transwell chambers with non-coated membranes (24-well insert, pore size)  (Corning Life Sciences)

Journal: Cells

Article Title: Radiation-Induced Overexpression of TGFβ and PODXL Contributes to Colorectal Cancer Cell Radioresistance through Enhanced Motility

doi: 10.3390/cells10082087

Figure Lengend Snippet: IR exposure enhanced EMT progression and migratory ability in CRC cells. ( a ) LoVo, SW480, HCT116, and DLD1 cells were exposed to IR at 5 Gy. After 24 h, cells were evaluated for migration ability using a transwell assay. The graph indicates the number of migrated cells. Scale bar, 100 μm. *** p < 0.005. ( b ) HCT116 and DLD1 cells were cultured with or without IR exposure (5 Gy) for 24 h. Cellular morphology was assessed using a microscope (magnification 20×). ( c ) HCT116 and DLD1 cells were exposed to IR at 5 Gy. Whole-cell lysates were subjected to Western blotting using antibodies against ZO1, E-cadherin, vimentin, and Snail. The β-actin signal was used as a loading control.

Article Snippet: To assess cell migration and invasiveness, irradiated cells were seeded into 24-well transwell chambers (Corning, NY, USA) with 8 μm pore polycarbonate membrane-coated inserts.

Techniques: Migration, Transwell Assay, Cell Culture, Microscopy, Western Blot, Control

Journal: Cells

Article Title: Radiation-Induced Overexpression of TGFβ and PODXL Contributes to Colorectal Cancer Cell Radioresistance through Enhanced Motility

doi: 10.3390/cells10082087

Figure Lengend Snippet: PODXL depletion inhibited invadopodia in CRC cells. ( a ) shCont and shPODXL cells were exposed to IR (5 Gy) and both migration and invasiveness were determined after 24 h using a transwell assay. The graphs show relative cell numbers. Scale bar, 100 μm. ** p < 0.001, and *** p < 0.005. ( b ) shCont- or shPODXL-transfected HCT116 cells were placed on FITC-coupled gelatin-coated coverslips and incubated for 48 h, either IR-untreated or IR-treated. Gelatin (green) degradation was visualized in areas devoid of FITC staining by fluorescent microscopy at a magnification of 20×. *** p < 0.005. ( c ) shCont- or shPODXL-transfected HCT116 and DLD1 cells were exposed to IR (5 Gy) and incubated for 24 h. Western blots were used to assess the expression patterns of PODXL, TGFβ, p-FAK, total FAK, ZO1, Snail, vimentin, and MMP2. The β-actin signal served as a loading control. shCont- or shPODXL-transfected HCT116 cells were IR-exposed (5Gy) or not, and then incubated for 24 h. In panel ( d ), cells were stained for p-FAK (red) and PODXL (green); in panel ( e ), the staining was for TGFβ (red) and ZO1 (green). DAPI was used for nuclear staining. The graph indicates fluorescence intensity from three independent experiments ± SD. * p < 0.05, ** p < 0.001, and *** p < 0.005.

Article Snippet: To assess cell migration and invasiveness, irradiated cells were seeded into 24-well transwell chambers (Corning, NY, USA) with 8 μm pore polycarbonate membrane-coated inserts.

Techniques: Migration, Transwell Assay, Transfection, Incubation, Staining, Microscopy, Western Blot, Expressing, Control, Fluorescence

Journal: Cells

Article Title: Radiation-Induced Overexpression of TGFβ and PODXL Contributes to Colorectal Cancer Cell Radioresistance through Enhanced Motility

doi: 10.3390/cells10082087

Figure Lengend Snippet: PODXL overexpression accelerated EMT progression and metastatic ability in CRC cells. ( a ) SW480 cells were transfected with control vector (pCMV6-XL3) or PODXL and then exposed to IR (5 Gy). Twenty-four hours after transfection, cell migration and invasiveness were determined using a transwell assay. The graph indicates relative cell numbers from three independent experiments. Scale bar, 100 μm. and *** p < 0.005. ( b ) pCMV6-XL3- or PODXL-expressing SW480 cells were assessed by Western blot for PODXL, ZO1, and p-FAK expression. The β-actin signal served as loading control. *** p < 0.005. ( c ) IF staining of PODXL (red) and ZO1 (green) in pCMV6-XL3- or PODXL-expressing SW480 cells. Nuclei were counterstained with DAPI. Scale bar, 100 μm. The graph shows fluorescence intensity ± SD. *** p < 0.005.

Article Snippet: To assess cell migration and invasiveness, irradiated cells were seeded into 24-well transwell chambers (Corning, NY, USA) with 8 μm pore polycarbonate membrane-coated inserts.

Techniques: Over Expression, Transfection, Control, Plasmid Preparation, Migration, Transwell Assay, Expressing, Western Blot, Staining, Fluorescence

Journal: Cells

Article Title: Radiation-Induced Overexpression of TGFβ and PODXL Contributes to Colorectal Cancer Cell Radioresistance through Enhanced Motility

doi: 10.3390/cells10082087

Figure Lengend Snippet: Combined galunisertib and IR treatment promoted antitumor activity in CRC cells. HCT116 cells were treated with galunisertib (10 µmol/L) alone, IR (5 Gy) alone, or combined for 24 h. ( a ) Western blot analysis was used to assess PODXL, vimentin, E-cadherin, and p-FAK expressions ( b ) HCT116 cells were treated with galunisertib (10 μM) alone, IR (5 Gy) alone, or combined for 24 h. Twenty-four hours after treatment, cell viability was estimated using a CCK-8 assay. The graph indicates cell viability from three independent experiments ± SD. *** p < 0.005. ( c ) C HCT116 cells were treated with galunisertib (10 µmol/L) alone, IR (5 Gy) alone, or combined for 21 days. The colony number was calculated from three replicate plates of three independent experiments; bars indicate SD. Representative images from 21 days post-plating are shown. ( d ) Cell migration was assessed using a transwell assay. The data shown are representative of three independent experiments. *** p < 0.005, ** p < 0.01, and * p < 0.05. ( e ) Representative images of the dissemination of HCT116 spheroids over a 3-day culture, in which cells were treated with IR, galunisertib, or combined treatment from day 0. Scale bar, 200 μm. The data shown are representative of three independent experiments. *** p < 0.005, ** p < 0.01, and * p < 0.05.

Article Snippet: To assess cell migration and invasiveness, irradiated cells were seeded into 24-well transwell chambers (Corning, NY, USA) with 8 μm pore polycarbonate membrane-coated inserts.

Techniques: Activity Assay, Western Blot, CCK-8 Assay, Migration, Transwell Assay