Journal: Cell Death & Disease

Article Title: Inhibitory role of proguanil on the growth of bladder cancer via enhancing EGFR degradation and inhibiting its downstream signaling pathway to induce autophagy

doi: 10.1038/s41419-022-04937-z

Figure Lengend Snippet: A 3D photocrosslinked microarray was used to immobilize proguanil, and then T24 lysates were distributed on the surface of the microarray. The captured target proteins by proguanil on the microarray surface were detected by SPRi and identified by LC-MS. B MOE molecular docking (using MOE14.0 software) was used to predict the binding site of proguanil and EGFR (PDB ID − 4HJO). C T24 were plated in 6-well plates (3.0 × 10 5 /well). When the cell density reaches 70–80%, cells were starved for 12 h and then treated with proguanil for 6 h, and the expression of p-EGFR and EGFR was detected by WB. D T24 were plated in 6-well plates (3.0 × 10 5 /well). When the cell density reaches 70–80%, cells were starved for 12 h and then treated with proguanil for 6 h, and the expression level of EGFR mRNA was detected by RT-PCR. E T24 were plated on glass disks in 12-well plates (2.0 × 10 4 /well). After 24 h, T24 cells were starved for 12 h and then treated with proguanil, and the changes of EGFR were detected by immunofluorescence. Data are representative of three independent experiments. Error bars represent means ± SD from triplicate experiments. Vehicle control means the concentration of DMSO lower than 0.3% (* P < 0.05, ** P < 0.01, *** P < 0.001, ns not significant).

Article Snippet: Bladder cancer cell lines, T24, J82, UMUC3, 5637, were obtained from iCell Bioscience Inc (Shanghai, China) and validated for authentication using the short tandem repeat (STR) method.

Techniques: Microarray, Liquid Chromatography with Mass Spectroscopy, Software, Binding Assay, Expressing, Reverse Transcription Polymerase Chain Reaction, Immunofluorescence, Control, Concentration Assay

Journal: Cell Death & Disease

Article Title: Inhibitory role of proguanil on the growth of bladder cancer via enhancing EGFR degradation and inhibiting its downstream signaling pathway to induce autophagy

doi: 10.1038/s41419-022-04937-z

Figure Lengend Snippet: A The expression levels of EGFR in different types of bladder cancer and adjacent tissues were detected by WB. B The TCGA database was used to analyze the correlation between EGFR expression and poor prognosis in bladder cancer patients. C , D Cells were plated in 6-well plates (3.0 × 10 5 /well) and lysed to collect protein or RNA when density reached 80–90%, the expression of EGFR and mRNA in 4 BC cells were analyzed by WB and RT-PCR. E Cells were plated in 96-well plates (6.0 × 10 3 /well), The proliferation of T24 and J82 cells was detected at specified times by MTT assay. EGFR stable knockdown ( F ) and overexpressed ( G ) cell lines were constructed by lentiviral vector plasmid as described in “Materials and methods”, then cells were plated in 6-well plates (3.0 × 10 5 /well) and lysed to collect protein when density reached 80–90%. The expression of EGFR was detected by WB. H , I Cells were plated in 96-well plates (6.0 × 10 3 /well), the proliferation of cell lines were detected at specified times by MTT assay. Data are representative of three independent experiments. Error bars represent means ± SD from triplicate experiments. Vehicle control means the concentration of DMSO lower than 0.3% (* P < 0.05, ** P < 0.01, *** P < 0.001).

Article Snippet: Bladder cancer cell lines, T24, J82, UMUC3, 5637, were obtained from iCell Bioscience Inc (Shanghai, China) and validated for authentication using the short tandem repeat (STR) method.

Techniques: Expressing, Reverse Transcription Polymerase Chain Reaction, MTT Assay, Knockdown, Construct, Plasmid Preparation, Control, Concentration Assay

Journal: Cell Death & Disease

Article Title: Inhibitory role of proguanil on the growth of bladder cancer via enhancing EGFR degradation and inhibiting its downstream signaling pathway to induce autophagy

doi: 10.1038/s41419-022-04937-z

Figure Lengend Snippet: A – C The inhibitory effects of proguanil on proliferation and migration of T24 and J82 cell lines were compared by MTT, colony formation and transwell assay. D – F The inhibitory effects of proguanil on proliferation and migration of shCtrl, shEGFR-1 and shEGFR-2 cell lines were compared by MTT, colony formation and transwell assay. G – I The inhibitory effects of proguanil on proliferation and migration of OC-J82 and OE-J82 were compared by MTT, colony formation and transwell assay. See “Materials and methods” for more experimental details. Data are representative of three independent experiments. Error bars represent means ± SD from triplicate experiments. Vehicle control means the concentration of DMSO lower than 0.3% (* P < 0.05, ** P < 0.01, *** P < 0.001).

Article Snippet: Bladder cancer cell lines, T24, J82, UMUC3, 5637, were obtained from iCell Bioscience Inc (Shanghai, China) and validated for authentication using the short tandem repeat (STR) method.

Techniques: Migration, Transwell Assay, Control, Concentration Assay

Journal: Cell Death & Disease

Article Title: Inhibitory role of proguanil on the growth of bladder cancer via enhancing EGFR degradation and inhibiting its downstream signaling pathway to induce autophagy

doi: 10.1038/s41419-022-04937-z

Figure Lengend Snippet: A , B T24 were plated in 6-well plates (2.0 × 10 5 /well) and transfected with corresponding siRNA as described in “Materials and methods”. When the cell density reaches 70–80%, cells were starved for 12 h and then treated with proguanil for 6 h. The effect of proguanil on the expression of EGFR was detected by WB. C After si Clathrin or si Caveolin-1 as described in “Materials and methods”, T24 were plated on glass disks in 12-well plates (2.0 × 10 4 /well), starved for 12 h and then treated with proguanil, and the changes of EGFR were detected by immunofluorescence. D When the density of T24 reaches 70–80% in 75 cm 2 vented culture flasks, cells starved for 12 h and then treated with proguanil. Then protein lysates were immunoprecipitated with EGFR antibody. The immunoprecipitate were tested by WB with antibodies to EGFR, Clathrin and Caveolin-1. E Cells were transfected with corresponding siRNA as described in “Materials and methods” and then plated in 96-well plates (6.0 × 10 3 /well), the bioactivity of proguanil after si Clathrin or si Caveolin-1 was detected by MTT assay. Data are representative of three independent experiments. Error bars represent means ± SD from triplicate experiments. Vehicle control means the concentration of DMSO lower than 0.3% (* P < 0.05, ** P < 0.01, *** P < 0.001, ns not significant).

Article Snippet: Bladder cancer cell lines, T24, J82, UMUC3, 5637, were obtained from iCell Bioscience Inc (Shanghai, China) and validated for authentication using the short tandem repeat (STR) method.

Techniques: Transfection, Expressing, Immunofluorescence, Immunoprecipitation, MTT Assay, Control, Concentration Assay

Journal: Cell Death & Disease

Article Title: Inhibitory role of proguanil on the growth of bladder cancer via enhancing EGFR degradation and inhibiting its downstream signaling pathway to induce autophagy

doi: 10.1038/s41419-022-04937-z

Figure Lengend Snippet: A When the density of T24 reaches 70–80% in 75 cm 2 vented culture flasks, cells starved for 12 h and then treated with proguanil. Then protein lysates were immunoprecipitated with EGFR antibody. The immunoprecipitates were tested by WB with antibodies to EGFR, c-Cbl and Ub. B When the density of T24 reaches 70–80% in 75 cm 2 vented culture flasks, cells starved for 12 h and then treated with proguanil. Then protein lysates were immunoprecipitated with Ub antibody. The immunoprecipitates were tested by WB with antibodies to EGFR, c-Cbl and Cav-1. C T24 were plated in 6-well plates (2.0 × 10 5 /well) and transfected with corresponding siRNA as described in “Materials and methods”. When the cell density reaches 70–80%, cells were starved for 12 h and then treated with proguanil for 6 h. The effect of proguanil on the expression of EGFR and c-Cbl were detected by WB. D After si c-Cbl as described in “Materials and methods”, T24 were plated on glass disks in 12-well plates (2.0 × 10 4 /well), starved for 12 h and then treated with proguanil for 6 h. The effects of proguanil on EGFR endocytosis and expression were detected by immunofluorescence. E Cells were transfected with corresponding siRNA as described in “Materials and methods” and then plated in 96-well plates (6.0 × 10 3 /well). The bioactivity of proguanil on cells after si c-Cbl was detected by MTT assay. Data are representative of three independent experiments. Error bars represent means ± SD from triplicate experiments. Vehicle control means the concentration of DMSO lower than 0.3% (* P < 0.05, ** P < 0.01, *** P < 0.001, ns not significant).

Article Snippet: Bladder cancer cell lines, T24, J82, UMUC3, 5637, were obtained from iCell Bioscience Inc (Shanghai, China) and validated for authentication using the short tandem repeat (STR) method.

Techniques: Immunoprecipitation, Transfection, Expressing, Immunofluorescence, MTT Assay, Control, Concentration Assay

Journal: Cell Death & Disease

Article Title: Inhibitory role of proguanil on the growth of bladder cancer via enhancing EGFR degradation and inhibiting its downstream signaling pathway to induce autophagy

doi: 10.1038/s41419-022-04937-z

Figure Lengend Snippet: A T24, J82 were plated in 6-well plates (3.0 × 10 5 /well). When the cell density reaches 70–80%, cells were treated with proguanil for 12 h, and the protein expressions of LC3 and Beclin-1 were detected by WB. B When the density of T24 and J82 reaches 70–80% in 75 cm 2 vented culture flasks, cells were treated with proguanil for 12 h. Subsequently, cells are then fixed with electron microscope fixative and observed under a transmission electron microscope (Red arrows indicate autophagosomes). C T24, J82 were plated on glass disks in 12-well plates (8.0 × 10 4 /well) and treated with proguanil for 12 h. Subsequently, cells washed with wash buffer and stained with MDC as described in “Materials and methods”, and then observed under an inverted fluorescence microscope. D T24, J82 were plated on glass disks in 12-well plates (2.0 × 10 4 /well), After 24 h, cells were treated with proguanil for 12 h. The effect of proguanil on LC3 was detected by immunofluorescence. E T24, OE-T24 were plated in 6-well plates (3.0 × 10 5 /well). When the cell density reaches 70–80%, cells were treated with proguanil for 12 h, the effect of proguanil on the expression of LC3 was detected by WB. F T24 were plated in 96-well plates (6.0 × 10 3 /well) and pretreated with 400 nm AZD6244 for 12 h before exposure to increasing concentrations of proguanil for an additional 72 h, the combined effect of proguanil and AZD6244 was detected by MTT assay. G Combination index (CI) among the combinations of two drugs was calculated using CompuSyn software. if CI > 1, it denotes antagonism; if CI < 1, it denotes synergism. CI values in all of combinations were less than 1, indicating synergism. H T24 were plated in 6-well plates (3.0 × 10 5 /well). When the cell density reaches 70–80%, cells were pretreated with 400 nm AZD6244 for 12 h before exposure to proguanil for an additional 12 h, the protein expressions of p-ERK1/2 and LC3 were detected by WB. Data are representative of three independent experiments. Error bars represent means ± SD from triplicate experiments. Vehicle control means the concentration of DMSO lower than 0.3% (* P < 0.05, ** P < 0.01, *** P < 0.001, ns not significant).

Article Snippet: Bladder cancer cell lines, T24, J82, UMUC3, 5637, were obtained from iCell Bioscience Inc (Shanghai, China) and validated for authentication using the short tandem repeat (STR) method.

Techniques: Microscopy, Transmission Assay, Staining, Fluorescence, Immunofluorescence, Expressing, MTT Assay, Software, Control, Concentration Assay

Journal: Cell Death & Disease

Article Title: Inhibitory role of proguanil on the growth of bladder cancer via enhancing EGFR degradation and inhibiting its downstream signaling pathway to induce autophagy

doi: 10.1038/s41419-022-04937-z

Figure Lengend Snippet: A T24, J82 were plated in 6-well plates (3.0 × 10 5 /well). When the cell density reaches 70–80%, cells were treated with 3-MA (5 mM) and proguanil for 12 h. Western blotting was used to detect the protein expression of LC3 and Beclin-1. B T24, J82 were plated on glass disks in 12-well plates (2.0 × 10 4 /well), After 24 h, cells were treated with 3-MA (5 mM) and proguanil, immunofluorescence was used to detect the protein expression of LC3. C Cells were plated in 96-well plates (6.0 × 10 3 /well), After 12 h, cells were treated with 3-MA (5 mM) and proguanil, the inhibitory effects of proguanil and 3-MA on proliferation were detected by MTT. D Cells were plated in 24-well plates (2.0 × 10 3 /well), After 12 h, cells were treated with 3-MA (5 mM) and proguanil, the he inhibitory effects of proguanil and 3-MA on proliferation were detected by colony formation assay. E The inhibitory effects of proguanil and 3-MA on migration were detected by transwell as described in “Materials and methods”. Data are representative of three independent experiments. Error bars represent means ± SD from triplicate experiments. Vehicle control means the concentration of DMSO lower than 0.3% (* P < 0.05, ** P < 0.01, *** P < 0.001, ns not significant).

Article Snippet: Bladder cancer cell lines, T24, J82, UMUC3, 5637, were obtained from iCell Bioscience Inc (Shanghai, China) and validated for authentication using the short tandem repeat (STR) method.

Techniques: Western Blot, Expressing, Immunofluorescence, Colony Assay, Migration, Control, Concentration Assay

Journal: Cell Death & Disease

Article Title: Inhibitory role of proguanil on the growth of bladder cancer via enhancing EGFR degradation and inhibiting its downstream signaling pathway to induce autophagy

doi: 10.1038/s41419-022-04937-z

Figure Lengend Snippet: A 10 7 T24 and shEGFR-2 T24 cell suspension were injected into the right and left axilla of mice in group 2 and group 3. When the tumor volume reached 70–100 mm 3 , mice in group 3 were treated with proguanil. After treated 14 days, tumors were removed and photographed. B The tumor volume of each mouse was measured every two days, and the mean volume of each group tumor was calculated to create the figure. C Statistical analysis of tumor weight. D Ki-67 was used to analyze the proliferation of xenograft tumor. E The expression of EGFR in tumor tissues was detected by immunohistochemistry. F Changes of each group mice weight. G HE staining of liver and kidney organs of each group mice. All in vitro experiments are representative of three independent experiments. Error bars represent means ± SD. Vehicle control(2% PEG-400 + 2% Tween-80 + 96% PBS). (* P < 0.05, ** P < 0.01, *** P < 0.001, ns not significant).

Article Snippet: Bladder cancer cell lines, T24, J82, UMUC3, 5637, were obtained from iCell Bioscience Inc (Shanghai, China) and validated for authentication using the short tandem repeat (STR) method.

Techniques: Suspension, Injection, Expressing, Immunohistochemistry, Staining, In Vitro, Control