Journal: bioRxiv

Article Title: The RNA helicases DDX5 and DDX17 facilitate neural differentiation of human pluripotent stem cells NTERA2

doi: 10.1101/2021.05.10.443309

Figure Lengend Snippet: NTERA2 cells were seeded at a density of 10,000 cell/cm 2 and induced differentiation by 10 μM retinoic acid for 32 days. The cultures were collected with a 4-days interval and analyzed by real-time PCR. NANOG and OCT4 were used as stem cell markers while PAX6, NESTIN, SOX1, GLAST, TUBB3 , and MAP2 were used as neural-specific markers. GAPDH was utilized as an internal control. Error bars represent SD; (n=3).

Article Snippet: The membrane was subjected to western blot analysis with antibodies against proteins of interest as following; anti-DDX5 (Abcam, ab126730, 1:10,000 dilution), anti-DDX17 (Proteintech, 19910-1-AP, 1:3,000 dilution), anti-OCT4 (Invitrogen, MA1-104, 1:1,000 dilution) and anti-TUBB3 (Origene, TA500047, 1:3,000 dilution), anti-GAPDH conjugated peroxidase (Proteintech, HRP-60004, 1:30,000 dilution), goat anti-rabbit IgG conjugated peroxidase (Cell Signaling, #7074, 1:5,000 dilution), and horse anti-mouse IgG conjugated peroxidase (Cell Signaling, #7076, 1:5,000 dilution).

Techniques: Real-time Polymerase Chain Reaction

Journal: bioRxiv

Article Title: The RNA helicases DDX5 and DDX17 facilitate neural differentiation of human pluripotent stem cells NTERA2

doi: 10.1101/2021.05.10.443309

Figure Lengend Snippet: NTERA2 cells were seeded at 10,000 cell/cm 2 and were induced to differentiate toward neural lineage by retinoic acid (RA) at 10 μM for the time course of 32 days. Samples were collected every 4 days for real-time PCR (A). GAPDH was utilized as an internal control. Error bars represent SD; (n=3). (B) Western blots of DDX5, DDX17, the stem cell marker OCT4, and the neural differentiation marker TUBB3. GAPDH was utilized as an internal control. (C) Immunofluorescent staining of undifferentiated culture and day 32 retinoic acid-induced neural derivatives was performed using anti-OCT4 and anti-TUBB3 antibodies (green), respectively. The cells were co-stained with anti-DDX5 or anti-DDX17 (red). DAPI was used to localize the nucleus. Scale bar = 50 μm.

Article Snippet: The membrane was subjected to western blot analysis with antibodies against proteins of interest as following; anti-DDX5 (Abcam, ab126730, 1:10,000 dilution), anti-DDX17 (Proteintech, 19910-1-AP, 1:3,000 dilution), anti-OCT4 (Invitrogen, MA1-104, 1:1,000 dilution) and anti-TUBB3 (Origene, TA500047, 1:3,000 dilution), anti-GAPDH conjugated peroxidase (Proteintech, HRP-60004, 1:30,000 dilution), goat anti-rabbit IgG conjugated peroxidase (Cell Signaling, #7074, 1:5,000 dilution), and horse anti-mouse IgG conjugated peroxidase (Cell Signaling, #7076, 1:5,000 dilution).

Techniques: Real-time Polymerase Chain Reaction, Western Blot, Marker, Staining

Journal: bioRxiv

Article Title: The RNA helicases DDX5 and DDX17 facilitate neural differentiation of human pluripotent stem cells NTERA2

doi: 10.1101/2021.05.10.443309

Figure Lengend Snippet: (A) The stem cells were silenced for individual RNA helicases or both using siRNA and were treated by retinoic acid for 16 days to derive early neural derivatives. (B) hPSCs were induced by retinoic acid to differentiate for 14 days to obtain early neural derivatives prior to RNA silencing. Upon knockdown of the RNA helicases, the neural derivatives were allowed to differentiate further in the presence of retinoic acid for another 14 days to derive late neural derivatives. Immunofluorescent staining of mature neurons and neural progenitors was performed using anti-TUBB3 (green) and anti-NESTIN (red) antibodies, respectively. The cells were counter stained with DAPI. Scale bar = 200 μm. Fluorescence micrographs of the early neural derivatives (A) and the late neural derivatives (B) were determined for the percentage of cells positive for NESTIN (C) or TUBB3 (D). Error bars represent SD; (n=3). Student t-test. ** p<0.01; **** p<0.0001.

Article Snippet: The membrane was subjected to western blot analysis with antibodies against proteins of interest as following; anti-DDX5 (Abcam, ab126730, 1:10,000 dilution), anti-DDX17 (Proteintech, 19910-1-AP, 1:3,000 dilution), anti-OCT4 (Invitrogen, MA1-104, 1:1,000 dilution) and anti-TUBB3 (Origene, TA500047, 1:3,000 dilution), anti-GAPDH conjugated peroxidase (Proteintech, HRP-60004, 1:30,000 dilution), goat anti-rabbit IgG conjugated peroxidase (Cell Signaling, #7074, 1:5,000 dilution), and horse anti-mouse IgG conjugated peroxidase (Cell Signaling, #7076, 1:5,000 dilution).

Techniques: Staining, Fluorescence

Journal: Cell

Article Title: iPSCs from a hibernator provide a platform for studying cold adaptation and its potential medical applications

doi: 10.1016/j.cell.2018.03.010

Figure Lengend Snippet: (A) Immunofluorescence of TUBB3 (red), polyglutamylated tubulin (poly-E-T; green), or delta2-tubulin (Δ2-T; blue) in cultured rat primary cortical neurons incubated at 4°C for various durations. Scale bar: 40 μm.

Article Snippet: p CMV6-Myc-DDK-Tubb3 , Origene , Cat# MR207181.

Techniques: Immunofluorescence, Cell Culture, Incubation

Journal: Cell

Article Title: iPSCs from a hibernator provide a platform for studying cold adaptation and its potential medical applications

doi: 10.1016/j.cell.2018.03.010

Figure Lengend Snippet: (A) Microtubule morphology in rat (n = 10 animals) retinal ganglion cells (RGCs) immunostained for TUBB3 (red). Scale bars: 20 μm.

Article Snippet: p CMV6-Myc-DDK-Tubb3 , Origene , Cat# MR207181.

Techniques:

Journal: Cell

Article Title: iPSCs from a hibernator provide a platform for studying cold adaptation and its potential medical applications

doi: 10.1016/j.cell.2018.03.010

Figure Lengend Snippet: (A) Western blots and quantification of TUBB3, poly-E-T and Δ2-T proteins in GS and human iPSC-neurons following 4- or 16-h cold exposure with or without TUBB3 overexpression (n = 5 experiments from 2 GS and 2 human cell lines; Student’s t-test between untreated controls with or without cold exposure, and between cold-exposed groups with or without the TUBB3 overexpression plasmid; ** p < 0.01; *** p < 0.001). See also Figure S2C.

Article Snippet: p CMV6-Myc-DDK-Tubb3 , Origene , Cat# MR207181.

Techniques: Western Blot, Over Expression, Plasmid Preparation

Journal: Cell

Article Title: iPSCs from a hibernator provide a platform for studying cold adaptation and its potential medical applications

doi: 10.1016/j.cell.2018.03.010

Figure Lengend Snippet: (A) Pre-treatment with BAM15 (0.1 μM; n = 17 experiments), PI (1:500; n = 24 experiments) or a combination of both (n = 20 experiments) preserved long neurites (poly-E-T: green; TUBB3: red; Δ2-T: blue) of human iPSC-neurons following 4-h incubation at 4°C. Scale bar: 40 μm. See also Figure S6.

Article Snippet: p CMV6-Myc-DDK-Tubb3 , Origene , Cat# MR207181.

Techniques: Incubation

Journal: Cell

Article Title: iPSCs from a hibernator provide a platform for studying cold adaptation and its potential medical applications

doi: 10.1016/j.cell.2018.03.010

Figure Lengend Snippet: Key Resources Table

Article Snippet: p CMV6-Myc-DDK-Tubb3 , Origene , Cat# MR207181.

Techniques: Recombinant, Cell Culture, Protease Inhibitor, Lysis, SYBR Green Assay, In Vitro, Transfection, Imaging, Software

Journal: PLoS ONE

Article Title: A TNF Receptor 2 Selective Agonist Rescues Human Neurons from Oxidative Stress-Induced Cell Death

doi: 10.1371/journal.pone.0027621

Figure Lengend Snippet: ( A ) Differentiated LUHMES cells were incubated with different concentrations of hydrogen peroxide (H 2 O 2 ) for one hour. Then cells were washed with medium und cultivated for additional 24 hours. Cell viability was measured using the MTT assay (n = 3; shown are the mean values of triplicate determinations ± SEM). ( B–E ) LUHMES cells were stimulated with H 2 O 2 (100 µM). After one hour the cells were washed with medium und regenerated for the indicated time intervals in medium with or without TNC-scTNF R2 (100 ng/ml). ( B ) LUHMES cells were regenerated for 24 hours and cell viability was measured using the MTT assay (n = 3, shown are the mean values ± SEM). ( C ) Cells were regenerated for one or three hours, fixed with 4%PFA, permeabilized with 0.1% Triton-X100 and β-III-tubulin was detected with specific antibodies. Cell nuclei were visualized using DAPI. Pictures are projections of eight optical sections (0.4 µm; bar = 50 µm). ( D ) Number of cells was determined by counting the nuclei (DAPI staining). ( E ) Cells were regenerated for one hour and apoptotic cells were identified by terminal deoxynucleotidyl transferase (TdT)-mediated dUTP-FITC nick end labeling (TUNEL). (D,E) At least 10 different image sections containing a minimum of 500 cells were used to determine the number of total and TUNEL-positive cells. *p values less than 0.05 (** p-value less than 0.001) were considered to be significant (n = 2, shown are the mean values ± SD).

Article Snippet: The antibody anti-6-his was from Biovision (San Francisco, CA), the antibodies against huTNFR2 (HP9003; MR2-1) were from Hbt (Uden, The Netherlands), the antibody against huTNF (AF-210-NA) was from R&D Systems (Wiesbaden, Germany) and the antibody against β-III-tubulin was purchased from Acris Antibodies (Hiddenhausen, Germany).

Techniques: Incubation, MTT Assay, Staining, End Labeling, TUNEL Assay