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, Control

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, Control

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, Control, 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: Control, Confocal Microscopy, Transfection

Journal: Journal of neurochemistry

Article Title: cDNA cloning and molecular characterization of human brain metalloprotease MP100: a beta-secretase candidate?

doi: 10.1046/j.1471-4159.1999.0721215.x

Figure Lengend Snippet: FIG. 2. Expression of active hMP100L. A: The long form of recombinant human MP100 showed increased MP100 activity compared with vector alone-transfected HEK cells using 150 mg of cell extract and b-APP peptide-pNA digestion. The activity was inhibited by the metalloprotease inhibitor o-phenanthroline (3 mM). B: MP100 western blotting confirmed strong overex- pression of recombinant MP100L (arrow).

Article Snippet: Culture and transfection of the HEK293 cell line (CRL 1573; ATCC) were done as described by Malherbe et al. (1994).

Techniques: Expressing, Recombinant, Activity Assay, Plasmid Preparation, Transfection, Western Blot

Journal: Journal of neurochemistry

Article Title: cDNA cloning and molecular characterization of human brain metalloprotease MP100: a beta-secretase candidate?

doi: 10.1046/j.1471-4159.1999.0721215.x

Figure Lengend Snippet: FIG. 4. Double MP100/b-APP immunofluorescence by confocal microscopy. A: Stable HEK-b-APP695 cells transiently coex- pressing hMP100L show colocalization of MP100 (Texas red- labeled) and b-APP (FITC-labeled) at certain locations, although considerable amounts of both proteins are also differently ex- pressed. The colocalized areas are visualized in white (as de- scribed in Materials and Methods) and marked by arrows. B: Human neuroblastoma IMR32 cells displaying endogenous MP100 (Texas red-labeled) and b-APP (FITC-labeled) show sim- ilar partial colocalization of the two proteins, whereas fractions of both proteins are noncolocalized.

Article Snippet: Culture and transfection of the HEK293 cell line (CRL 1573; ATCC) were done as described by Malherbe et al. (1994).

Techniques: Confocal Microscopy, Labeling

Journal: Nature Communications

Article Title: PharmacoSTORM nanoscale pharmacology reveals cariprazine binding on Islands of Calleja granule cells

doi: 10.1038/s41467-021-26757-z

Figure Lengend Snippet: a General workflow of the development of PharmacoSTORM probes, demonstrated by the example of a CB 1 R ligand. (I) The docking pose of the fluorescently tagged CB 1 R agonist (fluo-cannabinoid, yellow) in the CB 1 R (gray) ( PDB:5XRA ). The hydrogen bonds between the protein residues (red) and the probe are represented as yellow dashed lines. (II) In vitro cAMP assay for the functional characterization of fluo-cannabinoid activity on CB 1 R ( n = 5, logEC 50 = −7.48 nM, E max = 36.1%). Reference compound was WIN55,212-2 ( n = 4, logEC 50 = −8.03 nM, E max = 51.5%). (III) Confocal microscopic image of HEK 293 cells expressing GFP-CB 1 R, labeled with 100 nM fluo-cannabinoid; (IV) Comparison of confocal and STORM images depicting fluo-cannabinoid binding to CB 1 Rs on the same plasma membrane segment. b , e , h Surface representations of three PharmacoSTORM probes, bound to their respective protein targets that represent three major protein families. b GPCR:CB 1 R, ( PDB:5XRA , gray) with fluo-cannabinoid (yellow); e enzyme: MAGL ( PDB:6BQ0 , gray) with DH-463 (yellow); h ion channel: α7-nAchR ( PDB:4HQP , gray) with fluorescent α-bungarotoxin (yellow). c , f , i Competitive ligand-binding measurements by dual direct STORM imaging demonstrate the high specificity of PharmacoSTORM probes. Target proteins were visualized simultaneously by pharmacoprobe- and antibody-based labeling. c 100 nM fluo-cannabinoid and anti-GFP immunostaining for GFP-CB 1 R in the absence (vehicle) or presence of CB 1 R antagonist rimonabant (Rim, 10 µM). f 100 nM DH-463 and anti-GFP immunostaining for GFP-MAGL pretreated either with vehicle or the MAGL inhibitor JZL184 (JZL, 10 µM). i 1 ng/µl Alexa647-α-bungarotoxin and anti-HA immunostaining for α7-nAchR-HA pretreated either with vehicle or the α7-nAchR antagonist methyllycaconitine (MLA, 10 µM). d , g , j Scatter dot plots display the ratio of PharmacoSTORM and ImmunoSTORM localization points (LPs) in the absence or presence of the unlabeled competitive ligands. Data are normalized to average vehicle (Veh) pretreatment. Two-tailed Mann–Whitney tests were performed. d n = 5, P = 0.0079; g n = 4, P = 0.0286; j n = 4, P = 0.0286. Data are presented as mean ± SEM, n values indicate biologically independent experiments in all panels.

Article Snippet: HEK 293 and HEK 293T cells were obtained from American Type Culture Collection (ATCC CRL-1573 and CRL-3216 respectively).

Techniques: In Vitro, cAMP Assay, Functional Assay, Activity Assay, Expressing, Labeling, Comparison, Binding Assay, Clinical Proteomics, Membrane, Ligand Binding Assay, Imaging, Antibody Labeling, Immunostaining, Two Tailed Test, MANN-WHITNEY

Journal: Nature Communications

Article Title: PharmacoSTORM nanoscale pharmacology reveals cariprazine binding on Islands of Calleja granule cells

doi: 10.1038/s41467-021-26757-z

Figure Lengend Snippet: a The docking pose of the fluorescently tagged cariprazine (Fluo-CAR, yellow) in a D 3 R (gray) homology model (for details, see Supplementary Information). The hydrogen bonds between the key protein residues (Glu2.65, Asp3.32 in red) and the pharmacoprobe are represented as yellow dashed lines. b , c Functional effects of Fluo-CAR on D 3 R in BRET-based G i1 -activation assay. b Fluo-CAR antagonized the effect of the D 3 R selective agonist PD128907. Cells were incubated with Fluo-CAR or vehicle for 5 min before PD128907 application. Fluo-CAR pretreatment increased the EC 50 value of PD128907 from 1.047 nM to 256.1 nM ( n = 3). c Fluo-CAR alone acted as a weak partial agonist. Sigmoidal concentration-response curve was fitted (EC 50 = 44.1 nm). BRET ratios are expressed as the percentage of the signal of 1 µM PD128907 ( n = 3). d Representative confocal images of HA-D 3 R-expressing HEK 293 cells treated with Fluo-CAR (100 nM) following HA immunostaining. Fluo-CAR selectively binds those cells that express HA-D 3 R. e , f Competitive ligand-binding experiment assessed by quantitative dual direct STORM imaging. HA-D 3 R-expressing cells were incubated with either vehicle (Veh) or 10 µM SB277011-A (SB), a selective D 3 R antagonist. The ratio of PharmacoSTORM and ImmunoSTORM localization points (LPs) was normalized to vehicle pretreatment. SB277011-A pretreatment markedly reduced the Fluo-CAR signal ( n = 4, two-tailed Mann–Whitney U test, P = 0.0286). g Saturation binding assay of Fluo-CAR treatment measured within the plasma membrane. Relative (rel.) receptor occupancy (the ratio of PharmacoSTORM and ImmunoSTORM LPs) was expressed as the percentage of the signal of 1 µM Fluo-CAR, n values are (from left to the right) 4; 4; 3; 4; 3; 3; 3; 7. One-site sigmoidal binding curve was fitted (half-maximal occupancy was at 123 nM). h , i Nanoscale visualization of Fluo-CAR binding sites in the sub-nanomolar concentration range. The representative image is shown in ( h ). White arrows point to sparsely bound individual Fluo-CAR molecules. i Higher magnification of the lower concentration range from ( g ) is shown (two-tailed Mann–Whitney U test, P = 0.0294). Data are presented as mean ± SEM, n values indicate biologically independent experiments in all panels.

Article Snippet: HEK 293 and HEK 293T cells were obtained from American Type Culture Collection (ATCC CRL-1573 and CRL-3216 respectively).

Techniques: Functional Assay, Activation Assay, Incubation, Concentration Assay, Expressing, Immunostaining, Ligand Binding Assay, Imaging, Two Tailed Test, MANN-WHITNEY, Saturation Assay, Clinical Proteomics, Membrane, Binding Assay