Journal: Scientific Reports

Article Title: Histological analysis of sleep and circadian brain circuitry in cranial radiation-induced hypersomnolence (C-RIH) mouse model

doi: 10.1038/s41598-022-15074-0

Figure Lengend Snippet: In vitro radiosensitivity of astrocytic cell lines based on region of isolation and treatment timing. ( A ) Photomicrographs of SCN2.2 and CTXTNA2 cells, two astrocytic cell lines with similar morphology. B. Quantification of trypan blue postitive cells to determine cell viability 1, 6 and 24 h after 8 Gy radiation, there were significant main effects for time (F(1,18) = 96.834, p < 0.001), cell type (F(1,18) = 235.937, p < 0.001), and an interaction between time and cell type (F(2,17) = 151.927, < 0.001). Posthoc tests (tukey) were significant if p < 0.05. SCN2.2 cells (green bars) survived at significantly higher level 1 ( p < 0.001) and 6 h ( p = 0.024) after radiation when compared to the cortical astrocytes (purple bars). ( C ) Clonogenic assay to quantify survival fractions from 0 to 8 Gy in SCN and cortical cells. Again SCN2.2 cells (green line) had better overall survival after radiation when compared to CTXTNA2 cells (purple line). There were significant main effects of both cell line (F(1,24) = 33.228, p < 0.001) and radiation dose (F(5,24) = 402.558, p < 0.001), and an interaction between cell line and dose (F(5,24) = 6.172, p = 0.001). The dotted black line across the graph shows the dose modifying fractors (DMF 10 ) levels for both cells lines which was also significantly different between the two cell lines. ( D ) The chronotherapeutic experimental timeline, 1.0 × 10 6 cells were plated on day 0. Cells were then serum shocked with a 50% horse serum solution for 2 h at circadian time(CT) 8 (01:00, Day 2) or 20 (13:00, Day 1) on the subsequent days. All cells were irradiated on Day 2 at 11:00 and then colonies stained and counted after Day 5. ( E ) Clonogenic assay of SCN2.2 cells irradiated at CT8 (dotted line) and CT20 (solid line). Astrocytes irradiated at CT8 were significantly more sensitive to radiotherapy. There were significant main effects of time (F(1,24) = 7.041, p = 0.014) and dose (F(5,24) = 26.995, p < 0.001) but no interaction (F(5,24) = 1.081, p = 0.396). ( F ) Clonogenic assayof CTXTNA2 cells irradiated at CT8 (dotted line) and CT20 (solid line). Again, astrocytes irradiated at CT8 were significantly more sensitive to radiotherapy. There were significant main effect of time (F(1,24) = 14.099, p = 0.001) and dose (F(5,24) = 135.997, p < 0.001) but no interaction (F(5,24) = 1.349, p = 0.278). ( G ) ATM western analysis of both cell lines when samples were collected at two timepoint post-synchronization. Western blot of ATM (blue bands) and control β-actin (teal bands) in both cell lines show higher presence of ATM after serum shock entrainment (Left panel). A graph of the ATM/β-actin relative ratio (Right panel), shows that CTXTNA cells express more of the protein than SCN2.2 cells. Four replicates of each cell line at the 3 timepoints were compared, there were significant main effects of time (F(2,21) = 83.154, p < 0.001) and cell type (F(1,21) = 8.312, p = 0.009) and an interaction (F(2,21) = 5.230, p = 0.014). Posthoc tests (tukey) were significant if p < 0.05. Both cell lines had more protien during CT08 when compared to CT20 but only CTXTNA cells had significant differences between the two ( p = 0.007). Significance was defined as p < 0.05 and indicated by * or a, b, and c with different letters indicating significant differences.

Article Snippet: Membranes were stained using primary antibodies for ATM (1:1000; ab81292, abcam, MA) and β-actin (1:5000; ab8226, abcam, MA) for 24 h at 4°C, followed by 1 h at room temperature in secondary antibodies (1:5000) for IRDye 800CW Goat anti-Mouse (926-32210; Li-Cor Biotechnology, NE) and IRDye 680CW Goat anti-Rabbit (926-68071; Li-Cor Biotechnology, NE).

Techniques: In Vitro, Isolation, IF-P, Clonogenic Assay, Irradiation, Staining, Western Blot, Control

Journal: PLOS ONE

Article Title: Human pericytes degrade diverse α-synuclein aggregates

doi: 10.1371/journal.pone.0277658

Figure Lengend Snippet: (A) Electron microscopy of α-syn strains before and after fragmentation. (B) Live cell imaging of fluorescently tagged α-syn for 30 hours. (C) Immunofluorescent labelling of α-syn strains after 30 min in control pericytes with α-syn epitope-specific antibody AA103-108. (D) Schematic representation of experimental setup. (E) Loading of western blots. Lane 1 only contains pure α-syn aggregate, whereas lane 2–5 shows α-syn isolated from pericytes. (F) Detailed fluorescent western blot with α-syn epitope-specific antibodies detecting Ribbons with α-syn antibody AA103-108 (magenta), AA124-134 (yellow) and merge showing overlap of α-syn bands. Various bands are identified indicating full length (14.4kDa, cyan arrow) and cleaved α-syn (red and blue arrows). α-syn detection on individual western blots for Fibrils, Ribbons, fibrils65, fibrils91, fibrils110 and no-treatment control (PBS) with α-syn epitope specific antibodies showing full length α-syn (cyan arrow) and cleaved α-syn fragments. Fibrils110 lacks a full-length band as aggregate is made up of C-term cleaved α-syn (green arrow). (G) AA 103–108, (H) AA124-134, and (H) GAPDH blot corresponds to blot shown in G after antibody stripping and relabelling. Full blots shown in images. Confocal image with orthogonal views showing pericyte with internalised α-syn Fibrils after 24 hours (cyan arrows, K). Scale bars represent 100 nm (A), 20 μm (B,C), 10 μm (K).

Article Snippet: Antibodies raised against α-syn C-terminus (Mouse anti α-syn, Amino Acid (AA) 124–134 (1:1,000) and ab1903 (Abcam); Mouse anti α-syn 4B12, AA 103–108, IgG1 (1:2,000, MA1-90346, ThermoFisher), GAPDH (1:1,000, ab9484, Abcam).

Techniques: Electron Microscopy, Live Cell Imaging, Control, Western Blot, Isolation, Stripping Membranes

Journal: Nature Communications

Article Title: A reversible state of hypometabolism in a human cellular model of sporadic Parkinson’s disease

doi: 10.1038/s41467-023-42862-7

Figure Lengend Snippet: a Quantification of total mitochondrial mass and functional mitochondria in hNPCs or b DAns. Total mitochondrial mass was visualized using immunostainings with an antibody against the ATP synthase (ATP5F1A). Functional mitochondria with an active membrane potential were visualized using a MitoTracker probe (200 nM for 20 min). Images are exemplarily shown for hNPCs of clone i1E4-R1-003 (Ctrl), and iR66-R1-007 (sPD); for DAns of clone i1E4-R1-003 (mitoTracker—Ctrl), iR66-R1-007 (mitoTracker—sPD), i1JF-R1-018 (ATP5F1A—Ctrl), and iJ2C-R1-015 (ATP5F1A—sPD). Scale bar = 20 μm. c Violin plots highlight some morphological characteristics of mitochondria in hNPCs or d DAns: number of mitochondria (count), mitochondrial area, skeleton length, number of branch points, and mean intensity. On average, 112 (hNPCs—ATP5F1A), 160 (hNPCs—mitoTracker), 370 (DAns—ATP5F1A), 360 (DAns—mitoTracker) cells per clone were analyzed. e To assess alterations in the mitochondrial fusion machinery, expression of the mitofusions MFN1 , MFN2 , and OPA1 was quantified in hNPCs by RT-qPCR. Cells were cultivated on the energy substrates used in the Seahorse XF analysis (25 mM glucose or 5 mM pyruvate). f To assess alterations in the mitochondrial fission machinery, expression of DRP1 and its phosphorylation on Ser 616 was quantified in hNPCs by western blot. Protein levels were normalized to ACTB. g Western blots are exemplary shown for some hNPC clones. Boxplots display the median and range from the 25th to 75th percentile. Whiskers extend from the min to max value or to the most extreme data point which is no more than 1.5 times the interquartile range ( c , d ). Each dot represents one patient. n = 5 Ctrl and 7 sPD patient-derived cell clones, in triplicates. p- values were determined by linear mixed effects model ( c , d ); one-way ANOVA with Sidak’s Post hoc test ( p -values are provided together with the source data) ( e , f ). * p < 0.05; ** p < 0.01; *** p < 0.001. Source data are provided as a Source Data file.

Article Snippet: Primary antibodies NDUFB8 (459210, Novex; 1:500), Complex II- Subunit 30 (SDHB) (459230, Thermo Fisher Scientific; 1:500), UQCRC2 (ab14745, Abcam; 1:2500), MT-CO2 (ab110258, Abcam; 1:1000), ATP5F1A (ab14748, Abcam; 1:4000), DRP1 (5391, Cell Signaling; 1:1000), DRP1 phospho-Ser616 (4494, Cell Signaling; 1:1000), TUBA (GTX628802, Genetex; 1:20,000), ACTB (ABO145-200, OriGene; 1:2000), OGDHL (17110-1-AP, Proteintech, 1:5000), GAPDH (GTX627408 peroxidase coupled, Genetex, 1:20,000), SLC2A1 (MA5-31960, Invitrogen, 1:5000), SLC2A3 (PA5-72331, Thermo Fisher Scientific, 1:5000) were incubated in blocking buffer overnight at 4 °C.

Techniques: Functional Assay, Membrane, Expressing, Quantitative RT-PCR, Phospho-proteomics, Western Blot, Clone Assay, Derivative Assay

Journal: Frontiers in Cellular and Infection Microbiology

Article Title: Well-known surface and extracellular antigens of pathogenic microorganisms among the immunodominant proteins of the infectious microalgae Prototheca zopfii

doi: 10.3389/fcimb.2015.00067

Figure Lengend Snippet: Flow cytometry analyses for GAPDH expression on the surface of P. blaschkeae (SAG 2064), P. zopfii GT2 (SAG 2021), and P. zopfii GT1 (SAG 2063) . A representative image is shown. Cells in suspension were incubated with anti-GAPDH polyclonal antibodies, followed by incubation with Alexa-Fluor 488-labeled goat anti-rabbit IgG. As controls, cells were incubated only with the Alexa-Fluor 488-labeled goat anti-rabbit IgG to exclude any background. The experiment was repeated three independent times and results of one representative experiment are shown.

Article Snippet: Cells were incubated with 5–10 μg/ml polyclonal anti-GAPDH (Rockland, Limerick, USA) for 1 h at 37°C.

Techniques: Flow Cytometry, Expressing, Incubation, Labeling