Journal: International Journal of Biological Sciences

Article Title: Inhibitory role of TRIP-Br1 oncoprotein in anticancer drug-mediated programmed cell death via mitophagy activation

doi: 10.7150/ijbs.72138

Figure Lengend Snippet: Subcellular distribution of TRIP-Br1 protein in normal breast or cancer cell line and mitochondrial localization of TRIP-Br1. A-B Indicated cell lines were used in mitochondrial fractionation as described in Materials and Methods. TRIP-Br1 protein level in each fraction was quantified via immunoblotting analyses. The following proteins were used as cellular markers: HSP60 as a mitochondrial marker, γ-tubulin as a cytosolic marker, and histone H3 as a nuclear marker. Amount of protein loaded: 15 µg for total protein, 2.5 µg for mitochondrial (Mito), and 15 µg for cytosolic (Cyto) fraction. Data are expressed as mean ± SD (n = 3; ***, p < 0.001). C Indicated four cell lines were stained with TRIP-Br1 (green) for 24 h, incubated with 1 µM of Mitotracker (red) for 30 min, and fixed with 4% paraformaldehyde. DAPI (blue) was used as a nuclear marker. Resulting cells were visualized with a confocal microscope. D-E MCF7 and MCF10A cell lines were cultured for 24 h and then used for mitochondrial fractionation. TRIP-Br1 protein level in each fraction was determined by western blot analysis. Data are presented as mean ± SD (n = 3; ***, p < 0.001). F Mitochondrial fraction from MCF7 cells was incubated with 50 μg/mL of proteinase K in the absence or presence of 1% Triton X-100 for 30 min. Samples were subjected to western blotting analysis.

Article Snippet: MCF7 and MDA-MB-231 human breast cancer cell lines were purchased from the American Type Culture Collection (ATCC).

Techniques: Fractionation, Western Blot, Marker, Staining, Incubation, Microscopy, Cell Culture

Journal: International Journal of Biological Sciences

Article Title: Inhibitory role of TRIP-Br1 oncoprotein in anticancer drug-mediated programmed cell death via mitophagy activation

doi: 10.7150/ijbs.72138

Figure Lengend Snippet: Up-regulated TRIP-Br1 protein level in mitochondria in response to anticancer drug treatment and TRIP-Br1-mediated ROS suppression in cancer cells. A MCF7 cells were treated with three different anticancer drugs for indicated time periods and concentrations, followed by mitochondrial fractionation. B MCF7 cells were treated with STS at different concentrations (0.01~1 μM) for 90 min followed by mitochondrial fractionation. C MCF7 cells were treated with 0.1 μM of STS for 4 h. Reverse transcription (RT)-PCR analysis was performed with TRIP-Br1-specific primer pairs (See Materials and Methods) using β-actin as an internal control. D-F MCF7 cells (1×10 6 ) were cultured for 24 h and intracellular levels of ATP, MMP, and ROS were measured as indicated in Materials and Methods. Results are presented as mean ± SD (n = 5). G-I MCF7 and MCF10A cells were cultured for 24 h and then treated with STS or H 2 O 2 at indicated concentrations or time periods. Resulting cells were used for mitochondrial fractionation and ROS measurement. J-K Cells were pre-incubated with or without NAC, an ROS scavenger, for 30 min (5 mM for MCF7 and 1 mM for MDA-MB-231) and then treated with 0.1 μM of STS for 45 min. Each fraction of sample lysate was subjected to western blotting analysis. ROS levels were measured with the same sample. L-M MCF7 cells were treated with 0.1 μM of STS for 3 h. Cellular ROS ( L ) or mitochondrial ROS ( M ) levels were then measured. Data are presented as mean ± SD (n = 6).

Article Snippet: MCF7 and MDA-MB-231 human breast cancer cell lines were purchased from the American Type Culture Collection (ATCC).

Techniques: Fractionation, Reverse Transcription, Reverse Transcription Polymerase Chain Reaction, Control, Cell Culture, Incubation, Western Blot

Journal: International Journal of Biological Sciences

Article Title: Inhibitory role of TRIP-Br1 oncoprotein in anticancer drug-mediated programmed cell death via mitophagy activation

doi: 10.7150/ijbs.72138

Figure Lengend Snippet: Mitochondrial translocation of TRIP-Br1 via PP2A-mediated dephosphorylation upon STS treatment. A Phosphorylation status of TRIP-Br1 was analyzed using a Phos-tag SDS-PAGE gel. Lysates of MCF7 cells were prepared and then exposed to CIP for 1 h at 37°C, followed by 8 % SDS-PAGE with 20 μM of phos-tag and immunoblotting analysis. Conventional SDS-PAGE was also performed with the same lysate as a control. B MCF7 cells were treated with 0.1 μM STS for 90 min followed by subcellular fractionation. Fractionated samples were subjected to Phos-tag SDS-PAGE. C MCF7 cells were treated with STS (0.1 μM) and/or okadaic acid (50 nM) for 3 h. Fractionated samples were subjected to western blot analysis. D U2OS 4.3 osteosarcoma cells were pre-incubated with or without doxycycline (1 μg/mL) to induce TRIP-Br1 expression for 24 h before treatment with okadaic acid (50 nM) for 24 h. E MCF7 cells were treated with okadaic acid (50 nM) for 16 h and incubated with Mitotracker (red) for 30 min. TRIP-Br1 (green) and Mitotracker (red) were visualized under a fluorescence microscope. F MCF7 cells were exposed to C 2 -Ceramide (10 μM) in a time-dependent manner followed by mitochondrial fractionation.

Article Snippet: MCF7 and MDA-MB-231 human breast cancer cell lines were purchased from the American Type Culture Collection (ATCC).

Techniques: Translocation Assay, De-Phosphorylation Assay, Phospho-proteomics, SDS Page, Western Blot, Control, Fractionation, Incubation, Expressing, Fluorescence, Microscopy

Journal: International Journal of Biological Sciences

Article Title: Inhibitory role of TRIP-Br1 oncoprotein in anticancer drug-mediated programmed cell death via mitophagy activation

doi: 10.7150/ijbs.72138

Figure Lengend Snippet: Suppression of STS-mediated cell death by TRIP-Br1 via induction of autophagy . A-B MCF7 WT-TRIP-Br1 and MCF7 KD-TRIP-Br1 cells were cultured for 24 h and then treated with 0.1 or 0.5 µM of STS for 3 h or 24 h. Western blot was performed and β-actin was used as a loading control. Data are presented as mean ± SD (n = 3; **, p < 0.01, ***, p < 0.001). C MCF7 WT-TRIP-Br1 and MCF7 KD-TRIP-Br1 cells were cultured in 100 mm culture dishes for 24 h and then treated with 0.1 or 0.5 µM of STS for 3 h or 24 h, respectively. Cell morphology was observed using an optical microscope with an IS capture software (KI-400F; Korea Lab Tech, Seongnam, South Korea) at ×100 magnification. The experiment was repeated three times. Images represent the average cell morphology. D MCF7 WT-TRIP-Br1 and MCF7KD-TRIP-Br1 cells were pretreated with CQ (100 mM) for 6 h, followed by STS treatment at indicated concentrations for 24 h. Cell viability was measured using a CellTiter-Glo 3D cell viability assay kit. Data are expressed as mean ± SD (n = 4). E Morphological changes in MCF7 WT-TRIP-Br1 and MCF7 KD-TRIP-Br1 cells were observed after treatment with CQ (100 mM) for 24 h, followed by STS (0.1 μM) treatment for 24 h. F MCF7 WT-TRIP-Br1 and MCF7 KD-TRIP-Br1 cells were pre-treated with CQ (100 mM) for 3 h, followed by STS (0.1 μM) treatment for 24 h. Each protein level was detected via immunoblotting using corresponding antibody.

Article Snippet: MCF7 and MDA-MB-231 human breast cancer cell lines were purchased from the American Type Culture Collection (ATCC).

Techniques: Cell Culture, Western Blot, Control, Microscopy, Software, Viability Assay

Journal: International Journal of Biological Sciences

Article Title: Inhibitory role of TRIP-Br1 oncoprotein in anticancer drug-mediated programmed cell death via mitophagy activation

doi: 10.7150/ijbs.72138

Figure Lengend Snippet: Suppression of STS-mediated cell death by TRIP-Br1 via induction of mitophagy. A-B MCF7 WT-TRIP-Br1 and MCF7 KD-TRIP-Br1 cells were pre-treated with CQ (100 mM), followed by STS (0.1 μM) for 24 h. Each protein level was detected via immunoblotting using the corresponding antibody. C-D MCF7 WT-TRIP-Br1 and MCF7 KD-TRIP-Br1 cells were cultured in the absence or presence of STS (0.1 μM) for 3 h. Resulting cells were used for mitochondrial fractionation and western blotting analysis as mentioned in Materials and Methods. Data are expressed as mean ± SD (n = 3; **, p < 0.01; ***, p < 0.001). E Relative quantification of mtDNA amount in MCF7 WT-TRIP-Br1 and MCF7 KD-TRIP-Br1 cells was performed using real-time (RT)-PCR. Cells were pre-treated with or without 100 mM CQ for 6 h and then treated with 0.1 µM STS for 24 h. Relative mtDNA levels were normalized to GAPDH and a graphical representation of the summary data is presented. Data are expressed as mean ± SD (n = 3). F MCF7 WT-TRIP-Br1 and MCF7KD-TRIP-Br1 cells were grown in confocal dishes for 24 h. These cells were incubated with STS (0.1 μM) or CQ (100 mM) for 6 h. They were then incubated with 1 µM of Mitotracker dye for 30 min. Cells were stained with LC3 (yellow) and Mitotracker (red). Representative fluorescent images were visualized with a confocal microscope (scale bars, 20 μM). The co-localization of mitochondria and LC3 was measured by counting more than 100 cells in ImageJ per experiment for each group. Data represent the mean value of three independent experiments. H-I TRIP-Br1 wild-type MEF (MEF WT-TRIP-Br1 ) and TRIP-Br1-null MEF (MEF KO-TRIP-Br1 ) were cultured in confocal dishes for 24 h, followed by staining with Mitotracker (100 nM) for 30 min. Representative fluorescent images were visualized under an immunofluorescence microscope (scale bars, 20 μm). Mitochondria intensity with red staining was quantified using ImageJ.

Article Snippet: MCF7 and MDA-MB-231 human breast cancer cell lines were purchased from the American Type Culture Collection (ATCC).

Techniques: Western Blot, Cell Culture, Fractionation, Quantitative Proteomics, Quantitative RT-PCR, Incubation, Staining, Microscopy, Immunofluorescence

Journal: International Journal of Biological Sciences

Article Title: Inhibitory role of TRIP-Br1 oncoprotein in anticancer drug-mediated programmed cell death via mitophagy activation

doi: 10.7150/ijbs.72138

Figure Lengend Snippet: Enhanced TRIP-Br1-mediated mitophagy via up-regulation of lysosomal proteases cathepsins B and D. A-B MCF7 WT-TRIP-Br1 and MCF7 KD-TRIP-Br1 cells were treated with 0.1 µM STS for 3 h and 24 h. The precursor and mature forms of cathepsins B and D were determined via immunoblotting analysis. β-actin was used as an internal control. Data are presented as mean ± SD (n = 3). C MCF7 WT-TRIP-Br1 and MCF7 KD-TRIP-Br1 cells were cultured in confocal dishes for 24 h and then incubated with Lysotracker (Red) for 90 min. These cells were captured using a confocal microscope. Quantification of red fluorescence intensity of Lysotracker is shown. Values are expressed as mean ± SD of three independent experiments with each count of no less than 100 cells. D Both cell lines were cultured for 24 h and then treated with 0.1 μM STS for 3 h. Cells were harvested and lysosomal fractions were isolated as described in Materials and Methods. γ -Tubulin was used as a cytosolic marker.

Article Snippet: MCF7 and MDA-MB-231 human breast cancer cell lines were purchased from the American Type Culture Collection (ATCC).

Techniques: Western Blot, Control, Cell Culture, Incubation, Microscopy, Fluorescence, Isolation, Marker

Journal: iScience

Article Title: Disuse plasticity limits spinal cord injury recovery

doi: 10.1016/j.isci.2025.112180

Figure Lengend Snippet: Early disuse persists synaptic expression of GluA2-lacking AMPARs as changes in synapse numbers on ventral horn neurons and dendritic neuropils after spinal cord injury Overdrive of glutamate AMPARs is known to reflect maladaptive spinal cord plasticity in central nervous system trauma. , (A–E) Workflow diagram of the automated and unbiased high-resolution robotic confocal microscopy. To assess synaptic levels of glutamate AMPARs on dendritic fields and somata of large ventral horn neurons, the randomized microscopic detection and analysis were performed blindly by spinning disk confocal scan system at week 8 post-injury according to the established algorithm. , (A) Fluorescently labeled large ventral horn neurons (diameter >40 μm) indicated presumptive motorneurons and were detected centrally in the sampling window (80 × 80 μm) at 63× magnification. (B) A stack of high-resolution images was taken in the z-plane through a 650 nm filter for presynaptic synaptophysin (upper panel) and a 490 nm filter for postsynaptic AMPARs (lower panel) at each level separately (scale bar: 20 μm). (C) Scanned confocal z stacks were deblurred by 3-D blind iterative deconvolution, and then the total expression of synaptic colocalized AMPAR puncta was quantified using the established approach. (D) Single optical planes showing maximal synaptic colocalization of presynaptic synaptophysin and postsynaptic AMPARs on the somata were selected among the z stacks, and then the optical fraction images of the somatic membrane were generated to assess the synaptic AMPAR expression on the plasma membrane (left panel). Representative optical detection of the presynaptic vesicle, postsynaptic AMPAR subunit, and synaptic colocalization (white arrows) are shown in the enlarged image of the boxed region from the plasma membrane image (right panel; see Figure 5 ). (E) Schematic overview on synaptic colocalization of AMPAR subunits GluA1 and GluA2. The overlapping of presynaptic synaptophysin (magenta) and synaptic AMPAR subunit (GluA1 or GluA2; green) puncta indicates synaptic colocalization (white). (F) Representative merged 3-D confocal image of large ventral horn neurons showed postsynaptic GluA1 (green) and synaptophysin (magenta) positive presynaptic terminals on the soma with surrounding dendritic neuropils in the control subject (left panel). The enlarged image of the boxed region from (F, left panel) demonstrates relatively few synaptic colocalized GluA1 puncta (lower right panel, white) within the merge image (upper right panel) in the control subject. (G) Representative merged motorneuron from the early disuse subject demonstrating greater numbers of membrane and dendritic GluA1 colocalized to the synapses (left panel). The enlargement of the boxed region from (G, left panel) demonstrates high levels of synaptic colocalized GluA1 puncta (lower right panel, white) within the merge image (upper right panel) in the early disuse subject. Representative images of synaptic GluA2 puncta are shown in Figure S8 . (H) Quantification of synaptic colocalized AMPAR expression with synaptophysin though the confocal z stacks in the early disuse group ( n = 5; total 164 cells, total 11,971 optical planes for GluA1; total 138 cells, total 9,788 planes for GluA2) compared to the control group ( n = 6; total 163 cells, total 13,037 images for GluA1; total 156 cells, total 10,894 optical planes for GluA2) on the somata and dendritic neuropil. Random effects ANOVA controlling for non-independence of within-subject and within-section variability confirmed that early disuse significantly increased in synaptic colocalization of GluA1 (effect of early disuse condition: F (1,9) = 8.05, ∗ p = 0.019) but no significant difference in synaptic colocalization of GluA2 (effect of early disuse condition: F (1,9) = 0.074, p = 0.792). There was a non-significantly difference between both groups in the presynaptic synaptophysin and postsynaptic AMPAR subunit ( Figure S8 ). Representative examples reflect the group median. ∗ p < 0.05 by one-way ANOVA. All data are shown as means ± SEM. Scale bars in the lower magnification images represent 20 μm, and the scale bar in the higher magnification image represents 5 μm.

Article Snippet: Slides were washed repeatedly with PBS and then coverslipped with Vectashield mounting medium with DAPI (Vector Laboratories, Burlingame, CA). (2) Confocal image acquisition and 3-D blind iterative image deconvolution All images were acquired at the UCSF Nikon Imaging Center using a Ti inverted microscope (Nikon Instruments, Melville, NY) with a spinning disk confocal scan system (CSU-22; Yokogawa Corporation, Sugar Land, TX) and a Plan Apo VC 63× oil immersion objective with 1.4 numerical aperture (Nikon Instruments), electron multiplying CCD camera running a custom instance of NIS elements imaging software (Nikon Instruments).

Techniques: Expressing, Confocal Microscopy, Labeling, Sampling, Membrane, Generated, Clinical Proteomics, Control

Journal: iScience

Article Title: Disuse plasticity limits spinal cord injury recovery

doi: 10.1016/j.isci.2025.112180

Figure Lengend Snippet:

Article Snippet: Slides were washed repeatedly with PBS and then coverslipped with Vectashield mounting medium with DAPI (Vector Laboratories, Burlingame, CA). (2) Confocal image acquisition and 3-D blind iterative image deconvolution All images were acquired at the UCSF Nikon Imaging Center using a Ti inverted microscope (Nikon Instruments, Melville, NY) with a spinning disk confocal scan system (CSU-22; Yokogawa Corporation, Sugar Land, TX) and a Plan Apo VC 63× oil immersion objective with 1.4 numerical aperture (Nikon Instruments), electron multiplying CCD camera running a custom instance of NIS elements imaging software (Nikon Instruments).

Techniques: Recombinant, Protease Inhibitor, Blocking Assay, Plasmid Preparation, Enzyme-linked Immunosorbent Assay, Bicinchoninic Acid Protein Assay, Software, Imaging, Inverted Microscopy, Microscopy

Journal: Experimental eye research

Article Title: Casein kinase I inhibitor D4476 influences autophagy and apoptosis in chloroquine-induced adult retinal pigment epithelial-19 cells.

doi: 10.1016/j.exer.2022.109004

Figure Lengend Snippet: Fig. 1. Protective effect of D4476 and possible association with autophagy in chloroquine (CQ)-treated adult retinal pigment epithelial (ARPE)-19 (commercially available stable RPE cell line) and primary mouse RPE cells. Cytotoxicity of ARPE-19 cells treated with (A) varying concentrations of CQ (10–100 μM) alone for 24 h; (B) 100 μM CQ with vehicle or different D4476 concentrations (1–20 μM) for 24 h; (C) vehicle, 100 μM CQ, CQ+D4476 and 10 μM D4476 for different time-points. (D and E) Relative survival rate of ARPE-19 cells treated with casein kinase 1 (CK1) inhibitor, IC261, and selective activin receptor-like kinase (ALK)5 inhibitor, SB525334, instead of D4476 with the same combination of drugs for 24 h and (F) 100 μM CQ with vehicle and with 0.5 μM rapamycin (Rap) for 24 h. Cytotoxicity assessed using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay unless otherwise specified. (G) Phase-contrast and fluorescence photo micrographs of primary mouse RPE cells. After 3 weeks of culture, cells were stained with RPE65, marker protein of RPE, and Na+/K+-ATPase, marker of plasma membrane. Scale bar, 20 μm. (H and I) Relative survival rate of primary mouse RPE cells. Cells were treated with vehicle, 100 μM CQ, CQ+D4476, 10 μM D4476, CQ+IC261, 1 μM IC261, CQ+SB525334, 1 μM SB525334, CQ+Rap, and 0.5 μM Rap for 24 h. Mean ± standard deviation (SD), n = 3; ##P < 0.01 and ###P < 0.001 compared to vehicle and *P < 0.05, **P < 0.01, ***P < 0.001 compared to CQ alone. ns, not significant, compared to vehicle or CQ-treated cells.

Article Snippet: Antibodies against AKT (#14691), phosphorylated-AKT (p-AKT, #4060), Bcl-2-associated X protein (Bax, #2772), Bcl-2 (#15071), and Bcl-xL (#2764), Bcl-2 Homology 3 (BH3)-interacting domain death agonist (BID, #2002), cleaved poly-ADP ribose polymerase (PARP, #5625), Mouse monoclonal antibody (mAb) immunoglobulin G1 (IgG1) isotype control (#5415), lysosomal membrane-associated protein (LAMP)-1 (#15665), light chain 3 (LC3) A/B (#12741), mammalian target of rapamycin (mTOR, #2983), p-mTOR (#5536), p62 (#8025), phosphoinositide 3- kinase (PI3K, #3358), p-PI3K (#13857)), p-c-Jun N terminal kinase (JNK, #4668), p38 mitogen-activated protein kinase (MAPK) (#8690), p-p38 MAPK (#4511), and zonula occludens (ZO)-1 (#13663) were purchased from Cell Signaling Technology (Danvers, MA, USA).

Techniques: MTT Assay, Fluorescence, Staining, Marker, Clinical Proteomics, Membrane, Standard Deviation

Journal: Experimental eye research

Article Title: Casein kinase I inhibitor D4476 influences autophagy and apoptosis in chloroquine-induced adult retinal pigment epithelial-19 cells.

doi: 10.1016/j.exer.2022.109004

Figure Lengend Snippet: Fig. 2. Attenuation of chloroquine (CQ)-induced inhibition of autophagy by D4476 in adult retinal pigment epithelial (ARPE)-19 cells. (A) Phase-contrast photo micrograph and (B) confocal images of green fluorescent protein-light chaing 3 (GFP-LC3, green)-transfected cells after immunostaining with anti-lysosomal asso ciated membrane protein (LAMP)-1 (red) antibody. Cells were treated with vehicle, 100 μM CQ alone, 10 μM D4476 alone, or 10 μM D4476 + 100 μM CQ for 6 h. 4ʹ,6-Diamidino-2-phenylindole (DAPI, blue) was used to counterstain nuclei. Scale bar, 10 μm. Quantification of LC3-positive vacuole (C) size, (D) number (green), and (E) number of LC3-positive (green) vacuoles colocalized (yellow) to LAMP-1 (red). One-hundred cells per experimental condition were analyzed using fluo rescence microscopy. (F) Western blot and quantitative analyses of Beclin 1, p62, and LC3 A/B expression in ARPE-19 cell lysates 6 h after treatment with vehicle, 100 μM CQ alone, 10 μM D4476 + 100 μM CQ, 10 μM D4476 alone, 0.5 μM rapamycin (Rap) plus 100 μM CQ, and 0.5 μM Rap alone. α-Tubulin was used as a loading control. Mean ± standard deviation (SD), n = 3; #P < 0.05, ##P < 0.01, and ###P < 0.001 compared to vehicle; *P < 0.05 and **P < 0.01 compared to CQ alone. ns, not significant, compared to vehicle or Bafilomycin A1 (BA1) alone-treated cells.

Article Snippet: Antibodies against AKT (#14691), phosphorylated-AKT (p-AKT, #4060), Bcl-2-associated X protein (Bax, #2772), Bcl-2 (#15071), and Bcl-xL (#2764), Bcl-2 Homology 3 (BH3)-interacting domain death agonist (BID, #2002), cleaved poly-ADP ribose polymerase (PARP, #5625), Mouse monoclonal antibody (mAb) immunoglobulin G1 (IgG1) isotype control (#5415), lysosomal membrane-associated protein (LAMP)-1 (#15665), light chain 3 (LC3) A/B (#12741), mammalian target of rapamycin (mTOR, #2983), p-mTOR (#5536), p62 (#8025), phosphoinositide 3- kinase (PI3K, #3358), p-PI3K (#13857)), p-c-Jun N terminal kinase (JNK, #4668), p38 mitogen-activated protein kinase (MAPK) (#8690), p-p38 MAPK (#4511), and zonula occludens (ZO)-1 (#13663) were purchased from Cell Signaling Technology (Danvers, MA, USA).

Techniques: Inhibition, Transfection, Immunostaining, Membrane, Microscopy, Western Blot, Expressing, Control, Standard Deviation

Journal: Experimental eye research

Article Title: Casein kinase I inhibitor D4476 influences autophagy and apoptosis in chloroquine-induced adult retinal pigment epithelial-19 cells.

doi: 10.1016/j.exer.2022.109004

Figure Lengend Snippet: Fig. 5. Effect of D4476 on chloroquine (CQ)-induced interaction of Beclin 1 B-cell lymphoma 2 (Bcl-2) homology 3 (BH3) domain with Bcl-2 and cell proliferation- associated signaling in adult retinal pigment epithelial (ARPE)-19 cells. (A) immunoblotted or (B) immunoprecipitated ARPE-19 cells. Cells were immunoprecipitated with anti-mouse IgG, as an isotype control, or anti-Bcl-2 antibody, then immunoblotted with anti-Beclin 1 or anti-Bcl-2 antibody. (C and D) Densitometric analysis of Immunoblots with Beclin 1 and Bcl-2. Quantitative analysis performed using ImageJ software. (E) Western blot and (F) quantitative densitometric analyses of phosphorylated phosphoinositide 3-kinase (p-PI3K), PI3K, p-AKT, AKT, p-mechanistic target of rapamycin (Rap, mTOR), mTOR, p-p38 mitogen-activated protein kinase (MAPK), p38 MAPK, p-c-Jun N terminal kinase (JNK), and JNK in APRE-19 cells 6 h after treatment with vehicle, 100 μM CQ alone, 10 μM D4476 + 100 μM CQ, 10 μM D4476 alone, 0.5 μM Rap+100 μM CQ, and 0.5 μM Rap alone. Data are means ± standard deviation (SD), n = 3; #P < 0.05 and ##P < 0.01 compared to vehicle and *P < 0.05 and **P < 0.01 compared to CQ alone. ns, not significant, compared to vehicle or CQ alone.

Article Snippet: Antibodies against AKT (#14691), phosphorylated-AKT (p-AKT, #4060), Bcl-2-associated X protein (Bax, #2772), Bcl-2 (#15071), and Bcl-xL (#2764), Bcl-2 Homology 3 (BH3)-interacting domain death agonist (BID, #2002), cleaved poly-ADP ribose polymerase (PARP, #5625), Mouse monoclonal antibody (mAb) immunoglobulin G1 (IgG1) isotype control (#5415), lysosomal membrane-associated protein (LAMP)-1 (#15665), light chain 3 (LC3) A/B (#12741), mammalian target of rapamycin (mTOR, #2983), p-mTOR (#5536), p62 (#8025), phosphoinositide 3- kinase (PI3K, #3358), p-PI3K (#13857)), p-c-Jun N terminal kinase (JNK, #4668), p38 mitogen-activated protein kinase (MAPK) (#8690), p-p38 MAPK (#4511), and zonula occludens (ZO)-1 (#13663) were purchased from Cell Signaling Technology (Danvers, MA, USA).

Techniques: Immunoprecipitation, Control, Western Blot, Software, Standard Deviation

Journal: Experimental eye research

Article Title: Casein kinase I inhibitor D4476 influences autophagy and apoptosis in chloroquine-induced adult retinal pigment epithelial-19 cells.

doi: 10.1016/j.exer.2022.109004

Figure Lengend Snippet: Fig. 7. c-Jun N terminal kinase (JNK) inhibitor-induced reproduction of D4476 effects in chloroquine (CQ)-treated adult retinal pigment epithelial (ARPE)-19 cells. (A) Relative survival percentage of APRE-19 cells treated with 100 μM CQ alone, 100 μM CQ+10 μM SP600125, and 10 μM SP600125 alone for 24 h. (B) immu noblotted or (C) immunoprecipitated ARPE-19 cells. Cell lysates immunoprecipitated with anti-mouse Ig G or anti-B-cell lymphoma 2 (Bcl-2) antibody were immunoblotted with anti-Beclin 1 or anti-Bcl-2 antibody. Bars denote densitometric analysis of Immunoblots. (D) Western blots and (E and F) quantitative densi tometric analyses for phosphorylated mechanistic target of rapamycin (p-mTOR), mTOR, p-p38 mitogen-activated protein kinase (MAPK), p38 MAPK, p-JNK, JNK, light chain 3 (LC3) A/B, p62, and Bcl-xL in APRE-19 cells 6 h after treatment as in (A). Data are means ± standard deviation (SD), n = 3; #P < 0.05, ##P < 0.01, and ###P < 0.001 compared to vehicle and *P < 0.05, **P < 0.01, and ***P < 0.001 compared to CQ alone. ns, not significant, compared to vehicle or CQ alone.

Article Snippet: Antibodies against AKT (#14691), phosphorylated-AKT (p-AKT, #4060), Bcl-2-associated X protein (Bax, #2772), Bcl-2 (#15071), and Bcl-xL (#2764), Bcl-2 Homology 3 (BH3)-interacting domain death agonist (BID, #2002), cleaved poly-ADP ribose polymerase (PARP, #5625), Mouse monoclonal antibody (mAb) immunoglobulin G1 (IgG1) isotype control (#5415), lysosomal membrane-associated protein (LAMP)-1 (#15665), light chain 3 (LC3) A/B (#12741), mammalian target of rapamycin (mTOR, #2983), p-mTOR (#5536), p62 (#8025), phosphoinositide 3- kinase (PI3K, #3358), p-PI3K (#13857)), p-c-Jun N terminal kinase (JNK, #4668), p38 mitogen-activated protein kinase (MAPK) (#8690), p-p38 MAPK (#4511), and zonula occludens (ZO)-1 (#13663) were purchased from Cell Signaling Technology (Danvers, MA, USA).

Techniques: Immunoprecipitation, Western Blot, Standard Deviation

Journal: Experimental eye research

Article Title: Casein kinase I inhibitor D4476 influences autophagy and apoptosis in chloroquine-induced adult retinal pigment epithelial-19 cells.

doi: 10.1016/j.exer.2022.109004

Figure Lengend Snippet: Fig. 6. p38 Mitogen-activated protein kinase (MAPK) inhibitor-induced reproduction of D4476 effects in chloroquine (CQ)-treated adult retinal pigment epithelial (ARPE)-19 cells. (A) Relative survival rate of APRE-19 cells 24 h after treatment with vehicle, 100 μM CQ alone, 100 μM CQ+20 μM SB203580, 20 μM SB203580 alone. (B) immunoblotted or (C) immunoprecipitated ARPE-19 cells. Cell lysates immunoprecipitated using anti-mouse Ig G or anti-B-cell lymphoma 2 (Bcl-2) antibody were immunoblotted with anti-Beclin 1 and anti-Bcl-2 antibodies. Bars denote densitometric analyses of Immunoblots. (D) Western blot and (E and F) quantitative densitometric analyses of phosphorylated mechanistic target of rapamycin (p-mTOR), mTOR, p-p38 mitogen-activated protein kinase (MAPK), p38 MAPK, p-c-Jun N terminal kinase (JNK), JNK, light chain 3 (LC3) A/B, p62, and Bcl-2 extra-large (Bcl-xL) in APRE-19 cells 6 h after treatment as in (A). Data are means ± standard deviation (SD), n = 3; #P < 0.05, ##P < 0.01, and ###P < 0.001 compared to vehicle and *P < 0.05, **P < 0.01, and ***P < 0.001 compared to CQ alone. ns, not significant, compared to vehicle or CQ alone.

Article Snippet: Antibodies against AKT (#14691), phosphorylated-AKT (p-AKT, #4060), Bcl-2-associated X protein (Bax, #2772), Bcl-2 (#15071), and Bcl-xL (#2764), Bcl-2 Homology 3 (BH3)-interacting domain death agonist (BID, #2002), cleaved poly-ADP ribose polymerase (PARP, #5625), Mouse monoclonal antibody (mAb) immunoglobulin G1 (IgG1) isotype control (#5415), lysosomal membrane-associated protein (LAMP)-1 (#15665), light chain 3 (LC3) A/B (#12741), mammalian target of rapamycin (mTOR, #2983), p-mTOR (#5536), p62 (#8025), phosphoinositide 3- kinase (PI3K, #3358), p-PI3K (#13857)), p-c-Jun N terminal kinase (JNK, #4668), p38 mitogen-activated protein kinase (MAPK) (#8690), p-p38 MAPK (#4511), and zonula occludens (ZO)-1 (#13663) were purchased from Cell Signaling Technology (Danvers, MA, USA).

Techniques: Immunoprecipitation, Western Blot, Standard Deviation

Journal: Experimental eye research

Article Title: Casein kinase I inhibitor D4476 influences autophagy and apoptosis in chloroquine-induced adult retinal pigment epithelial-19 cells.

doi: 10.1016/j.exer.2022.109004

Figure Lengend Snippet: Fig. 8. Schematic representation of D4476 effects on crosstalk between autophagy and apoptosis in chlo roquine (CQ)-treated adult retinal pigment epithelial (ARPE)-19 cells. D4476 inhibits CQ-induced increase of mechanistic target of rapamycin (mTOR), c-Jun N terminal kinase (JNK), and p38 mitogen-activated protein kinase (MAPK) activities. This inhibitory ef fect alters CQ-mediated interaction between Beclin 1 and B-cell lymphoma 2 (Bcl-2) via the Bcl-2 homol ogy 3 (BH3) domain, resulting in release of Beclin 1 from Bcl-2 and activating autophagy. Beclin 1, light chain 3 (LC3) A/B, and p62 are all associated with autophagosome formation and autophagy flux. D4476 may play an important role in the gateway of intersection between autophagy and apoptosis during CQ-induced toxicity, mitigating damage through reinstating cellular homeostasis.

Article Snippet: Antibodies against AKT (#14691), phosphorylated-AKT (p-AKT, #4060), Bcl-2-associated X protein (Bax, #2772), Bcl-2 (#15071), and Bcl-xL (#2764), Bcl-2 Homology 3 (BH3)-interacting domain death agonist (BID, #2002), cleaved poly-ADP ribose polymerase (PARP, #5625), Mouse monoclonal antibody (mAb) immunoglobulin G1 (IgG1) isotype control (#5415), lysosomal membrane-associated protein (LAMP)-1 (#15665), light chain 3 (LC3) A/B (#12741), mammalian target of rapamycin (mTOR, #2983), p-mTOR (#5536), p62 (#8025), phosphoinositide 3- kinase (PI3K, #3358), p-PI3K (#13857)), p-c-Jun N terminal kinase (JNK, #4668), p38 mitogen-activated protein kinase (MAPK) (#8690), p-p38 MAPK (#4511), and zonula occludens (ZO)-1 (#13663) were purchased from Cell Signaling Technology (Danvers, MA, USA).

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