480 and WiDr with DMSO or RSL3 treatment was analyzed by AddModuleScore tool. P values were calculated by Wilcox.test. (K and L) The ferroptosis suppressor score of hypoxia cluster in SW480 and WiDr treated with DMSO or RSL3 was shown. P values were calculated by Wilcox.test.

Article Snippet: The human MSS CRC cell lines SW480, HT-29, and WiDr, human HEK293T, and mouse MSS CRC cell line CT26 were obtained from American Type Culture Collection (ATCC).

Techniques: Protein-Protein interactions, Activity Assay

Journal: Redox Biology

Article Title: Targeting HIF-1α promotes ferroptosis and boosts antitumor immunity in MSS colorectal cancer

doi: 10.1016/j.redox.2026.104151

Figure Lengend Snippet: HIF-1α nuclear distribution increased in RSL3-resistant CT26 cells and significantly promoted tumourigenicity and metastasis. (A) The diagram demonstrated the procedure for sphere formation. (B and C) The representative images of sphere formation in MSS CRC cells and quantification analysis of sphere number derived from SW480, HT-29, and WiDr. (D) HIF-1α expression in WiDr was detected by western blotting. (E) qPCR analyzed the indicated gene expression involved in glycolysis in WiDr spheres. (F) Cell viability of parental CT26 and RSL3-resistant CT26 (Re-CT26). (G) Cytoplasm and nuclear HIF-1α expression in CT26. α-tubulin was used as an internal reference for the cytoplasm, and Histone 3 was used as a nuclear reference. (H) qPCR analyzed the indicated gene expression involved in glycolysis in CT26. (I) Image of subcutaneous tumors derived from parental CT26 and Re-CT26. (J) Tumor volume determined by formula: 0.52 × Long × width 2 . (K)Tumor weight of subcutaneous tumors. (L) Bioluminescent images in liver metastatic models. (M) Images of liver metastasis derived from parental CT26 and Re-CT26. (N) Number of liver nodules in liver metastasis models. (O) Representative hematoxylin and eosin (H&E) and immunohistochemical staining images from liver metastasis models. Scale bar: 25 μm. (P) Quantification of immunohistochemical staining results shown in (O). Data are shown as means ± SD. ∗ P < 0.05; ∗∗ P < 0.01; ∗∗∗ P < 0.001; ns: not significant. Two-way ANOVA in (J), others unpaired two-tailed Student's t -test.

Article Snippet: The human MSS CRC cell lines SW480, HT-29, and WiDr, human HEK293T, and mouse MSS CRC cell line CT26 were obtained from American Type Culture Collection (ATCC).

Techniques: Derivative Assay, Expressing, Western Blot, Gene Expression, Immunohistochemical staining, Staining, Two Tailed Test

Journal: Redox Biology

Article Title: Targeting HIF-1α promotes ferroptosis and boosts antitumor immunity in MSS colorectal cancer

doi: 10.1016/j.redox.2026.104151

Figure Lengend Snippet: P4HA1 was a major factor regulated by HIF-1α and was enriched in ferroptosis-resistant cells. (A) Venn diagram screening 10 genes commonly induced by hypoxia and the glycolysis pathway in SW480 and WiDr by single-cell sequencing. (B) Gene expression heatmap showed the distribution of genes in different clusters in the presence or absence of RSL3. D: DMSO, R: RSL3. (C) Correlation between ferroptosis suppressor score and P4HA1 in MSS CRC containing 119 patients using Pearson's method. (D) Kaplan-Meier plots of RFS in MSS colon cancer patients according to P4HA1 expression. (E) The correlation between HIF-1α and P4HA1 was evaluated by Spearman's analysis in a colon adenocarcinoma cohort of 457 patients. (F) Relative P4HA1 mRNA expression after HIF-1α knockdown, detected by qPCR. (G) Relative P4HA1 protein expression after HIF-1α knockdown, detected by Western blot. (H) Relative P4HA1 mRNA expression after HIF-1α overexpression, detected by qPCR. (I) Relative P4HA1 protein expression after HIF-1α overexpression, detected by Western blot. (J) Relative P4HA1 expression, detected by qPCR. (K) HIF-1α binding motif predicted from JASPAR. (L) The prospective binding site of HIF-1α on the promoter of P4HA1. (M) ChIP assay of HIF-1α and IgG in parental CT26 cells or Re-CT26 cells, followed by qPCR for the binding sequences.

Article Snippet: The human MSS CRC cell lines SW480, HT-29, and WiDr, human HEK293T, and mouse MSS CRC cell line CT26 were obtained from American Type Culture Collection (ATCC).

Techniques: Single Cell, Sequencing, Gene Expression, Expressing, Knockdown, Western Blot, Over Expression, Binding Assay

Journal: Redox Biology

Article Title: Targeting HIF-1α promotes ferroptosis and boosts antitumor immunity in MSS colorectal cancer

doi: 10.1016/j.redox.2026.104151

Figure Lengend Snippet: HIF-1α inhibition enhanced ferroptosis inducer sensitivity in MSS CRC cells. (A and B) Cell viability of HT-29 or SW480 treated with RSL3, BAY 87-2243 alone or in combination treatment. (C and D) DCFH-DA oxidation in HT-29 or SW480 treated with RSL3, BAY 87-2243 alone or in combination treatment were quantified using flow cytometry with DCFH-DA probe. (E and F) Lipid peroxidation levels of HT-29 or SW480 treated with RSL3, BAY 87-2243 alone or in combination treatment were detected using flow cytometry with C11-BODIPY 581/591 (FITC channel; excitation/emission: 488/510 nm). (G) MDA levels in cells subjected to the indicated treatment. (H) GSH levels in cells subjected to the indicated treatment. Results are shown as means ± SD. ∗ P < 0.05 ; ∗∗ P < 0.01 ; ∗∗∗ P < 0.001 ; ∗∗∗∗ P < 0.0001 . P values were calculated by one-way ANOVA.

Article Snippet: The human MSS CRC cell lines SW480, HT-29, and WiDr, human HEK293T, and mouse MSS CRC cell line CT26 were obtained from American Type Culture Collection (ATCC).

Techniques: Inhibition, Flow Cytometry

Journal: The Journal of Experimental Medicine

Article Title: MYCT1–IFITM2/3 interaction links endothelial endolysosomal trafficking to white adipose tissue expansion

doi: 10.1084/jem.20251497

Figure Lengend Snippet: MYCT1 is a transmembrane phosphoglycoprotein that interacts with IFITM2/3, related to Fig. 5 . (A) The MYCT1 protein is highly conserved across vertebrates. An alignment of human MYCT1 protein sequence with those of the indicated species, amino acid conservation is color-coded as indicated in the legend below. Percentages next to species indicate amino acid sequence identity (left) and homology (right) in comparison with human sequence. (B) Short MYCT1 isoform is predominant in ECs. Cells were transduced with Ad- GFP (control) or Ad- MYCT1 (187-aa isoform with C-terminal V5 tag) adenoviruses. Western blot analysis for the indicated proteins. (C) Workflow for mass spectrometry experiments. MYCT1-negative SW480 colon cancer cells were used to exclude nonspecific interactors pulled down by MYCT1 IgG. n = 2 independent experiments. (D) Venn diagram showing how the short list of MYCT1 interactors was selected. (E) Interaction between MYCT1 and IFITM2/3 was analyzed by PLA in ECs. siRNA-mediated knockdown of either protein confirmed specificity of PLA signal. Staining of ECs for PLA dots (gray), VE-cadherin (magenta), and DNA (blue). Scale bar, 50 µm. (F) Quantification of the number of PLA dots per cell in control, MYCT1 KD , and IFITM2/3 KD cells. n = 3 independent experiments; 500–1,500 cells were analyzed per condition for each experiment; mean ± SD; one-way ANOVA with Dunnett’s multiple comparisons, P = 0.036 (*) for MYCT1 knockdown effect. (G) Positive control (VE-cadherin::β-catenin) and negative control (MYCT1 antibody alone) for PLA signal in human brain sections. PLA dots (gray) and staining of ECs for VE-cadherin or Pecam1 (magenta) and DNA (blue). Arrowhead, colocalization of PLA dots with vascular marker. Scale bar, 20 µm. Source data are available for this figure: .

Article Snippet: 293T, SW480, and LLC cells (ATCC) were cultured in complete DMEM (D5030; Sigma-Aldrich) supplemented with 10% FBS, and 1% penicillin/streptomycin.

Techniques: Sequencing, Comparison, Transduction, Control, Western Blot, Mass Spectrometry, Knockdown, Staining, Positive Control, Negative Control, Marker

Journal: The Journal of Experimental Medicine

Article Title: MYCT1–IFITM2/3 interaction links endothelial endolysosomal trafficking to white adipose tissue expansion

doi: 10.1084/jem.20251497

Figure Lengend Snippet: MYCT1 is a transmembrane phosphoglycoprotein that interacts with IFITM2/3, related to Fig. 5 . (A) The MYCT1 protein is highly conserved across vertebrates. An alignment of human MYCT1 protein sequence with those of the indicated species, amino acid conservation is color-coded as indicated in the legend below. Percentages next to species indicate amino acid sequence identity (left) and homology (right) in comparison with human sequence. (B) Short MYCT1 isoform is predominant in ECs. Cells were transduced with Ad- GFP (control) or Ad- MYCT1 (187-aa isoform with C-terminal V5 tag) adenoviruses. Western blot analysis for the indicated proteins. (C) Workflow for mass spectrometry experiments. MYCT1-negative SW480 colon cancer cells were used to exclude nonspecific interactors pulled down by MYCT1 IgG. n = 2 independent experiments. (D) Venn diagram showing how the short list of MYCT1 interactors was selected. (E) Interaction between MYCT1 and IFITM2/3 was analyzed by PLA in ECs. siRNA-mediated knockdown of either protein confirmed specificity of PLA signal. Staining of ECs for PLA dots (gray), VE-cadherin (magenta), and DNA (blue). Scale bar, 50 µm. (F) Quantification of the number of PLA dots per cell in control, MYCT1 KD , and IFITM2/3 KD cells. n = 3 independent experiments; 500–1,500 cells were analyzed per condition for each experiment; mean ± SD; one-way ANOVA with Dunnett’s multiple comparisons, P = 0.036 (*) for MYCT1 knockdown effect. (G) Positive control (VE-cadherin::β-catenin) and negative control (MYCT1 antibody alone) for PLA signal in human brain sections. PLA dots (gray) and staining of ECs for VE-cadherin or Pecam1 (magenta) and DNA (blue). Arrowhead, colocalization of PLA dots with vascular marker. Scale bar, 20 µm. Source data are available for this figure: .

Article Snippet: SW480 cells , ATCC , CCL-228.

Techniques: Sequencing, Comparison, Transduction, Control, Western Blot, Mass Spectrometry, Knockdown, Staining, Positive Control, Negative Control, Marker