Journal: Cell Death & Disease

Article Title: Integrated multi-omics profiling of treatment-naive cervix uteri premalignant lesions and cervical squamous cell carcinoma reveals ecosystem and drivers underlying cervical cancer progression

doi: 10.1038/s41419-026-08960-2

Figure Lengend Snippet: A CLDN1 expression was detected in CLDN1-overexpressed human cervical epithelial cells (CECs) by Western blotting. B ISG15 expression was verified in ISG15-overexpressed CECs by Western blotting. C Proliferation of CLDN1-overexpressed human CECs was assessed by MTT assay. D Proliferation of ISG15-overexpressed human CECs was detected by MTT assay. E Proliferation of SiHa cells with or without human cervical fibroblasts co-culture was assessed by MTT assay. F , G Colony formation of SiHa cells with or without coculture human cervical fibroblasts. H Western blot analysis was performed to determine the levels of p-FGFR1 (Y654) and p-AKT (S473) in SiHa cells under conditions with or without cervical fibroblasts co-culture. I Molecular docking model indicated the binding between FGF1 and ISG15. (docking score = -242.92; confidence score: 0.8651). J Interaction of FGF1 with ISG15 was verified by SPR assay (KD (M) = 1.442E-8, FGF1 concentration: 0.078, 0.15625, 0.3125, 1.25, 2.5 µM). K Immunoprecipitation assay of FGF1 and ISG15 in C-33A and SiHa cells. L Real-time PCR analysis of FGF1 mRNA levels after ISG15 overexpression. M Western blotting assay of FGF1 protein levels following ISG15 overexpression in HEK293, C-33A, and CaSki cells. N C-33A and CaSki cells transfected with empty vector or ISG15-encoding plasmid were treated with cycloheximide (CHX) for the indicated times. O Western blotting was used to measure FGF1 expression following 3-MA and MG132 treatment. P , Q Lysates from cells expressing control shRNA or MG132-treated ISG15 knockdown cells were immunoprecipitated to assess FGF1-ISG15 interaction. R Lysates from cells expressing an empty vector or ISG15, MG132-treated cells were immunoprecipitated to detect the binding affinity. S Total lysates derived from HEK293 cells co-transfected with different plasmids for 48 h and subsequently treated with MG132 for 6-8 h were immunoprecipitated to detect the binding affinity. T Proliferation of cervical cancer cells treated with graded concentrations of PD161570 was assessed by MTT assay. U Colony formation assays of cervical cancer cells treated with different concentrations of PD161570 . V Expression of key markers in the FGF1/FGFR1/PI3K/AKT signaling pathway was examined by Western blotting in CLDN1-, ISG15-overexpressed, and PTGDS-knockdown cells. W The diagram of the interaction between inflammatory cancer-associated fibroblasts and tumor cells in the progression of cervical squamous cell carcinoma. Statistical significance was determined by Student’s t -test ( C – F , L ) and one-way ANOVA ( T , U ). The p- value < 0.05 was considered statistically significant ( p < 0.05: *, p < 0.01: **, p < 0.001: ***, p < 0.0001: ****).

Article Snippet: The reagents we used in this study were as follows: PI3Kα inhibitor alpelisib (HY-15244, MedChemExpress), pan-AKT inhibitor capivasertib (HY-15431, MedChemExpress), mTOR1 inhibitor everolimus (HY-10218, MedChemExpress), cycloheximide (HY-12320, MedChemExpress), FGF1 receptor inhibitor PD161570 (HY-100434, MedChemExpress), proteasome and calpain inhibitor MG132 (HY-13259, MedChemExpress), autophagy inhibitor 3-Methyladenine (HY-19312, MedChemExpress), Recombinant human ISG15 protein (I245-31H, Sino Biological), Recombinant human FGF1 protein (ab91374, abcam).

Techniques: Expressing, Western Blot, MTT Assay, Co-Culture Assay, Binding Assay, SPR Assay, Concentration Assay, Immunoprecipitation, Real-time Polymerase Chain Reaction, Over Expression, Transfection, Plasmid Preparation, Control, shRNA, Knockdown, Derivative Assay