Journal: Cancers

Article Title: Mitochondrial ROS Produced in Human Colon Carcinoma Associated with Cell Survival via Autophagy

doi: 10.3390/cancers14081883

Figure Lengend Snippet: Autophagy is induced in MT cells. ( A ) Mutant UQCRB-expressing cell lines were examined by Western blotting using anti-UQCRB and anti-Myc antibodies. ( B ) HEK293 and MT cells treated with either 10% serum-containing medium or serum-free medium for 24 h and then analyzed by Western blot analysis using antibodies against LC3B and p62. shows treatment of either 10% serum-containing medium or serum-free medium for 4 h. ( C ) Confocal microscopy images of HEK293 and MT cells treated with serum-free medium for 24 h after immunostaining with anti-LC3 antibody. Scale bar, 50 μm. ( D ) Western blot analysis results of LC3B and p62 levels in cells after treatment with 5 μM chloroquine (CQ) treatment for 0–6 h. ( E ) HEK293 and MT cells were treated with either deionized water (DIW) or CQ for 6 h. Cell extracts were subjected to Western blotting using LC3B and p62 antibodies. The densitometric value of LC3-II was quantitated based on Western blot data and normalized to LC3-I levels. ( F ) Confocal microscopy images of HEK293 and MT cells transfected with mRFP-GFP-LC3 for 24 h. Scale bar, 20 μm. ( G ) Numbers of mRFP-LC3 puncta were counted in HEK293 and MT cells ( n = 8). The relative band intensities value was normalized to β-actin (loading control). The normalized protein levels are shown under each band. Data are presented as the mean ± SD; statistical significance was assessed using an unpaired t -test. ns, p > 0.05.

Article Snippet: Cell lines were purchased commercially by American Type Culture Collection (ATCC), CCD18co (CCL-1459TM, ATCC), HCT116 (CCL-247 TM, ATCC), HEK293 (CRL-1573.3 TM, ATCC).

Techniques: Mutagenesis, Expressing, Western Blot, Confocal Microscopy, Immunostaining, Transfection

Journal: Cancers

Article Title: Mitochondrial ROS Produced in Human Colon Carcinoma Associated with Cell Survival via Autophagy

doi: 10.3390/cancers14081883

Figure Lengend Snippet: TRPML1-induced lysosomal Ca 2+ release and TFEB activation are increased in MT cells. HEK293 and MT cells were transfected with GCaMP3-TRPML1 encoding a lysosome-specific Ca 2+ probe, 40 µM ML-SA1, for 120 s. ( A ) Confocal microscopy images of GCaMP green fluorescence showing HEK293 and ( B ) MT cells. ML-SA1 treatment for 120 s. ( C ) Confocal microscopy images of HEK293 and MT cells transfected with EGFP-TFEB and treated with the DMSO control or A1938 for 6 h. ( D ) The number of cells with a nuclear (Nuc) or cytoplasmic (Cyt) TFEB localization ratio is represented in the graph. Scale bar, 20 μm. Statistical significance was assessed using Student’s t -test. ** p < 0.01.

Article Snippet: Cell lines were purchased commercially by American Type Culture Collection (ATCC), CCD18co (CCL-1459TM, ATCC), HCT116 (CCL-247 TM, ATCC), HEK293 (CRL-1573.3 TM, ATCC).

Techniques: Activation Assay, Transfection, Confocal Microscopy, Fluorescence

Journal: Cancers

Article Title: Mitochondrial ROS Produced in Human Colon Carcinoma Associated with Cell Survival via Autophagy

doi: 10.3390/cancers14081883

Figure Lengend Snippet: Lysosome activity in MT cells with or without A1938 treatment. ( A ) Confocal microscopy images and ( B ) numbers of acidic lysosome red puncta were counted in HEK293 and MT cells ( n = 15). The cells were treated with DMSO as a control or 30 µM A1938 for 24 h and then stained with LTR. Scale bar, 20 μm. ( C ) Confocal microscopy images of MT cells treated with DMSO as a control or 30 µM A1938 for 24 h, then the live cells were stained with 2 μg/mL acridine orange for 25 min and fixed. Scale bar, 20 μm. ( D ) Images of HEK293 and MT cell extracts treated with the DMSO control or 30 µM A1938 for 24 h in a serum-free medium, then subjected to Western blot analysis using antibodies against LAMP1. ( E ) Intensities of the LAMP1 immunoblot bands were normalized to β-actin expression. Western blot analysis out of three independent experiments. Statistical significance was assessed using Student’s t -test. ns, p > 0.05, * p < 0.05, *** p < 0.001, **** p < 0.0001.

Article Snippet: Cell lines were purchased commercially by American Type Culture Collection (ATCC), CCD18co (CCL-1459TM, ATCC), HCT116 (CCL-247 TM, ATCC), HEK293 (CRL-1573.3 TM, ATCC).

Techniques: Activity Assay, Confocal Microscopy, Staining, Western Blot, Expressing

Journal: Cancers

Article Title: Mitochondrial ROS Produced in Human Colon Carcinoma Associated with Cell Survival via Autophagy

doi: 10.3390/cancers14081883

Figure Lengend Snippet: A1938 regulates autophagy flux by suppression of mROS generation in MT cells. ( A ) Cells treated with A1938 for 24 h in a serum-free medium. The relative band intensities value was normalized to β-actin (loading control). The normalized protein levels are shown under each band. ( B ) Confocal microscopy images of HEK293 and MT cells transfected with mRFP-GFP-LC3 and treated with DMSO, A1938, or Mito-Tempo (mTP) for 24 h. Scale bar, 20 μm. Quantification of data indicating red puncta (autolysosome) versus yellow puncta (autophagosome) is shown in ( C ). Values are the means ± SEM; statistical significance was assessed using a paired t -test. n = 10 cells, ns, p > 0.05, * p < 0.05, ** p < 0.01, *** p < 0.001.

Article Snippet: Cell lines were purchased commercially by American Type Culture Collection (ATCC), CCD18co (CCL-1459TM, ATCC), HCT116 (CCL-247 TM, ATCC), HEK293 (CRL-1573.3 TM, ATCC).

Techniques: Confocal Microscopy, Transfection