Journal: Scientific Reports

Article Title: FTY720 in resistant human epidermal growth factor receptor 2-positive breast cancer

doi: 10.1038/s41598-021-04328-y

Figure Lengend Snippet: Xenograft mouse model of HCC1954 cells showing the efficacy of FTY720 with or without trastuzumab. ( a ) Mice bearing HCC1954 xenografts were randomized into four treatment groups (n = 9 for each group) when tumor size reached 150–200 mm 3 . Mice were treated with PBS, trastuzumab, FTY720, or FTY720 plus trastuzumab. Xenografts were harvested 24 h after the last treatment. Each plot indicates the mean increase in tumor volume from the first day of treatment, whereas error bars indicate the standard error of the mean. Differences between the four groups were analyzed using ANOVA with mixed-effect model. Differences in control versus FTY720 and control versus FTY720 plus trastuzumab were statistically significant. P value: * P < 0.05 and ** P < 0.01. ( b ) Tumor tissue sections were evaluated using TUNEL assays (green) and also counterstained with Hoechst 33,342 dye (blue, indicating nuclei). They were then examined using fluorescence microscopy. The top three tissue sections with the most abundant green signals in each group were presented. ( c ) Using the same tissue sections, cell membranes were outlined using HER2-antibody conjugating secondary antibody (red) and then examined using fluorescence microscopy. Bar, 100 μm. F plus T: FTY720 plus trastuzumab.

Article Snippet: After counterstaining with Hoechst 33,342 dye (blue, staining nuclei, Merck KGaA) and HER2 antibody/PE-Texas Red-conjugated secondary antibody (red, plasma membrane, Merck KGaA), the cells were examined using fluorescence microscopy .

Techniques: Control, TUNEL Assay, Fluorescence, 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: 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: OncoTargets and Therapy

Article Title: Red Sea Sponge Callyspongia siphonella Extract Induced Growth Inhibition and Apoptosis in Breast MCF-7 and Hepatic HepG-2 Cancer Cell Lines in 2D and 3D Cell Cultures

doi: 10.2147/OTT.S467083

Figure Lengend Snippet: The effect of C. siphonella extract on apoptosis and necrosis using flow cytometric analysis. Representative flow cytometric dot plots of ( A ) MCF-7 and ( B ) HepG-2 cells were treated with DMSO, IC 50 , and 2X IC 50 of C. siphonella extract. Graphical presentation shows the percentage of MCF-7 cells and HepG-2 cells. Data are displayed as mean ± SD. *Significant difference at p < 0.05. **Significant difference at p < 0.01. ***Significant difference at p < 0.001. ****Significant difference at p < 0.0001.

Article Snippet: The PI/Hoechst 33,342 apoptosis detection kit from Solarbio, China, was utilized for this purpose.

Techniques:

Journal: Pharmaceutics

Article Title: Bacterial Cellulose as a Potential Bio-Scaffold for Effective Re-Epithelialization Therapy

doi: 10.3390/pharmaceutics13101592

Figure Lengend Snippet: Immunofluorescence staining of human adipose stem cells cultured on bacterial cellulose scaffold for 14 days. ( a , b ) Octamer-binding protein 4 (OCT-4) and OCT-4/Hoechst overlay expression, respectively. ( c , d ) Nestin and Nestin/Hoechst overlay expression, respectively. ( e , f ) Sex determining region Y-box 9 (SOX-9) and SOX-9/Hoechst overlay expression, respectively (scale bar = 200 µm).

Article Snippet: Hoechst 33,342 (1:5000; AnaSpec, Fremont, CA, USA) was then added to the samples for 15 min for nuclei visualization.

Techniques: Immunofluorescence, Staining, Cell Culture, Binding Assay, Expressing