Fig 2: CD44 overexpressing cells have a more aggressive phenotype and improved response to INK128(A) PLC/PRF/5 and Huh7 cells were stably transfected with CD44 expressing vector or empty vector (EV) control. CD44 and GAPDH levels were measured by immunoblotting. (B) Cell growth curves of Huh7CD44 and Huh7EV cells. (C) In vitro migration of Huh7CD44 and Huh7EV cells. (D) In vitro migration of Huh7CD44 and Huh7EV and PLC/PRF/5CD44 and PLC/PRF/5EV cells. (E) Huh7CD44, Huh7EV, PLC/PRF/5CD44 and PLC/PRF/5EV cells were treated with 500 nM INK128 or vehicle control for 72 h. Cell proliferation was measured by a WST-1 assay. (F) Huh7 and PLC/PRF/5 expressing CD44 or EV control cells were treated with sorafenib for 72 h and cell viability was determined using WST-1 reagent. **p < 0.01, Student's t-test.

Fig 3: Effect of CDK5RAP3 in gastric CSCs.a Photos and graph of AGS and KATOIII cells transduced with CDK5RAP3 or Vector and grew in spheroid formation conditions. Scale bar, 50 μm. b Western blot analysis and immunofluorescence staining demonstrating levels of CDK5RAP3, CD44, Sox2, Oct4, Nanog and c-Myc in spheroid cells of AGS and KATOIII transduced with CDK5RAP3 or Vector. c Graphs and photos of migration and invasion assays for AGS and KATOIII spheroid cells transduced with CDK5RAP3 or Vector. d Photos and graph of NCI-N87 and SNU668 cells transduced with sh.CDK5RAP3 or sh.Scr and grown in spheroid formation conditions. Scale bar, 50 μm. e Photos and graphs of migration and invasion assays for AGS and KATOIII in spheroids transduced with CDK5RAP3 or Vector. f Photos and graphs of migration and invasion assays for NCI-N87 an dSNU668 in spheroids transduced with sh.CDK5RAP3 or sh.Scr. β-actin was used as a loading control. *p < 0.05 compared to control.

Fig 4: Two distinct phenotypes of SCLC cell line NCI-H69. (A) The arrows indicated the bright-field images of H69A and H69S cells. Magnification, ×200. (B) H69A and H69S cells were cultured for 48 h and then stained with trypan blue. Cell proliferation was analyzed using Countstar. (C) H69A and H69S cells were each cultured for 24 and 48 h and then stained with trypan blue. Adherent cells and suspended cells were collected. Cell numbers were counted using Countstar. (D) H69A and H69S cells were collected and western blotting was used to detect the expression of SYP and CD44. GAPDH was the internal standard. (E) Quantitative analysis of (D): H69A was the control (100%). Data are presented as the mean ± standard deviation of three independent experiments. (F) Western blotting was used to detect the expression of ASCL1, NEUROD1, POU2F3, YAP1 and GAPDH. (G) Quantitative analysis of (F): H69A was the control (100%). Data are presented as the mean ± standard deviation of three independent experiments. *P<0.05. SCLC, small cell lung cancer; SYP, synaptophysin; ASCL1, achaete-scute homologue 1; NEUROD1, neurogenic differentiation factor 1; POU2F3, POU class 2 homeobox 3; YAP1, YES-associated protein 1.

Fig 5: The effect of CSF2 inhibitors on drug sensitivity and the conversion of the non-NE phenotype to the NE phenotype. H69A cells were treated with different concentrations of butoconazole nitrate (0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 and 11 µg/ml) and cell viability was detected with Alamar Blue after treatment for (A) 24 h and (B) 48 h. (C) H69A cells treated with 3 and 7 µg/ml butoconazole nitrate. The cells were collected after treatment for 24 and 48 h. The expression of CSF2 and GAPDH were analyzed by using western blotting. (D) Quantitative analysis of (C), mock-treated H69A cells were used as the control (100%). Data are presented as the mean ± standard deviation of three independent experiments. (E) H69A cells were treated with butoconazole nitrate (7 µg/ml) combined with anlotinib (0, 0.5, 1, 1.5, 2, 3, 4 and 6 µg/ml), (F) CS2164 (0, 1, 2, 2.5, 3, 4, 6 and 10 µg/ml) and (G) RAD001 (0, 1, 2, 4, 6, 8, 10 and 20 µg/ml) for 24 h. Cell viability was tested by using Alamar Blue. (H) H69A cells were treated with butoconazole nitrate (3 and 7 µg/ml) for 24 h. Western blotting was used to detect the expression of CSF2, SYP, CD44, YAP1 and GAPDH. (I) Quantitative analysis of (H), mock-treated H69A cells were used as the control (100%). Data are presented as the mean ± standard deviation of three independent experiments. *P<0.05. CSF2, colony-stimulating factor 2; NE, neuroendocrine; CSF2, colony-stimulating factor 2; SYP, synaptophysin; YAP1, YES-associated protein 1.