Journal: The Journal of Biological Chemistry

Article Title: A surge of cytosolic calcium dysregulates lysosomal function and impairs autophagy flux during cupric chloride–induced neuronal death

doi: 10.1016/j.jbc.2023.105479

Figure Lengend Snippet: Biochemical features of autophagy in MN9D cells treated with CuCl 2 . Cells were treated with or without 250 μM CuCl 2 for the indicated time periods. A , immunoblot analyses were performed using anti-LC3 or anti-p62 antibodies. Anti-GAPDH antibody was utilized as a loading control. The relative intensities of LC3-II ( B ) and p62 ( C ) signals at the indicated time points were measured using ImageJ software, normalized by GAPDH signal, and expressed as fold change relative to untreated control (value = 1). Data are shown as the mean ± S.D of three independent experiments. ∗ p < 0.05; ∗∗ p < 0.01; ∗∗∗ p < 0.001. D , cells treated with 250 μM CuCl 2 for 15 h were subjected to immunocytochemical localization of LC3 ( green ) and p62 ( red ). Nuclei were counterstained with Hoechst 33258 ( blue ). Cells were examined under a confocal microscope. Merged images are shown to the right . Scale bar represents 10 μm. The number ( E ) and area ( F ) of LC3 and the area of p62 ( G ) puncta per cell were quantified using ImageJ software. Data are expressed as fold change relative to untreated control (value = 1) and shown as the mean ± S.D of three independent experiments. ∗∗ p < 0.01. H , cellular lysates were subjected to immunoblot analyses using the indicated antibodies. After normalization against the intensity of total protein, the relative intensities of the phosphorylated forms of mTOR ( I ), p-AMPK ( J ), p-Akt ( K ), and p-p70S6K ( L ) are expressed as fold change relative to untreated controls (value = 1). Data are shown as the mean ± S.D of three independent experiments. ∗∗ p < 0.01; ∗∗∗ p < 0.001; NS, not significant.

Article Snippet: Nuclear counterstaining was carried out using the DNA helix intercalating dye Hoechst 33,258 (1 μg/ml; Molecular Probes, H-1398).

Techniques: Western Blot, Control, Software, Microscopy

Journal: The Journal of Biological Chemistry

Article Title: A surge of cytosolic calcium dysregulates lysosomal function and impairs autophagy flux during cupric chloride–induced neuronal death

doi: 10.1016/j.jbc.2023.105479

Figure Lengend Snippet: CuCl 2 -mediated blockade of autophagy flux. MN9D cells were treated with or without 250 μM CuCl 2 for 15 h in the presence or absence of 25 nM Baf.A1 for the final 6 h. A , cell lysates were subjected to autophagy flux assay by monitoring and comparing levels of LC3-II and p62. The relative intensity of normalized LC3-II ( B ) and p62 ( C ) signals measured using ImageJ software expressed as fold change relative to untreated control (value = 1). Data are shown as the mean ± S.D of three independent experiments. Two-way ANOVA followed by Tukey’s post hoc test was performed. ∗ p < 0.05; ∗∗ p < 0.01; ∗∗∗ p < 0.001; NS, not significant. D , immunocytochemical analyses were performed using anti-LC3 ( green ) followed by nuclei counterstaining with Hoechst 33258 ( blue ). Fluorescent images were obtained from the confocal examination. Merged images are shown to the right . Scale bar represents 10 μm. The number ( E ) and area ( F ) of LC3 puncta per cell were quantified using ImageJ software. Data are expressed as fold change relative to untreated control (value = 1) and shown as the mean ± S.D of three independent experiments. Two-way ANOVA followed by Tukey’s post hoc test was performed. ∗∗∗ p < 0.001; NS, not significant. G , MN9D cells were transfected with an mRFP-EGFP–tagged LC3B probe for 24 h and then treated with 250 μM CuCl 2 for 15 h. After fixation, nuclei were counterstained with Hoechst 33258 ( blue ). Fluorescent images were acquired using confocal microscopy. Merged images are shown to the right . Scale bar represents 10 μm. H , quantification of the number of yellow (mRFP + -EGFP + ) and red (mRFP + -EGFP − ) puncta from the merged images was performed using ImageJ software. Data are shown as the mean ± S.D of three independent experiments. ∗∗∗ p < 0.001.

Article Snippet: Nuclear counterstaining was carried out using the DNA helix intercalating dye Hoechst 33,258 (1 μg/ml; Molecular Probes, H-1398).

Techniques: Flux Assay, Software, Control, Transfection, Confocal Microscopy

Journal: The Journal of Biological Chemistry

Article Title: A surge of cytosolic calcium dysregulates lysosomal function and impairs autophagy flux during cupric chloride–induced neuronal death

doi: 10.1016/j.jbc.2023.105479

Figure Lengend Snippet: Lysosomal dysfunction induced by CuCl 2 treatment. MN9D cells were treated with or without 250 μM CuCl 2 for 15 h. A , immunocytochemical analyses were performed using anti-LAMP1 ( red ) followed by nuclei counterstaining with Hoechst 33258 ( blue ). Merged images are shown to the right . Scale bar represents 20 μm. B , the number of LAMP1 puncta per cell was quantified using ImageJ software. Data are shown as the mean ± S.D of three independent experiments. NS, not significant. C , immunoblot analyses were performed using anti-LAMP1, anti-cathepsin D, or anti-LC3 antibodies. The cleaved band of cathepsin D (c-Cat D) was detected by an anti-cathepsin D antibody. The relative intensities of LAMP1 ( D ) and c-Cat D ( E ) signals were measured using ImageJ software, normalized by GAPDH signal, and expressed as fold change relative to untreated control (value = 1). Data are shown as the mean ± S.D of three independent experiments. ∗ p < 0.05; NS, not significant. F , after treatment with 250 μM CuCl 2 for 15 h or 500 nM Torin-1 for 24 h, cells were subjected to immunocytochemical analyses after probing with anti-LC3 ( green ) or anti-LAMP1 ( red ) followed by nuclei counterstaining with Hoechst 33258 ( blue ). Merged images are shown to the right . Scale bar represents 10 μm. G , quantification of puncta colocalized with LC3 and LAMP1 was performed. Data are shown as the mean ± S.D of three independent experiments. NS, not significant. H , MN9D cells treated with or without 250 μM CuCl 2 for 15 h were stained with Magic Red cathepsin B (Cat B). Nuclei were counterstained with Hoechst 33258 ( blue ). Merged images are shown to the right . Scale bar represents 20 μm. I , the relative intensity of each fluorescent signal of Cat B was quantified using ImageJ software. Data are shown as the mean ± S.D of three independent experiments. ∗∗∗ p < 0.001. J , MN9D cells treated with or without 250 μM CuCl 2 for 15 h and probed with LysoTracker Red. Representative confocal images are provided. Nuclei were counterstained with Hoechst 33258 ( blue ). Merged images are shown to the right . Scale bar represents 20 μm. K , the relative intensity of each fluorescent signal of LysoTracker Red was quantified using ImageJ software. Data are shown as the mean ± S.D of three independent experiments. ∗ p < 0.05.

Article Snippet: Nuclear counterstaining was carried out using the DNA helix intercalating dye Hoechst 33,258 (1 μg/ml; Molecular Probes, H-1398).

Techniques: Software, Western Blot, Control, Staining

Journal: The Journal of Biological Chemistry

Article Title: A surge of cytosolic calcium dysregulates lysosomal function and impairs autophagy flux during cupric chloride–induced neuronal death

doi: 10.1016/j.jbc.2023.105479

Figure Lengend Snippet: Cytosolic Ca 2+ -dependent impairment of autophagy flux in MN9D cells following CuCl 2 treatment. A , immunoblot analyses were performed using anti-CB300 antibody using cell lysates obtained from stable MN9D/Neo and MN9D/CaBP cells. Anti-GAPDH antibody was utilized as a loading control. B – O , MN9D/Neo or MN9D/CaBP cells were treated with or without 250 μM CuCl 2 for 15 h. B , cells were stained with 3 μM Fluo-3 ( green ). Representative confocal images are provided. Nuclei were counterstained with Hoechst 33258 ( blue ). Merged images are shown to the right . Scale bar represents 20 μm. C , MTT reduction assay was performed to assess cell viability, which was expressed as a percentage over untreated matching control (value = 1). Data are shown as the mean ± S.D. of three independent experiments. Two-way ANOVA followed by Tukey’s post hoc test was performed. ∗∗∗ p < 0.001. D , immunoblot analyses were performed using anti-LC3, anti-fodrin, or anti-p62 antibodies. The calpain-cleaved band of fodrin (c-fodrin) was detected by an anti-fodrin antibody. Relative intensities of LC3-II ( E ) and p62 ( F ) signals were measured using ImageJ software, normalized by the intensity of GAPDH signal, and expressed as fold change relative to untreated control (value = 1). Data are shown as the mean ± S.D of three independent experiments. Two-way ANOVA followed by Tukey’s post hoc test was performed. ∗∗∗ p < 0.001; NS, not significant. G , immunocytochemical analyses were performed using anti-LC3 ( green ) and anti-p62 ( red ), and nuclei were counterstained with Hoechst 33258 ( blue ). Representative confocal images are provided. Merged images are shown to the right . Scale bar represents 10 μm. The number ( H ) and area ( I ) of LC3 and area of p62 ( J ) puncta per cell were quantified using ImageJ software. Data are shown as the mean ± S.D of three independent experiments. Two-way ANOVA followed by Tukey’s post hoc test was performed. ∗∗ p < 0.01; ∗∗∗ p < 0.001. K , cellular lysates were subjected to immunoblot analyses using the indicated antibodies. After normalization against the intensity of total protein, relative intensities of the phosphorylated forms of mTOR ( L ), p-AMPK ( M ), p-Akt ( N ), and p-p70S6K ( O ) are expressed as fold change relative to untreated control (value = 1). Data are shown as the mean ± S.D of three independent experiments. Two-way ANOVA followed by Tukey’s post hoc test was performed. (( N ) ANOVA p value is 0.229215) ∗ p < 0.05; ∗∗ p < 0.01; ∗∗∗ p < 0.001; NS, not significant.

Article Snippet: Nuclear counterstaining was carried out using the DNA helix intercalating dye Hoechst 33,258 (1 μg/ml; Molecular Probes, H-1398).

Techniques: Western Blot, Control, Staining, MTT Reduction Assay, Software

Journal: The Journal of Biological Chemistry

Article Title: A surge of cytosolic calcium dysregulates lysosomal function and impairs autophagy flux during cupric chloride–induced neuronal death

doi: 10.1016/j.jbc.2023.105479

Figure Lengend Snippet: Rescue from CuCl 2 -induced dysregulated lysosomal function via buffering cytosolic Ca 2+ . MN9D/Neo or MN9D/CaBP cells were treated with or without 250 μM CuCl 2 for 15 h. A , immunocytochemical analyses were performed using anti-LAMP1 ( red ) and nuclei were counterstained with Hoechst 33258 ( blue ). Representative confocal images are provided. Merged images are shown to the right . Scale bar represents 20 μm. B , relative intensity of LAMP1 per cell was quantified using ImageJ software. Data are shown as the mean ± S.D. of three independent experiments. Two-way ANOVA was performed ( p value is 0.401729). NS, not significant. C , cellular lysates were subjected to immunoblot analyses using the indicated antibodies. Relative intensities of LAMP1 ( D ) and c-Cat D ( E ) signals were measured using ImageJ software, normalized by the intensity of GAPDH signal, and expressed as fold change relative to untreated control (value = 1). Data are shown as the mean ± S.D of three independent experiments. Two-way ANOVA followed by Tukey’s post hoc test was performed (( D ) ANOVA p value is 0.318773). ∗∗∗ p < 0.001; NS, not significant. F , after CuCl 2 treatment, cells were stained with Magic Red Cat B. Nuclei were counterstained with Hoechst 33258 ( blue ). Merged images are shown to the right . Representative confocal images are provided. Scale bar represents 20 μm. G , the relative intensity of each fluorescent signal of Cat B per cell was quantified using ImageJ software. Data are shown as the mean ± S.D. of three independent experiments. Two-way ANOVA followed by Tukey’s post hoc test was performed. ∗∗∗ p < 0.001; NS, not significant. H , after CuCl 2 treatment, cells were stained with LysoTracker Red. Nuclei were counterstained with Hoechst 33258 ( blue ). Merged images are shown to the right . Representative confocal images are provided. Scale bar represents 20 μm. I , the relative intensity of each fluorescent signal of LysoTracker Red per cell was quantified using ImageJ software. Data are shown as the mean ± S.D of three independent experiments. Two-way ANOVA followed by Tukey’s post hoc test was performed. ∗∗∗ p < 0.001; NS, not significant.

Article Snippet: Nuclear counterstaining was carried out using the DNA helix intercalating dye Hoechst 33,258 (1 μg/ml; Molecular Probes, H-1398).

Techniques: Software, Western Blot, Control, Staining

Journal: The Journal of Biological Chemistry

Article Title: A surge of cytosolic calcium dysregulates lysosomal function and impairs autophagy flux during cupric chloride–induced neuronal death

doi: 10.1016/j.jbc.2023.105479

Figure Lengend Snippet: Autophagic flux impairment and lysosomal dysfunction induced by CuCl 2 treatment in cortical neuronal cells. Primary cultures of cortical neurons established as described in Experimental Procedures were treated with or without 250 μM CuCl 2 for 18 h. A , immunoblot analyses were performed using the indicated antibodies. B , immunocytochemical localization analysis was performed using anti-LC3 ( green ) and anti-p62 ( red ) antibodies followed by counterstaining with Hoechst 33258 ( blue ). Merged images are shown to the right . Representative confocal images are provided. Scale bar represents 10 μm. C , MTT reduction assay was performed to assess cell viability, which was expressed as a fold change relative to untreated control cell (value = 1). Data are shown as the mean ± S.D of four independent experiments. ∗∗∗ p < 0.001. D , immunoblot analyses were performed using the indicated antibodies in the presence or absence of 50 nM Baf.A1 for the final 4 h. E , immunocytochemical analyses were performed using anti-LAMP1 ( red ) followed by nuclei counterstaining with Hoechst 33258 ( blue ). Merged images are shown to the right . Scale bar represents 20 μm. F and G , after CuCl 2 treatment, cells were stained with ( F ) Magic Red cathepsin B (Cat B) or ( G ) LysoTracker Red. Nuclei were counterstained with Hoechst 33258 ( blue ). Merged images are shown to the right . Representative confocal images are provided. Scale bar represents 20 μm. All data represent of three independent experiments.

Article Snippet: Nuclear counterstaining was carried out using the DNA helix intercalating dye Hoechst 33,258 (1 μg/ml; Molecular Probes, H-1398).

Techniques: Western Blot, MTT Reduction Assay, Control, Staining

Journal: The Journal of Biological Chemistry

Article Title: A surge of cytosolic calcium dysregulates lysosomal function and impairs autophagy flux during cupric chloride–induced neuronal death

doi: 10.1016/j.jbc.2023.105479

Figure Lengend Snippet: Rescue of CuCl 2 -induced impairment of autophagic flux by buffering of drug-induced surge of cytosolic Ca 2+ in cortical neuronal cells. Cortical neuronal cells were incubated with 250 μM CuCl 2 for 15 h in the presence or absence of 40 μM BAPTA-AM. A , immunoblot analyses were performed using the indicated antibodies. B , immunocytochemical localization analysis was performed using anti-LC3 ( green ) and anti-p62 ( red ) antibodies followed by counterstaining with Hoechst 33258 ( blue ). Merged images are shown to the right . Representative confocal images are provided. Scale bar represents 10 μm. C , MTT reduction assay was performed to assess cell viability, which was expressed as a percentage over untreated control cells (value = 1). Data are shown as the mean ± S.D of three independent experiments. ∗∗ p < 0.01; ∗∗∗ p < 0.001. D , immunocytochemical analyses were performed using anti-LAMP1 ( red ) followed by nuclei counterstaining with Hoechst 33258 ( blue ). Merged images are shown to the right . Scale bar represents 20 μm. E and F , after CuCl 2 treatment, cells were stained with ( E ) Magic Red cathepsin B (Cat B) or ( F ) LysoTracker Red. Nuclei were counterstained with Hoechst 33258 ( blue ). Merged images are shown to the right . Scale bar represents 20 μm. All data represent of three independent experiments.

Article Snippet: Nuclear counterstaining was carried out using the DNA helix intercalating dye Hoechst 33,258 (1 μg/ml; Molecular Probes, H-1398).

Techniques: Incubation, Western Blot, MTT Reduction Assay, Control, Staining

Journal: PLoS ONE

Article Title: Development of highly sensitive and low-cost DNA agarose gel electrophoresis detection systems, and evaluation of non-mutagenic and loading dye-type DNA-staining reagents

doi: 10.1371/journal.pone.0222209

Figure Lengend Snippet: (a) Detection by UV (312 nm)-transilluminator system (STAGE-One, AMZ System Science) and Blook TM (470 nm, Bio-Helix); (b) Detection by blue-LED light (470 nm) excitation. SC-54 filter was used as longpass emission filter; (c) Detection by combination of cyan LED light (490–495 nm) excitation and a shortpass filter (510 nm). SC-54 filter was used as longpass emission filter. Each DNA-staining reagent was represented by underlined letters. DNA ladder markers were loaded by successive dilution. Lane 1, standard volume (5 μL (500 ng), 1 volume); lane 2, 1/2 volume; lane 3, 1/3 volume; lane 4, 1/6 volume; lane 5, 1/10 volume; lane 6, 1/15 volume; lane 7, 1/20 volume; lane 8, 1/30 volume.

Article Snippet: A 1Kb DNA Ladder RTU (Bio-Helix, 100 ng/μL) was used as DNA ladder marker (10, 8.0, 6.0, 5.0, 4.0, 3.0, 2.5, 2.0, 1.5, 1.0, 0.75, 0.50, 0.25 kbp).

Techniques: Staining

Journal: PLoS ONE

Article Title: Development of highly sensitive and low-cost DNA agarose gel electrophoresis detection systems, and evaluation of non-mutagenic and loading dye-type DNA-staining reagents

doi: 10.1371/journal.pone.0222209

Figure Lengend Snippet: (a) Detection of DNA fragments stained by EZ-Vision; (b) Detection of DNA fragments stained with Safelook Load-White. EZ-Vision and Safelook Load-White were excited by black light (~360 nm) or UV (365 nm) transilluminator system (STAGE-2000, AMZ System Science). Each excitation system is represented with boxed letters. SC-46 was used as longpass emission-filter for black light system, and the accessory filter of STAGE-2000 was used as an emission filter for UV (365 nm)-transilluminator system (STAGE-2000). DNA ladder markers were loaded by successive dilution. Lane 1, standard volume (5 μL (500 ng), 1 volume); lane 2, 1/2 volume; lane 3, 1/3 volume; lane 4, 1/6 volume; lane 5, 1/10 volume; lane 6, 1/15 volume; lane 7, 1/20 volume; lane 8, 1/30 volume.

Article Snippet: A 1Kb DNA Ladder RTU (Bio-Helix, 100 ng/μL) was used as DNA ladder marker (10, 8.0, 6.0, 5.0, 4.0, 3.0, 2.5, 2.0, 1.5, 1.0, 0.75, 0.50, 0.25 kbp).

Techniques: Staining

Journal: PLoS ONE

Article Title: Development of highly sensitive and low-cost DNA agarose gel electrophoresis detection systems, and evaluation of non-mutagenic and loading dye-type DNA-staining reagents

doi: 10.1371/journal.pone.0222209

Figure Lengend Snippet: (a) Detection by blue-LED light (470 nm) excitation. SC-54 filter was used as longpass emission filter; (b) Detection by combination of cyan LED light (490–495 nm) excitation and a shortpass filter (510 nm). SC-54 filter was used as longpass emission filter. DNA ladder markers were loaded by successive dilution. Lane 1, standard volume (5 μL (500 ng), 1 volume); lane 2, 1/2 volume; lane 3, 1/3 volume; lane 4, 1/6 volume; lane 5, 1/10 volume; lane 6, 1/15 volume; lane 7, 1/20 volume; lane 8, 1/30 volume.

Article Snippet: A 1Kb DNA Ladder RTU (Bio-Helix, 100 ng/μL) was used as DNA ladder marker (10, 8.0, 6.0, 5.0, 4.0, 3.0, 2.5, 2.0, 1.5, 1.0, 0.75, 0.50, 0.25 kbp).

Techniques: