Journal: Human Mutation

Article Title: CLEC4G Promotes Pancreatic Cancer Progression by Suppressing Cathepsin B‐Mediated Ferroptosis: Evidence From Mendelian Randomization Study and Experimental Validation

doi: 10.1155/humu/7359548

Figure Lengend Snippet: CLEC4G expression in pancreatic cancer cell lines and the effects of CLEC4G knockdown on malignant phenotypes. (A) Basal CLEC4G protein levels in the indicated pancreatic cancer cell lines were assessed by western blotting. AsPC‐1, MIA PaCa‐2, and BxPC‐3 cells showed relatively high CLEC4G expression and were selected for subsequent functional analyses. (B) Knockdown efficiency of CLEC4G in AsPC‐1, MIA PaCa‐2, and BxPC‐3 cells was confirmed by western blotting after siRNA transfection. (C) Cell proliferation curves of AsPC‐1, MIA PaCa‐2, and BxPC‐3 cells transfected with control siRNA (siNC) or siRNA targeting CLEC4G (siCLEC4G). (D) Colony formation assays of AsPC‐1, MIA PaCa‐2, and BxPC‐3 cells following CLEC4G knockdown. (E) Transwell migration assays of AsPC‐1, MIA PaCa‐2, and BxPC‐3 cells after CLEC4G silencing (Scale − bars = 200 μ m). (F) Transwell invasion assays of AsPC‐1, MIA PaCa‐2, and BxPC‐3 cells after CLEC4G silencing (Scale − bars = 50 μ m). Data are presented as mean ± SD. ∗∗ p < 0.01, ∗∗∗ p < 0.001.

Article Snippet: PC cells AsPC‐1, MIA PaCa‐2, BxPC‐3, Panc‐1, and SW1990 were purchased from the American Type Culture Collection (ATCC).

Techniques: Expressing, Knockdown, Western Blot, Functional Assay, Transfection, Control, Migration

Journal: Human Mutation

Article Title: CLEC4G Promotes Pancreatic Cancer Progression by Suppressing Cathepsin B‐Mediated Ferroptosis: Evidence From Mendelian Randomization Study and Experimental Validation

doi: 10.1155/humu/7359548

Figure Lengend Snippet: CLEC4G knockdown increases CTSB expression and enhances ferroptosis‐associated changes in pancreatic cancer cells. (A) Western blot analysis of CLEC4G and CTSB in MIA PaCa‐2 and AsPC‐1 cells after transfection with siNC or siCLEC4G. Total CTSB was quantified as the combined densitometric intensity of pro‐CTSB and mature CTSB normalized to the loading control. (B) RT‐qPCR analysis of CTSB mRNA expression after CLEC4G knockdown. (C) Fluorescence imaging of intracellular oxidative stress and labile iron‐associated signals in MIA PaCa‐2 and AsPC‐1 cells after CLEC4G knockdown. Scale bars = 50 μ m. (D, E) Flow cytometry profiles and quantification of labile Fe 2+ and ROS‐associated fluorescence in AsPC‐1 and MIA PaCa‐2 cells transfected with siNC or siCLEC4G. Data are presented as mean ± SD. ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001.

Article Snippet: PC cells AsPC‐1, MIA PaCa‐2, BxPC‐3, Panc‐1, and SW1990 were purchased from the American Type Culture Collection (ATCC).

Techniques: Knockdown, Expressing, Western Blot, Transfection, Control, Quantitative RT-PCR, Fluorescence, Imaging, Flow Cytometry

Journal: Human Mutation

Article Title: CLEC4G Promotes Pancreatic Cancer Progression by Suppressing Cathepsin B‐Mediated Ferroptosis: Evidence From Mendelian Randomization Study and Experimental Validation

doi: 10.1155/humu/7359548

Figure Lengend Snippet: Inhibition of CTSB reverses CLEC4G knockdown‐induced ferroptosis and suppressive effects on tumor cell growth and motility. (A) Flow cytometry measurement of intracellular labile Fe 2+ in AsPC‐1 pancreatic cancer cells transfected with control siRNA (siNC), CLEC4G siRNA (siCLEC4G), or combined CLEC4G and CTSB siRNAs (siCLEC4G + siCTSB). CLEC4G knockdown increases Fe 2+ levels compared with control, whereas concurrent CTSB knockdown reduces Fe 2+ levels toward control (Scale − bars = 50 μ m). (B) Flow cytometry measurement of lipid reactive oxygen species (ROS) under the same conditions. CLEC4G knockdown elevates lipid ROS relative to control, and CTSB coknockdown mitigates this increase, returning ROS levels closer to control. (C) Cell proliferation assay (72‐h viability) under the three treatment conditions. CLEC4G knockdown alone reduces cell proliferation compared with control, whereas coknockdown of CTSB leads to higher proliferation than CLEC4G knockdown alone (partial restoration toward control levels). (D) Colony formation assay for AsPC‐1 cells under the three treatments. CLEC4G knockdown yields fewer and smaller colonies than control; in contrast, dual knockdown results in more colonies than CLEC4G knockdown alone. (E) Cell migration assay. CLEC4G knockdown decreases the number of migrating cells relative to control, whereas adding CTSB knockdown increases cell migration compared with CLEC4G knockdown alone. (F) Cell invasion assay (Scale − bars = 200 μ m). CLEC4G knockdown reduces cell invasiveness, and dual knockdown of CLEC4G + CTSB leads to greater invasion than CLEC4G knockdown alone (Scale − bars = 200 μ m).

Article Snippet: PC cells AsPC‐1, MIA PaCa‐2, BxPC‐3, Panc‐1, and SW1990 were purchased from the American Type Culture Collection (ATCC).

Techniques: Inhibition, Knockdown, Flow Cytometry, Transfection, Control, Proliferation Assay, Colony Assay, Cell Migration Assay, Migration, Invasion Assay