Journal: Cell Death Discovery

Article Title: PGC-1a mediated mitochondrial biogenesis promotes recovery and survival of neuronal cells from cellular degeneration

doi: 10.1038/s41420-024-01953-0

Figure Lengend Snippet: A Representative bright field (BF) and immunofluorescence images of 5% EtOH treatment (vol/vol, 1 and 3 h) induced neurite retraction, and its regeneration after washing EtOH (Washed, 4 and 20 h) in neuronal PC12 cells. Green: neuron-specific β-III tubulin; Blue: DAPI. Scale bar: 50 µm. B , C Quantification results of mean neurite length per cell and the percentage of cells with neurites. *** P < 0.001, vs. control group; ## P < 0.01, ### P < 0.001, vs. EtOH 3 h group. EtOH ethanol.

Article Snippet: For immunostaining, cells were incubated with primary antibodies against neuron-specific β-III tubulin (MAB1195, R&D systems, USA), LC3B (NB100-2220, Novus Biologicals, USA), TOM20 (42406S, Cell Signaling Technology), and Drp1 (8570S, Cell Signaling Technology) overnight at 4 °C.

Techniques: Immunofluorescence, Control

Journal: Journal of Neuroinflammation

Article Title: Neuronal maturation reduces the type I IFN response to orthobunyavirus infection and leads to increased apoptosis of human neurons

doi: 10.1186/s12974-019-1614-1

Figure Lengend Snippet: Committed neurons are susceptible to LACV-induced apoptosis. Representative confocal images of ( a , f , and k ) mock or ( b – e , g – j, and l – o ) 3 dpi LACV-infected COs immunohistochemically labeled with LACV (green), neuronal phenotyping antibodies (white), activated poly caspases (magenta), and nuclei (blue). Images are grouped by row using the neuronal phenotyping antibody with Sox2 being top, DCX middle, and βIII tubulin bottom. The three middle columns are images of single channels overlaid on nuclei from 3 dpi LACV-infected COs that are labeled accordingly. The far-right column ( e , j , and o ) is a combination of all four labels. The insets in e , j , and o are enlarged images of the highlighted yellow boxes in each panel. The corresponding yellow arrows in the associated individual label panels highlight the cell of interest shown in the inset. The images in the a , f , and k mock column are a combination of all four labels. All images were taken with a × 63 objective and are maximum intensity projects of 3 μm z -stacks taken with a 0.5 μm step. Scale bar in B = 20 μm and applies to all panels

Article Snippet: Antibodies used: Trustain FcXTM (Biolegend #422301, 1:1000), Sox2 (Millipore #FCMAB112, 1:50), DCX (BD Pharmigen #561505, 1:50), and βIII tubulin (R&D Systems #IC1195C, 1:50).

Techniques: Infection, Labeling

Journal: Journal of Neuroinflammation

Article Title: Neuronal maturation reduces the type I IFN response to orthobunyavirus infection and leads to increased apoptosis of human neurons

doi: 10.1186/s12974-019-1614-1

Figure Lengend Snippet: Flow cytometry analysis of neuronal cells from mock- and LACV-infected COs. Mock- ( a – d ) and LACV-infected ( e – h ) COs were non-enzymatically digested into a single cell suspension and analyzed via flow cytometry as described in the “ ” section. Two representative examples are shown. Live cells were identified and interrogated for expression of activated poly-caspases ( y -axis) and LACV expression ( x -axis). a , e Active caspase and LACV staining from the whole live cell population. Gating within the whole live cell population for Sox2 ( b , f ), DCX ( c , g ) and βIII tubulin-positive cells allowed for examination of active poly-caspase and LACV staining within each neuronal population. Proportions of LACV-infected ( i ), activated poly-caspase ( j ), and LACV-infected/activated poly-caspase double-positive ( k ) neuronal cells within mock (closed circles) or infected (open squares) COs are shown. A two-way ANOVA with a Sidak’s multiple comparisons test was used to determine significance. ** p < 0.001, **** p < 0.0001

Article Snippet: Antibodies used: Trustain FcXTM (Biolegend #422301, 1:1000), Sox2 (Millipore #FCMAB112, 1:50), DCX (BD Pharmigen #561505, 1:50), and βIII tubulin (R&D Systems #IC1195C, 1:50).

Techniques: Flow Cytometry, Infection, Suspension, Expressing, Staining

Journal: Journal of Neuroinflammation

Article Title: Neuronal maturation reduces the type I IFN response to orthobunyavirus infection and leads to increased apoptosis of human neurons

doi: 10.1186/s12974-019-1614-1

Figure Lengend Snippet: Type I IFN signaling induction in committed neurons increases cell survival. Cell viability of mock- or LACV-infected COs treated with either a IFNα2 and IFNα4 concomitantly or b IFNβ1 individually were measured using a resazurin reduction-based assay. Data is plotted as an average percent of base fluorescence measured at 590 nm for each CO. Mock IFN-treated samples are indicated by open circles, LACV vehicle-treated samples are indicated by open squares and LACV IFN-treated samples are indicated by open triangles. Fluorescence for each sample at each time point was read in triplicate at the indicated time point. A two-way ANOVA with a Sidak’s multiple comparisons test was used to determine significance. * p < 0.05. Data are representative of n = 3 mock IFNα2/4, n = 6 LACV Vehicle, n = 3 LACV IFNα2/4, n = 6 mock IFNβ1 and n = 6 LACV IFNβ1 treated COs. c Supernatants from mock (black circles), LACV IFNβ1 (green triangles), LACV IFNα2/4 (blue triangles), or LACV vehicle (red squares) treated COs were collected daily and assayed for viral RNA via qRT PCR. Data are presented as LACV RNA expression relative to an experimentally determined PFU standard. A two-way repeated measure ANOVA with a Dunnett’s multiple comparison test was performed on the antilog of the experimental values to establish differences between LACV vehicle and IFN treated samples. ** p < 0.01, * p < 0.05. *Color denotes which condition differed relative to LACV vehicle. d Quantification of immunohistochemical labeling for Sox2, DCX, and βIII tubulin in sections from the same COs shown in b are plotted as a percent positive signal of organoid area. A Kruskal-Wallis one-way ANOVA test with multiple comparisons was used to determine significance. *** p < 0.005. Representative sections of βIII tubulin staining in whole COs treated with e mock INFβ1, f LACV vehicle, or g LACV IFNβ1 are shown

Article Snippet: Antibodies used: Trustain FcXTM (Biolegend #422301, 1:1000), Sox2 (Millipore #FCMAB112, 1:50), DCX (BD Pharmigen #561505, 1:50), and βIII tubulin (R&D Systems #IC1195C, 1:50).

Techniques: Infection, Fluorescence, Quantitative RT-PCR, RNA Expression, Comparison, Immunohistochemical staining, Labeling, Staining