Journal: Molecules

Article Title: Inhibition of MORC2 Mediates HDAC4 to Promote Cellular Senescence through p53/p21 Signaling Axis

doi: 10.3390/molecules27196247

Figure Lengend Snippet: Knockdown of MORC2 inhibits HCT-116 and Lovo cell proliferation and clone formation. ( A ) GSEA indicates that MORC2-associated DEGs were enriched in the cell cycle. ( B ) HCT-116 and Lovo cells were transfected with indicated shRNA, and the protein expression was analyzed with indicated antibodies. ( C ) CCK8 assays were used to assess the DNA synthesis of shCON, shMORC2#1, and shMORC2#2 groups with HCT-116 and Lovo cells. ( D , E ) Edu assay was used to assess the DNA synthesis of shCON, shMORC2#1, and shMORC2#2 groups with HCT-116 cells. The percentage of EdU-positive cells in shCON, shMORC2#1, and shMORC2#2 groups. ( F ) Colony formation assay showing clone formation capacity of shCON, shMORC2#1, and shMORC2#2 groups. ( G ) Each group colony number was counted and relative colony numbers in shMORC2#1 and shMORC2#2 were compared to the shCON group. ** p < 0.01, **** p < 0.0001.

Article Snippet: HCT-116 cells were obtained from Procell Co., Ltd. HCT-116 and HEK293 cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal bovine serum (FBS), under 37 °C and 5% CO 2 conditions.

Techniques: Transfection, shRNA, Expressing, DNA Synthesis, EdU Assay, Colony Assay

Journal: Molecules

Article Title: Inhibition of MORC2 Mediates HDAC4 to Promote Cellular Senescence through p53/p21 Signaling Axis

doi: 10.3390/molecules27196247

Figure Lengend Snippet: Knockdown of MORC2 induces cell senescence. ( A ) PPI network of the top 10 interacting genes related to MORC2. ( B ) GSEA indicates that MORC2-associated DEGs were enriched in cell senescence. ( C ) The single gene co-expression heat map presented the significant relationship among MORC2, HDAC4, IL-1α, and IL-1β. ( D ) HCT-116 and Lovo cells are transfected with shMORC2, and the protein expression is analyzed with indicated antibodies. ( E ) p53 promoter-luciferase activity was measured. ( F ) mRNA expression of p16, IL-1α, and IL-1β in HCT-116 cells. ( G – J ) The levels of IL-6 and IL-8 secreted by HCT-116 and Lovo cells after transfecting with shMORC2. ( K ) SA-β-gal staining analysis in HCT-116 and Lovo cells transfected with shMORC2. ( L ) Relative fold change of the SA-β-gal staining ratio in each shMORC2 group was calculated and compared to shCON group’s SA-β-gal staining ratio. * p < 0.05, ** p < 0.01, *** p < 0.001.

Article Snippet: HCT-116 cells were obtained from Procell Co., Ltd. HCT-116 and HEK293 cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal bovine serum (FBS), under 37 °C and 5% CO 2 conditions.

Techniques: Expressing, Transfection, Luciferase, Activity Assay, Staining

Journal: Molecules

Article Title: Inhibition of MORC2 Mediates HDAC4 to Promote Cellular Senescence through p53/p21 Signaling Axis

doi: 10.3390/molecules27196247

Figure Lengend Snippet: Knockdown of MORC2 inhibits tumor growth. ( A ) The growth curve of mice xenografts formed by shCON and shMORC2#1 cells ( n = 5). ( B ) Tumor weights after sacrificing the mice ( n = 5). ( C ) Mice xenografts formed by HCT-116 with or without MORC2 intervention. ( D , E ) IHC staining and statistical analysis of xenografts using antibodies against ki67, MORC2, p53, and HDAC4. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001.

Article Snippet: HCT-116 cells were obtained from Procell Co., Ltd. HCT-116 and HEK293 cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal bovine serum (FBS), under 37 °C and 5% CO 2 conditions.

Techniques: Immunohistochemistry

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

Journal: Oncology Letters

Article Title: Cisplatin resistance by induction of aldo-keto reductase family 1 member C2 in human bladder cancer cells

doi: 10.3892/ol.2013.1768

Figure Lengend Snippet: AKR1C2 protein expression in HT1376-CisR cells was markedly increased in comparison with the parental cells. AKR1C2 small interfering RNA reduced expression by ~80% in HT1376-CisR cells. AKR1C2 and β-tubulin exhibit discrete bands of the same molecular weight (AKR1C2, 37 kDa; β-tubulin, 51 kDa). AKR1C2, aldo-keto reductase family 1 member C2; CisR, cisplatin-resistant.

Article Snippet: The human HT1376 bladder cancer cell line used in this study was purchased from DS Pharma Biomedical (Osaka, Japan).

Techniques: Expressing, Comparison, Small Interfering RNA, Molecular Weight

Journal: Oncology Letters

Article Title: Cisplatin resistance by induction of aldo-keto reductase family 1 member C2 in human bladder cancer cells

doi: 10.3892/ol.2013.1768

Figure Lengend Snippet: Effect of AKR1C2 expression on cisplatin IC 50 values in parental and HT1376-CisR cells. Cells were treated with various cisplatin concentrations for 72 h, and then quantified using a cell counter. Each assay was performed in triplicate. Cell survival in the absence of cisplatin was set as 100%. (A) Silencing AKR1C2 restored HT1376-CisR cell response to cisplatin. (B) Inhibition of AKR1C2 by 100 μM 5β-cholanic acid restored the HT1376-CisR response to cisplatin. * P<0.05, vs. HT1376-CisR. Bars indicate standard deviation. AKR1C2, aldo-keto reductase family 1 member C2; CisR, cisplatin-resistant.

Article Snippet: The human HT1376 bladder cancer cell line used in this study was purchased from DS Pharma Biomedical (Osaka, Japan).

Techniques: Expressing, Inhibition, Standard Deviation

Journal: Oncology Letters

Article Title: Cisplatin resistance by induction of aldo-keto reductase family 1 member C2 in human bladder cancer cells

doi: 10.3892/ol.2013.1768

Figure Lengend Snippet: Effect of cisplatin on intracellular ROS in HT1376 cells. Exposure to cisplatin increased the levels of intracellular ROS in HT1376 cells in a dose-dependent manner. * P<0.05, vs. HT1376 cells cultured without cisplatin. Bars indicate standard deviation. ROS, reactive oxygen species.

Article Snippet: The human HT1376 bladder cancer cell line used in this study was purchased from DS Pharma Biomedical (Osaka, Japan).

Techniques: Cell Culture, Standard Deviation

Journal: Oncology Letters

Article Title: Cisplatin resistance by induction of aldo-keto reductase family 1 member C2 in human bladder cancer cells

doi: 10.3892/ol.2013.1768

Figure Lengend Snippet: Relative values of intracellular ROS measured using a 2,7-dichlorodihydrofluorescein diacetate probe. (A) Basal intracellular ROS levels in HT1376, HT1376-CisR and HT1376-CisR cells transiently transfected with AKR1C2 small interfering RNA [HT1376-CisR-AKR1C2(−)]. * P<0.05 and $ P<0.05, vs. HT1376 and HT1376-CisR cells cultured without cisplatin, respectively. (B) Effect of 10 −4 M cisplatin exposure on intracellular ROS in these cells. (C) Effect of 5 μM menadione on intracellular ROS in these cells. * P<0.05 vs. control cells cultured without cisplatin or menadione. Bars indicate standard deviation. AKR1C2, aldo-keto reductase family 1 member C2; CisR, cisplatin-resistant; ROS, reactive oxygen species.

Article Snippet: The human HT1376 bladder cancer cell line used in this study was purchased from DS Pharma Biomedical (Osaka, Japan).

Techniques: Transfection, Small Interfering RNA, Cell Culture, Control, Standard Deviation