Fig 2: The catalytic activity is responsible for the oncogenic roles of LOXL2.A Schematic representation of wild-type LOXL2, deletion mutant (?LOXL2), and double point mutant (H626Q/H628Q) with the HA-tag. B Western blotting showing the indicated LOXL2 variants stably expressed in PDAC cells using anti-LOXL2 and anti-HA antibodies. C Intracellular hydrogen peroxide measured in PDAC cells stably transfected with the indicated LOXL2 variants. D Western blotting showing hydroxylated HIF1a (HIF1a-OH) expression in PDAC cells stably transfected with the indicated LOXL2 variants treated with 10 µM MG132 for 6 h under normoxic and hypoxic conditions. E Western blotting showing HIF1a expression in PDAC cells stably transfected with the indicated LOXL2 variants under normoxic and hypoxic conditions. F Extracellular acid ratio (ECAR) in PDAC cells stably transfected with the indicated LOXL2 variants. G CCK-8 assay of PDAC cells stably transfected with the indicated LOXL2 variants. H, I Transwell migration (H) and invasion (I) assays of PDAC cells stably transfected with the indicated LOXL2 variants. J Western blotting showing HIF1a expression in LOXL2-overexpressing and vector control cells treated with 10 mM N-acetylcysteine (NAC) for 24 h under normoxic and hypoxic conditions. *p < 0.05, **p < 0.01, ns No significance.

Fig 3: LOXL2 expression profile in cervical cancer tissues. (A) Normalized expression of LOXL2 in the normal cervix and primary cancer using three expression profiling data (GSE63514, GSE7803, and GSE9750). P-values were calculated by Student's t-test Mean and SD were shown. (B) qPCR of LOXL2 relative expression in paired adjacent normal cervix and cancer tissues. Mean and SEM (n = 3) were shown. P-values were calculated by Two-way ANOVA test. #Means patients number. (C) Scoring of LOXL2 in the stained tissues. P-values were calculated by Chi-square test. (D) Representative images of IHC staining of LOXL2 in the adjacent normal cervix and primary cancer. (E) The correlation of LOXL2 expression and DNA methylation of LOXL2 promoter from MEXPRESS (19, 20), including 317 cervical squamous cell carcinoma and endocervical adenocarcinoma from TCGA datasets. The statistics (correlation coefficient (r) and P-value) on the right show how LOXL2 expression and DNA methylation of promoter were negatively correlated (* <0.05, ** <0.01, *** <0.001).

Fig 4: LOXL2 stabilizes HIF1a protein.A Western blotting showing HIF1a expression in LOXL2 knockdown and control cells under normoxic and hypoxic conditions. B Western blotting showing HIF1a expression in LOXL2-overexpressing and vector control cells under normoxic and hypoxic conditions. C, D HIF1a stability was examined by western blotting in LOXL2 knockdown and control cells. The cells were exposed to hypoxia for 6 h followed by incubation with 20 µg/ml cycloheximide (CHX) for the indicated times. E, F HIF1a stability was examined by western blotting in LOXL2-overexprssing and vector control cells. The cells were exposed to hypoxia for 6 h followed by incubation with 20 µg/ml CHX for the indicated times. G Western blotting showing HIF1a expression in LOXL2 knockdown and control cells treated with or without 10 µM MG132 for 6 h under normoxic and hypoxic conditions. H Western blotting showing HIF1a expression in LOXL2-overexpressing and vector control cells treated with or without 10 µM MG132 for 6 h under normoxic and hypoxic conditions.

Fig 5: Differential expression analysis of MIR29A, LOX, LOXL2, and VEGFA in HCC patients as compared to normal individuals. (A) Heatmap of MIR29A expression level in normal tissue and HCC, wherein 51 and 375 TCGA datasets were respectively accessed in the OncoMir Cancer Database (OMCD). MIR16 expression level served as normalization control. ** p < 0.01 between normal and HCC group. The raw data of TCGA samples retrieved from OMCD are shown in Supplementary File S1. (B) Serum MIR29A expression level of HCC patient and non-cancer individuals retrieved from the GSE113740 dataset. MIR16 expression level served as the normalization control. *** p < 0.001 between the non-cancer (Non) and HCC group. (C) The number (%) of HCC patients presenting a lower value than normal individuals at the cut-off of maximum, mean + 1SD, mean, mean-1SD, and minimum value of normal serum. (D) Receiver operating characteristic (ROC) curves for non-cancer and HCC cohorts based on serum MIR29A levels from the GSE113740 dataset. The gene expression level of LOX (E), LOXL2 (F), and VEGFA (G) in HCC and normal tissue retrieved from TCGA datasets. ** p < 0.01, *** p < 0.001 between the normal and HCC group. Panel E, F and G were adapted form UALCAN, which is an open assessed web server.