Journal: Frontiers in Pharmacology

Article Title: Resibufogenin Targets the ATP1A1 Signaling Cascade to Induce G2/M Phase Arrest and Inhibit Invasion in Glioma

doi: 10.3389/fphar.2022.855626

Figure Lengend Snippet: RB inhibits GBM cell invasion partially by elevating intracellular Ca 2+ levels to suppress the Src/FAK/Paxillin focal adhesion pathway. (A) Intersections of genes with downregulated expression in P3#GBM and U251 cells treated with 4 μM RB compared with control cells. (B) KEGG pathway enrichment analysis of intersecting genes with downregulated expression. (C) P3#GBM, U251 and A172 glioma cells were treated with RB for 48 h. Western blot analysis was used to determine focal adhesion-associated protein expression levels. (D) Intracellular Ca 2+ was measured by Fluo-4/AM with flow cytometry. (E) Graphic representation of the mean fluorescence intensity of Fluo-4/AM shown in (D) . (F) The numbers of invasive GBM cells were shown in . (G) Western blot analysis showed that blocking Ca 2+ with RR treatment for 48 h rescued RB-induced inhibition of invasion in U251 and A172 cells. Three independent experiments were performed with data shown as the mean ± SEM by unpaired Student’s t test (E) or one-way ANOVA (F) . * p < 0.05; ** p < 0.01 and *** p < 0.001.

Article Snippet: Fluo-4/AM (MCE) was added to the cells after they had been treated with diluted DMSO or RB (2 or 4 μM) for 48 h. The solution was kept in the dark at room temperature for 30 min.

Techniques: Expressing, Control, Western Blot, Flow Cytometry, Fluorescence, Blocking Assay, Inhibition

Journal: International Journal of Biological Sciences

Article Title: The MCU-MECOM Axis Orchestrates Glioblastoma Progression by Remodeling Mitochondrial Dynamics and Quality Control via MAMs

doi: 10.7150/ijbs.127940

Figure Lengend Snippet: MCU is overexpressed in high-grade glioma and promotes MAMs reinforcement and calcium dysregulation. (A) Pan-cancer bioinformatics analysis of MCU expression levels. Violin plots display MCU mRNA levels in tumor (red) versus normal (blue) tissues across various malignancies. The red boxes highlight the glioma cohorts (GBM and LGG). (B) Schematic illustration of the acquisition and experimental workflow for clinical specimens (Control, LGG, and HGG). (C) IHC images and corresponding quantitative analysis of MCU expression in Control, LGG, and HGG tissues. (D) Western blot analysis and relative protein quantification of MCU and VDAC1 in clinical lysates. (E) PPI network analysis (STRING) highlighting mitochondrial calcium transport-related molecules. (F) Schematic representation of primary cell cultures established from human astrocytes (Control), LGG, and HGG specimens. (G) Representative immunofluorescence images and quantification of mitochondrial calcium levels using the Rhod-2 AM probe in primary cultured cells. (H) Representative immunofluorescence images and quantification of ER calcium levels using the Mag-Fluo-4 AM probe. (I) Ultrastructural analysis of MAMs via TEM. Left: Representative TEM images showing mitochondria (green/blue/red) and ER (yellow) contacts. Middle: Schematic defining the quantitative MAMs parameters. Right: Quantitative analysis of MAMs-positive mitochondria (%), MAMs contact length (μm), and shortest vertical distance (nm) between ER and mitochondria. (J) Western blot analysis and relative protein quantification of the MAMs tethering complex proteins IP3R1 and GRP75 across clinical grades. (K) Representative confocal immunofluorescence images and quantitative colocalization analysis (Pearson's correlation coefficient) of Calnexin (green) and TOMM20 (red). Nuclei were counterstained with DAPI (blue). (L) Schematic model illustrating the progression of MAMs structural reinforcement and subsequent mitochondrial calcium overload in HGG compared to LGG. Data are presented as mean ± SD. * P < 0.05; ** P < 0.01; *** P < 0.001; **** P < 0.0001.

Article Snippet: Mitochondrial calcium and ER calcium in primary cells, U87MG, and GSCs were detected by using the indicator Rhod-2 AM (HY-101896, MedChemExpress, USA) and the indicator Mag-Fluo-4 AM (HY-D1498, MedChemExpress, USA) respectively (detailed in ).

Techniques: Expressing, Control, Western Blot, Immunofluorescence, Cell Culture

Journal: International Journal of Biological Sciences

Article Title: The MCU-MECOM Axis Orchestrates Glioblastoma Progression by Remodeling Mitochondrial Dynamics and Quality Control via MAMs

doi: 10.7150/ijbs.127940

Figure Lengend Snippet: MCU disruption impairs MRC remodeling through MAMs-dependent calcium shuttling. (A) Representative TEM images and quantitative analysis of MRC abundance (highlighted in green) across clinical grades. Analyzed mitochondria numbers: Control (n = 390), LGG (n = 544), HGG (n = 546). (B) Quantitative analysis of MMP in clinical specimens using JC-1 staining (ratio of red to green fluorescence). (C) Quantitative analysis of global ROS levels in clinical specimens. (D) Representative TEM images and quantitative analysis of MRC abundance (green) in U87MG cells following MCU knockdown. Analyzed mitochondria numbers: sh-MCU-NC (n = 108), sh-MCU (n = 115). (E) Flow cytometry analysis and quantification of MMP in U87MG cells following MCU knockdown. (F) Flow cytometry analysis and quantification of intracellular ROS levels in U87MG cells following MCU knockdown. (G) Representative confocal images and quantitative analysis of mitochondria-specific superoxide production using the MitoSOX probe (red) in U87MG cells. Nuclei were counterstained with DAPI (blue). (H) Ultrastructural analysis of MAMs in U87MG cells via TEM. Left: Representative images showing mitochondria (green/blue) and ER (yellow) contacts. Right: Quantitative analysis of MAMs-positive mitochondria, contact length, and shortest vertical distance. (I) Western blot analysis and relative protein quantification of MAMs tethering proteins IP3R1 and GRP75 in U87MG cells following MCU knockdown. (J) Representative confocal immunofluorescence images and quantitative colocalization analysis (Pearson's correlation coefficient) of Calnexin (green) and TOMM20 (red). Nuclei were counterstained with DAPI (blue). (K) Representative confocal images and quantification of mitochondrial calcium levels using the Rhod-2 AM probe (red) in U87MG cells. (L) Representative confocal images and quantification of ER calcium levels using the Mag-Fluo-4 AM probe (yellow) in U87MG cells. Data are presented as mean ± SD.

Article Snippet: Mitochondrial calcium and ER calcium in primary cells, U87MG, and GSCs were detected by using the indicator Rhod-2 AM (HY-101896, MedChemExpress, USA) and the indicator Mag-Fluo-4 AM (HY-D1498, MedChemExpress, USA) respectively (detailed in ).

Techniques: Disruption, Control, Staining, Fluorescence, Knockdown, Flow Cytometry, Western Blot, Immunofluorescence

Journal: The Journal of Neuroscience

Article Title: Integrating Temperature with Odor Processing in the Olfactory Bulb

doi: 10.1523/JNEUROSCI.0571-15.2015

Figure Lengend Snippet: Responses of the γ-glomerulus upon stimulation. A, Overview of the Xenopus olfactory bulb with the different layers and cell types indicated. The positions of the lateral, medial, and small glomerular clusters are labeled. The γ-glomerulus is situated close to the posterior and ventral border of the glomerular layer within the small cluster and is marked in blue. The top right side shows an image of a tadpole. The red rectangle indicates the area used for sample preparation. Scale bar, 2 mm. On the bottom right side, a scheme of the experiment including the glomerular afferents of the right ventral olfactory bulb is provided. Olfactory sensory neurons electroporated with Fluo-4 dextran are shown in green. B, Raw Fluo-4 fluorescence signal before stimulation. C, Top, Responses of primary olfactory projections in the ventral olfactory bulb upon stimulation with different temperature changes (I–IV) or a mixture of amino acids (VI). The images show the spatial map of the ΔF/F. Bottom, Time courses of responses of the γ-glomerulus (Glγ) and glomeruli in the lateral cluster (lat), along with the underlying temperature stimuli (ΔT), plotted as deviations from ambient temperature. D, Summary of normalized temperature response curves of the γ-glomerulus at two ambient temperatures of 22–24°C (green, n = 17 animals) and 18–20°C (blue, n = 9 animals). The points show the responses of individual animals, and the rectangles indicate the average responses with SD. The sigmoid curves represent the average fit for each ambient temperature. E, Representative response of the γ-glomerulus upon prolonged stimulation (temperature step, ΔT = −3.5°C). Dashed line, exponential fit. F, Odorant responses of the glomeruli in the lateral cluster as a function of temperature (n = 5 animals). γ, Glγ, γ-glomerulus; ob, olfactory bulb; oe, olfactory epithelium; on, olfactory nerve; lat, lateral cluster; C, caudal; L, lateral; M, medial; R, rostral. Scale bar, 20 μm.

Article Snippet: Briefly, a patch pipette (resistance, 5–8 MΩ; tip diameter, 1–2 μm) was filled with Ringer's solution containing 500 μ m Fluo-4 AM, 5% DMSO (v/v), 1% Pluronic F-127 (w/v), and 500 μ m MK571 (Alexis Biochemicals).

Techniques: Labeling, Sample Prep, Fluorescence

Journal: The Journal of Neuroscience

Article Title: Integrating Temperature with Odor Processing in the Olfactory Bulb

doi: 10.1523/JNEUROSCI.0571-15.2015

Figure Lengend Snippet: Temperature sensitivity and chemosensitivity in the olfactory bulb. A, Temperature response of the postsynaptic neuropil of a γ-glomerulus and two mitral cells. Dashed curves, Boltzmann fit. B, Integration of temperature and odor sensitivity in individual mitral cells. Left, Maximum projection of a 30-μm-thick volume of the olfactory bulb stained with Fluo-4 AM. Arrowheads highlight six mitral cells. Scale bar, 50 μm. Right, Ca2+ responses of the six mitral cells to a mixture of amino acids, a temperature drop (ΔT = −1.1°C), or the control with ambient temperature Ringer's solution. The red and blue bars under the traces indicate the application of amino acids (100 μm) and cold Ringer's solution (T = 0°C), respectively. C, Response patterns of the small glomerular cluster and surrounding mitral cells to temperature and chemical stimulation. Presynaptic Alexa Fluor staining is shown in green (arrow, γ-glomerulus; I). Postsynaptic Fluo-8 AM staining was performed through bolus loading. Stimulus sequence: cold Ringer's solution, cold Ringer's solution, l-histidine, cold Ringer's solution. II, Activity correlation map for the postsynaptic Ca2+ responses. Red, correlation to a pure His response; cyan, correlation to pure temperature drop responses (reference traces as inset). III, Regions responding to both stimuli (yellow). All images are maximum projections of the same 36-μm-thick volume. Scale bar, 20 μm. D, ΔF/F traces representing different mitral cells from the measurement in C. The relative response strength ranges from pure thermosensitivity to exclusive histidine sensitivity over various degrees of dual sensitivity. The bars under the traces depict the stimulus application in the same manner as in B.

Article Snippet: Briefly, a patch pipette (resistance, 5–8 MΩ; tip diameter, 1–2 μm) was filled with Ringer's solution containing 500 μ m Fluo-4 AM, 5% DMSO (v/v), 1% Pluronic F-127 (w/v), and 500 μ m MK571 (Alexis Biochemicals).

Techniques: Staining, Sequencing, Activity Assay

Journal: The Journal of Neuroscience

Article Title: Integrating Temperature with Odor Processing in the Olfactory Bulb

doi: 10.1523/JNEUROSCI.0571-15.2015

Figure Lengend Snippet: Postsynaptic response of a γ-glomerulus upon temperature stimulation in the olfactory epithelium. A, Maximum intensity projections (dorsoventral, I; mediolateral, II; rostrocaudal, III) of the ventral olfactory bulb after bolus injection of Fluo-4 AM (green). OSN axons originating from the contralateral olfactory epithelium (red, Alexa Fluor 568) enter the imaged volume at the ventrocaudal rim, protrude rostrodorsally (II) and form a glomerular tuft in the region of the γ-glomerulus. B, Stimulation (ΔT = −1.3°C) at the ipsilateral olfactory epithelium. C, The contralaterally projecting fibers indicate the region of the γ-glomerulus. D, ΔF/F peak response of postsynaptic elements upon cold stimulus occurs in the same region. Arrow, Neuropil; arrowheads, mitral cells. E–G, Stimulation (ΔT = −3.3°C) at the contralateral olfactory epithelium of the same brain preparation. The ipsilateral nerve was cut (E). F, The contralaterally projecting fibers indicate the region of the γ-glomerulus. G, ΔF/F peak response of postsynaptic elements upon cold stimulus occurs in the same region. Arrow, Neuropil; arrowhead, mitral cell; γ, γ-glomerulus. Scale bars, 20 μm.

Article Snippet: Briefly, a patch pipette (resistance, 5–8 MΩ; tip diameter, 1–2 μm) was filled with Ringer's solution containing 500 μ m Fluo-4 AM, 5% DMSO (v/v), 1% Pluronic F-127 (w/v), and 500 μ m MK571 (Alexis Biochemicals).

Techniques: Injection