AXL3 knockdown induces apoptosis of MCL cells. (A) K562 and (C) JeKo-1 cells were transduced with a doxycycline-inducible shAXL construct to block AXL expression. K562 and JeKo-1 control vector and shAXL cells were treated with an increasing dose of doxycycline for 96 hours. (A-D) Quantification of AXL expression by western blot after doxycycline treatment. (E) Quantification of cell number after doxycycline induction of shAXL. No effect on cell proliferation was observed for the control and shAXL cells. Experiments were performed in triplicate and the means ± standard deviations are shown. (F) Apoptosis on JeKo-1 shAXL or K562 shAXL cells was evaluated by annexin V/propidium iodide (PI) staining after 96 hours of doxycycline treatment. Experiments were performed in triplicates, and a representative flowchart is shown. (G) JeKo-1 cells were transfected with AXL3 siRNA and AXL protein expression was assessed by western blot (left). Quantification studies (right) showed that AXL expression was decreased in MCL after treatment with AXL siRNA as compared with cells treated with scrambled siRNA (P < .003). Experiments were performed in triplicates and the means ± standard deviations are shown. (H) Treatment of the JeKo-1 MCL cell line with AXL siRNA significantly decreased the number of cells in comparison with cells treated with scrambled siRNA, as assessed by using the 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium (MTS) assay (P < .0254) and (I) cell counting; the differences were statistically significant (P < .0122). (J) JeKo-1 cells were treated with scrambled or AXL3 siRNA and cell death was determined by trypan blue assay (P < .021). (K) The morphological changes were examined by light microscopy. Scale bar, 100 μM. Cell shrinkage and formation of apoptotic bodies were considered as apoptotic cells (original magnification ×200). (L) Using western blot, cleaved PARP was detectable in JeKo-1 cells treated with AXL3 siRNA (left). Quantification of PARP cleavage after AXL3 siRNA treatment (right). (M) Nucleolar morphological changes were observed under a fluorescence microscope after Hoechst-33342 staining. Scale bar, 20 μM. Condensed or fragmented nuclei were considered as apoptotic cells (original magnification ×400). Arrows indicate apoptotic cells. (N) AXL3 expression was downregulated using a CRISPRi method. sgRNAs targeting the AXL3 promoter were design to be induced by doxycycline treatment. Effect on AXL3 protein expression was monitored by western blot. Quantification of AXL3 expression in comparison with control cells. Experiments were performed in triplicates and the means ± standard deviations are shown. (O) Effect of AXL3 CRISPRi on cell proliferation was determined by cell counting. Experiments were performed in triplicates and the means ± standard deviations are shown.

AXL3 knockdown induces apoptosis of MCL cells. (A) K562 and (C) JeKo-1 cells were transduced with a doxycycline-inducible shAXL construct to block AXL expression. K562 and JeKo-1 control vector and shAXL cells were treated with an increasing dose of doxycycline for 96 hours. (A-D) Quantification of AXL expression by western blot after doxycycline treatment. (E) Quantification of cell number after doxycycline induction of shAXL. No effect on cell proliferation was observed for the control and shAXL cells. Experiments were performed in triplicate and the means ± standard deviations are shown. (F) Apoptosis on JeKo-1 shAXL or K562 shAXL cells was evaluated by annexin V/propidium iodide (PI) staining after 96 hours of doxycycline treatment. Experiments were performed in triplicates, and a representative flowchart is shown. (G) JeKo-1 cells were transfected with AXL3 siRNA and AXL protein expression was assessed by western blot (left). Quantification studies (right) showed that AXL expression was decreased in MCL after treatment with AXL siRNA as compared with cells treated with scrambled siRNA (P < .003). Experiments were performed in triplicates and the means ± standard deviations are shown. (H) Treatment of the JeKo-1 MCL cell line with AXL siRNA significantly decreased the number of cells in comparison with cells treated with scrambled siRNA, as assessed by using the 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium (MTS) assay (P < .0254) and (I) cell counting; the differences were statistically significant (P < .0122). (J) JeKo-1 cells were treated with scrambled or AXL3 siRNA and cell death was determined by trypan blue assay (P < .021). (K) The morphological changes were examined by light microscopy. Scale bar, 100 μM. Cell shrinkage and formation of apoptotic bodies were considered as apoptotic cells (original magnification ×200). (L) Using western blot, cleaved PARP was detectable in JeKo-1 cells treated with AXL3 siRNA (left). Quantification of PARP cleavage after AXL3 siRNA treatment (right). (M) Nucleolar morphological changes were observed under a fluorescence microscope after Hoechst-33342 staining. Scale bar, 20 μM. Condensed or fragmented nuclei were considered as apoptotic cells (original magnification ×400). Arrows indicate apoptotic cells. (N) AXL3 expression was downregulated using a CRISPRi method. sgRNAs targeting the AXL3 promoter were design to be induced by doxycycline treatment. Effect on AXL3 protein expression was monitored by western blot. Quantification of AXL3 expression in comparison with control cells. Experiments were performed in triplicates and the means ± standard deviations are shown. (O) Effect of AXL3 CRISPRi on cell proliferation was determined by cell counting. Experiments were performed in triplicates and the means ± standard deviations are shown.

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