Journal: Biomolecules & Therapeutics

Article Title: Late Passage Cultivation Induces Aged Astrocyte Phenotypes in Rat Primary Cultured Cells

doi: 10.4062/biomolther.2020.175

Figure Lengend Snippet: Late passage astrocytes show reduced mitochondrial membrane potential and ATP production. (A) Astrocytes were conducted for mitochondrial membrane potential by fluorescence staining of TMRM. Representative images were obtained by the IncuCyte ZOOM microscope software 2015A (Essen Bioscience). The data were measured by IncuCyte software (Essen Bioscience). The representative graph shows the total TMRM object integrated intensity (RCU x μm 2 /image) and the total TMRM object area (μm 2 /image). Bars represent the mean ± SEM (n=3). **indicates p <0.01, and ***indicates p <0.001 vs. vehicle group. (B) The mitochondrial OCR of astrocytes was experimented using Cell Mito Test Kit and Agilent Seahorse XFe96 Analyzer (Agilent Technologies). The representative graphs were visualized using Wave Desktop 2.6 software (Agilent Technologies). The graphs represent the basal level, spare respiratory capacity, proton leak, and ATP production level. OCRs are mean ± SEM (n=3), normalized to the third measurement point and expressed as % baseline OCR. *indicates p <0.05 and, ***indicates p <0.001 vs. P1 astrocytes.

Article Snippet: We detected the red fluorescence using the IncuCyte ZOOM system (Essen Bioscience) by imaging each well every 30 min for 12 h and analyzed the red particles using the IncuCyte ZOOM microscope software 2015A (Essen Bioscience).

Techniques: Membrane, Fluorescence, Staining, Microscopy, Software

Journal: Molecular Medicine Reports

Article Title: Effect of OLIG1 on the development of oligodendrocytes and myelination in a neonatal rat PVL model induced by hypoxia-ischemia

doi: 10.3892/mmr.2014.3028

Figure Lengend Snippet: Observation of the effects of hypoxia-ischemia (HI) on oligodendrocytes (OLs) by transmission electron microscopy (magnification, ×25,000). (A and C) In the normoxia group, the morphology and structure of OLs is visible, with a large, full nucleus, and a complete nuclear membrane, indicated by arrows. (B) In the HI group, the cell morphology and structure is highly irregular, with cell atrophy observed, altered nuclear structure, and cell necrosis at day 3. (D) By day 21, the OLs have obtained an irregular structure and show serious cell degeneration and necrosis.

Article Snippet: The sections were then blocked with 10% goat serum for 30 min at 37°C, and incubated with the mouse monoclonal primary antibodies anti-oligodendrocyte marker O4 and anti-OLIG1 (both at 1:100 dilution; R&D Systems, Minneapolis, MN, USA) overnight at 4°C.

Techniques: Transmission Assay, Electron Microscopy, Membrane

Journal: International Journal of Molecular Sciences

Article Title: DNA Methyltransferase 1 (DNMT1) Acts on Neurodegeneration by Modulating Proteostasis-Relevant Intracellular Processes

doi: 10.3390/ijms21155420

Figure Lengend Snippet: DNMT1 acts on retrograde vesicle transport. ( a ) GO terms found significantly enriched for genes that were up-regulated upon Dnmt1 deletion in adult Pvalb-Cre -expressing cortical interneurons determined by RNA sequencing after fluorescence activated cell (FAC)-sorting of Pvalb-Cre / tdTomato / Dnmt1 wild type (WT) and knockout (KO) mice (Benjamini-adjusted, n = 9 WT and n = 12 KO mice). ( b ) Schematic representation of a cell, depicting the directions of intracellular transport in the neurite-like processes. ( c ) Representative microphotographs of the tracking of cluster of differentiation 63 (CD63)-green fluorescent protein (GFP)-labeled vesicles during live cell imaging in neuroblastoma (N2a) cells transfected with Cd63-GFP plasmid together with control (Ctrl) siRNA ( n = 14 cells and 34 vesicles) or Dnmt1 siRNA ( n = 14 cells and 38 vesicles). The velocity of the transported vesicles is quantified in ( d ) (two-tailed Student’s t -test, * p < 0.05). ( e ) Representative microphotographs of the tracking of lysosomal-associated membrane protein 1 (LAMP1)-mCherry-positive vesicles during live cell imaging in N2a cells transfected with Lamp1-mCherry plasmid together with Ctrl siRNA ( n = 20 cells and 38 vesicles) or Dnmt1 siRNA ( n = 17 cells and 37 vesicles). The velocity of the transported vesicles is quantified in ( f ) (Two-tailed Student’s t -test, *** p < 0.001). Ctrl = control, KO = knockout, LAMP1 = lysosomal-associated membrane protein 1, N2a = neuroblastoma cells, siR = siRNA, WT = wild type. Scale bars: 10 µm in ( c , e ).

Article Snippet: For co-transfection we applied 30 nM Dnmt1 siRNA (#sc-35203, Santa Cruz Biotechnology), 15 nM control siRNA (Block-iT Alexa Fluor red (#14750100) or Block-iT green ((#2013) fluorescent oligo, Thermo Fisher Scientific, Waltham, MA, USA) and plasmid DNA (pLAMP1-mCherry, 200 ng/μL; CD63-pEGFP C2, 200 ng/μL; 1× GFP-pEGFP N3-HTT, 260 ng/μL) with Lipofectamine 2000 (Thermo Fisher Scientific), as described in the protocol of the manufacturer.

Techniques: Expressing, RNA Sequencing, Fluorescence, Knock-Out, Labeling, Live Cell Imaging, Transfection, Plasmid Preparation, Control, Two Tailed Test, Membrane

Journal: International Journal of Molecular Sciences

Article Title: DNA Methyltransferase 1 (DNMT1) Acts on Neurodegeneration by Modulating Proteostasis-Relevant Intracellular Processes

doi: 10.3390/ijms21155420

Figure Lengend Snippet: Dnmt1 knockdown ameliorated the cell survival upon mutant HTT-induced cytotoxicity. ( a ) Representative microphotographs of start and end points of the 12 h time interval used to perform the HTT cytotoxicity assay in N2a cells expressing GFP-labeled mutant HTT (green). Cells were transfected with either Ctrl or Dnmt1 siRNA. ( b ) Cell survival rate quantification of N2a cells expressing GFP-labeled mutant HTT and transfected with Ctrl siRNA ( n = 4 experiments, n = 274 cells) or Dnmt1 siRNA ( n = 4 experiments, n = 378 cells) over the time course of 12 h in the HTT cytotoxicity assay (two-way ANOVA, *** p < 0.001). ( c ) Comparison of the survival rates of cells analyzed in ( b ) at the final 12 h timepoint (two-tailed Student’s t -test, *** p < 0.001). ( d ) Representative microphotographs of start and end points of 12 h interval used to perform the HTT cytotoxicity assay in CB cells expressing GFP-labeled mutant HTT (green). Cells were transfected with either Ctrl or Dnmt1 siRNA. ( e ) Cell survival rates of CB cells expressing GFP-labeled mutant HTT and transfected with Ctrl siRNA ( n = 100 cells) or Dnmt1 siRNA ( n = 132 cells) over the time course of 12 h in the HTT cytotoxicity assay (two-way ANOVA, *** p < 0.001). ( f ) Comparison of the survival rates of cells analyzed in ( e ) at the 12 h timepoint (two-tailed Student’s t -test, *** p < 0.001). N2a = neuroblastoma cells, CB = cerebellar granule cells, Ctrl = control, HTT = Huntingtin, siR = siRNA, BF = brightfield. Scale bars: 50 µm in ( a , d ).

Article Snippet: For co-transfection we applied 30 nM Dnmt1 siRNA (#sc-35203, Santa Cruz Biotechnology), 15 nM control siRNA (Block-iT Alexa Fluor red (#14750100) or Block-iT green ((#2013) fluorescent oligo, Thermo Fisher Scientific, Waltham, MA, USA) and plasmid DNA (pLAMP1-mCherry, 200 ng/μL; CD63-pEGFP C2, 200 ng/μL; 1× GFP-pEGFP N3-HTT, 260 ng/μL) with Lipofectamine 2000 (Thermo Fisher Scientific), as described in the protocol of the manufacturer.

Techniques: Knockdown, Mutagenesis, Cytotoxicity Assay, Expressing, Labeling, Transfection, Comparison, Two Tailed Test, Control

Journal: International Journal of Molecular Sciences

Article Title: DNA Methyltransferase 1 (DNMT1) Acts on Neurodegeneration by Modulating Proteostasis-Relevant Intracellular Processes

doi: 10.3390/ijms21155420

Figure Lengend Snippet: Dnmt1 knockdown led to a faster aggregate formation of mutant HTT in CB cells. ( a ) Representative microphotographs of CB cells expressing GFP-labeled mutant HTT (green) and transfected with either Ctrl or Dnmt1 siRNA, showing the temporal differences in the aggregate formation of mutant HTT. ( b ) Quantification of the start and the duration of aggregate formation depicted in ( a ) for cells transfected with Ctrl siRNA ( n = 27 cells) or Dnmt1 siRNA ( n = 33 cells; two-sided Student’s t -test, * p < 0.05). BF = bright field, Ctrl = control, HTT = Huntingtin, siR = siRNA. Scale bars: 20 µm in ( a ).

Article Snippet: For co-transfection we applied 30 nM Dnmt1 siRNA (#sc-35203, Santa Cruz Biotechnology), 15 nM control siRNA (Block-iT Alexa Fluor red (#14750100) or Block-iT green ((#2013) fluorescent oligo, Thermo Fisher Scientific, Waltham, MA, USA) and plasmid DNA (pLAMP1-mCherry, 200 ng/μL; CD63-pEGFP C2, 200 ng/μL; 1× GFP-pEGFP N3-HTT, 260 ng/μL) with Lipofectamine 2000 (Thermo Fisher Scientific), as described in the protocol of the manufacturer.

Techniques: Knockdown, Mutagenesis, Expressing, Labeling, Transfection, Control

Journal: International Journal of Molecular Sciences

Article Title: DNA Methyltransferase 1 (DNMT1) Acts on Neurodegeneration by Modulating Proteostasis-Relevant Intracellular Processes

doi: 10.3390/ijms21155420

Figure Lengend Snippet: DNMT1 acts on autophagy. ( a ) Heat-map of significantly upregulated genes associated with the GO term autophagy in FAC-sorted cortical interneurons of Pvalb - Cre / tdTomato / Dnmt1 KO mice compared to cells of Pvalb - Cre / tdTomato / Dnmt1 WT mice. Expression level determined by RNA-sequencing (* p < 0.05, Benjamini-adjusted, n = 9 WT and n = 12 KO mice). ( b ) Schematic illustration of the autophagy pathway beginning with the formation of an autophagosome and depicting the transition to an amphisome, which fuses with a lysosome to form the autolysosome, where particles are degraded. ( c ) Protein levels of DNMT1, microtubule-associated protein 1 light chain 3B-I (LC3B-I), and LC3B-II in Ctrl and Dnmt1 siRNA-treated N2a cells in the autophagy assay analyzed by Western blot. Cells were cultured in culture medium or starving solution (Earle’s balanced salt solution (EBSS)) and partly treated with an autophagy inhibitor (bafilomycin A1). ( d ) Quantification of protein levels of the conditions shown in ( c ) normalized to beta-actin (bACTIN) and in relation to the control ( n = 7 experiments; two-tailed Student’s t -test, * p < 0.05, ** p < 0.01, *** p < 0.001). DEG = differentially expressed genes, FC = fold change, bACTIN = beta-actin, Baf. A1 = bafilomycin A1, Ctrl = control, EBSS = Earle’s balanced salt solution, LC3B-I/II = microtubule-associated protein 1 light chain 3B-I/II, N2a = neuroblastoma cells, siR = siRNA.

Article Snippet: For co-transfection we applied 30 nM Dnmt1 siRNA (#sc-35203, Santa Cruz Biotechnology), 15 nM control siRNA (Block-iT Alexa Fluor red (#14750100) or Block-iT green ((#2013) fluorescent oligo, Thermo Fisher Scientific, Waltham, MA, USA) and plasmid DNA (pLAMP1-mCherry, 200 ng/μL; CD63-pEGFP C2, 200 ng/μL; 1× GFP-pEGFP N3-HTT, 260 ng/μL) with Lipofectamine 2000 (Thermo Fisher Scientific), as described in the protocol of the manufacturer.

Techniques: Expressing, RNA Sequencing, Western Blot, Cell Culture, Control, Two Tailed Test

Journal: Oncogene

Article Title: RB loss determines selective resistance and novel vulnerabilities in ER-positive breast cancer models

doi: 10.1038/s41388-022-02362-2

Figure Lengend Snippet: A Heat map representing the relative growth rate of MCF7 and T47D cells in the presence of drug library at two different concentrations (100 nM and 500 nM) from cluster 1. B Western blot analysis on the indicated proteins from MCF7 and T47D cells that were treated with everolimus (250 nM) and alpelisib (250 nM) up to 48 H. C Western blot analysis to investigate the effect of alpelisib (250 nM) on cell cycle proteins from T47D and T47D-D1/K4 that were treated up to 48 H. D Live cell imaging to monitor the growth of MCF7-WT, MCF7-D1/K4, T47D-WT, and T47D-D1/K4 cells treated with different pairwise combination of palbociclib (50 nM), fulvestrant (100 nM), everolimus (125 nM) and alpelisib (125 nM). Error bars represent mean and SD from triplicates. Experiments were done at two independent times. E Western blot on the indicated proteins from MCF7-WT, MCF7-D1/K4, T47D-WT and T47D-D1/K4 that were treated with palbociclib (50 nM) + fuvlestrant (100 nM) and alpelisib (125 nM) + fulvestrant (100 nM) for 48 h. F Growth curves illustrating the effect of indicated combination treatments involving palbociclib (50 nM), fulvestrant (100 nM), everolimus (125 nM) and alpelisib (125 nM) in MCF7-RB-del and T47D-RB-del cells. Mean and SD were calculated from triplicates. G Western blot analysis to demonstrate the differential effect of palbociclib (50 nM) + Everolimus (125 nM) and palbociclib (50 nM)+ alpelisib (125 nM) on cell cycle proteins. H Heat map to demonstrate the synergistic effect between the combination of palbociclib with everolimus (Palb/Evero) and alpelisib (Palb/Alpe) on BrdU incorporation at the indicated doses in MCF-WT, MCF7-RB-del, T47D-WT, and T47D-RB-del cells. The values in heat map represent the mean from triplicates. BrdU incorporation was determined in CAMA-1 WT and CAMA-1-RB-del cells treated with the palbociclib (palbo) in combination with everolimus (evero) and fulvestrant (fulve) at indicated concentrations for 72 h. Error bars represent SD from triplicates (*p < 0.05, **p < 0.01 as determined by two-way ANOVA). I PFS of patients receiving CDK4/6i in combination with endocrine therapy with tumors harboring WT RB (n = 68) and RB-deletion mutation (n = 3). All p values were indicated.

Article Snippet: Palbociclib, Alisertib, and Birinapant were purchased from MedChemExpress (NJ, USA).

Techniques: Drug discovery, Western Blot, Live Cell Imaging, BrdU Incorporation Assay, Mutagenesis

Journal: Oncogene

Article Title: RB loss determines selective resistance and novel vulnerabilities in ER-positive breast cancer models

doi: 10.1038/s41388-022-02362-2

Figure Lengend Snippet: A Heat maps representing the relative cell viability of MCF7-WT, MCF7-RB-del, T47D-WT and T47D-RB-del cells in the presence of drug library (100 nM) following the pretreatment with birinapant (250 nM) from clusters 1 & 2. B Cell viability assays from MCF7 and T47D WT/RB-del that were treated with birinapant in combination with the indicated drugs at the indicated concentrations up to 6 days. Mean and SD were calculated from triplicates. Experiment was done at 2 independent times. (*p < 0.05, **p < 0.01 as determined by two-way ANOVA). C Crystal violet staining from MCF7-WT, MCF7-RB-del, T47D-WT and T47D-RB-del cells that were treated with Alpelisib (100 nM) in combination with DMSO or birinapant (500 nM). Cells were allowed to form colonies up to 15 days. D Live cell imaging to monitor the proliferation of MCF7-WT and RB-del cells that were treated with DMSO, birinapant (500 nM) and palbociclib (50 nM) + fulvestrant (100 nM) ± birinapant. The live cell imaging was terminated, and the cells were subjected to CTG assay to determine the viable cells. Mean and SD were calculated from 6 replicates. (**p < 0.01, ***p < 0.001 as determined by two-way ANOVA).

Article Snippet: Palbociclib, Alisertib, and Birinapant were purchased from MedChemExpress (NJ, USA).

Techniques: Drug discovery, Staining, Live Cell Imaging, CTG Assay

Journal: Frontiers in Immunology

Article Title: Unveiling spatial complexity in solid tumor immune microenvironments through multiplexed imaging

doi: 10.3389/fimmu.2024.1383932

Figure Lengend Snippet: Immunophenotyping panel for multiplexed tissue imaging of cancer.

Article Snippet: CD49α , REA1106 , 11 , 130-119-305 , FITC , Miltenyi Biotec.

Techniques: Imaging