Fig 1: Biological verification of the results of proteomic and N-glycoproteomic study. (A,B) Representative images of the Transwell migration (A) and invasion assay (B) of A549 and A549/DDP cells, and the quantitative number of migration and invasion cells. (C,D) WB assay of LRRC8A (C) and MRP1 (D) in ACM and DCM groups. (E) The amount of Pt in A549 and A549/DDP cells measured by ICP-MS. Error bars represent the mean ± SD; ** p < 0.01, **** p < 0.0001, Student’s t-test.
Fig 2: Effects of LRRC8A expression and metastases in lymph nodes on the survival time of colorectal cancer patients. (A and B) Expression of LRRC8A proteins in colon cancer adjacent and cancer tissues, respectively. (C and D) LRRC8A proteins in rectal cancer adjacent tissue and cancer tissue, respectively. (E and H) Average intensity of LRRC8A protein in the cancer tissue and the corresponding adjacent tissue of colorectal cancer patients with elevated and low expressed LRRC8A. (F) Colon cancer patients with elevated expression of LRRC8A in cancer tissue had poorer survival than patients with low expression of LRRC8A. (G) Colon cancer patients with metastases in the lymph nodes (node positive) had a shorter survival time than that of patients without metastases in the lymph nodes. (I and J) Graphs respectively demonstrate the influences of high expression of LRRC8A and positive lymph nodes on the survival of patients with rectal cancer. **P<0.01. LRRC8A, leucine-rich repeat-containing 8A.
Fig 3: Expression of LRRC8A proteins in the main organs of the human digestive system. (A-D) LRRC8A proteins were abundantly expressed in the stratified squamous epithelium of the esophagus and the columnar epithelium and glands of the stomach, colon and rectum, which were primarily located on the cell membrane. (E and F) LRRC8A proteins were distributed in the liver and pancreas. LRRC8A, leucine-rich repeat-containing 8A.
Fig 4: Downregulation of LRRC8A inhibits the EGF-induced migration of human colon cancer HCT116 cells. (A and B) Expression of LRRC8A protein was significantly decreased after HCT116 cells were transfected with LRRC8A siRNA for 72 h. (C-E) The typical time courses of hypotonicity-activated currents in non-treated (Ctrl), negative siRNA-treated, and LRRC8A siRNA-treated cells, respectively; currents induced by the hypotonic solutions could be inhibited by 20 µM tamoxifen and 100 µM NPPB chloride channel blockers, respectively. (F) The I–V relationships of chloride currents in the hypotonic solutions. (G) Images of EGF-induced cell migration captured under an inverted fluorescence microscope. The 2 upper rows are the images indicating that the wounds were scratched on the monolayer cells, referred to as 0 h. The two bottom rows present the migration of cells incubated in medium containing 10 ng/ml EGF for 48 h (referred to as 48 h; GFP, green fluorescent protein). (H) The migration distances of cells in the control, lentiviral scramble shRNA and lentiviral LRRC8A shRNA groups. n=3 experiments; **P<0.01 vs. control. LRRC8A, leucine-rich repeat-containing 8A.
Fig 5: Downregulation of LRRC8A inhibits the tumorigenesis of HCT116 cells in the nude mouse. (A) Images of HCT116 cell tumorigenesis captured by the in vivo non-invasive small animal molecular imaging system; the scramble and LRRC8A shRNA lentivirus-infected HCT116 cells were subcutaneously implanted at the nude mouse armpits of the right and left forelimb, respectively. (B) Sizes of cancer nodules dissected from the armpits of the right and left forelimbs after mice were fed for 3 weeks. (C) Mean body weights of mice during a consecutive 3 weeks after HCT116 cells were injected. (D and E) Radiant efficiencies and weights of cancer nodules in the scramble and LRRC8A shRNA groups, respectively. (F) Cancer nodule sections stained with H&E, TUNEL and CD31 in the scramble and LRRC8A shRNA groups; red arrows indicate the vessels. (G) Statistical analysis of vessel density. (H and I) Expression of LRRC8A protein in cancer nodules originating from the scramble or LRRC8A shRNA lentivirus-infected HCT116 cells. *P<0.05; **P<0.01. LRRC8A, leucine-rich repeat-containing 8A; H&E, hematoxylin and eosin.
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