Journal: Molecular and Cellular Pediatrics

Article Title: O-GlcNAcylation expression predicts a favorable prognosis and mitigates malignant phenotypes via MYCN suppression in neuroblastoma

doi: 10.1186/s40348-026-00218-3

Figure Lengend Snippet: Thiamet G reduces neuroblastoma malignancy similarly to retinoic acid. SK-N-BE and SK-N-DZ cells were treated with Thiamet G (10 µM) or retinoic acid (RA; 10 µM) for the indicated times. Control cells were treated with vehicle alone. A Western blot analysis of O-GlcNAc expression in SK-N-BE cells treated for 48 h. GAPDH was used as the internal loading control. The numbers below the blots indicate the relative intensity ratio of O-GlcNAc normalized to GAPDH. B MTT assay analysis showing significantly decreased cell proliferation in Thiamet G- and RA-treated groups compared to the control. Data are presented as the mean cell viability fold change ± SD relative to the control at day 0 (n = 3; *** p < 0.001). C Representative images from the transwell invasion assay. The bar graph represents the mean number of invaded cells ± SD (n = 3; p < 0.01). Scale bar: 100 μm. D Neurite outgrowth analysis with representative immunofluorescence staining of NFH (red) and O-GlcNAc (green) in cells treated for 3 days. Images were acquired by confocal microscopy. Nuclei were stained with DAPI (blue). Quantification of fluorescence intensity is shown in the right panel. Data represent mean ± SD (n = 3; * p < 0.01). Scale bar: 100 μm. RA, retinoic acid; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; SD, standard deviation; NFH, neurofilament heavy chain; DAPI, 4′,6-diamidino-2-phenylindole

Article Snippet: Cells were seeded at a density of 5 × 10 3 cells per well in chamber slides (SPL Life Sciences), fixed with 4% PFA, permeabilized with 0.25% Triton X100, blocked in PBS containing 2% bovine serum albumin (Bio-Rad) and applied with primary antibodies to O-GlcNAc (Cat. No. LS‐B2534, LSBio, dilution [1:200]) and NFH (Cat. No. 171 104, Synaptic Systems, dilution [1:500]).

Techniques: Control, Western Blot, Expressing, MTT Assay, Transwell Invasion Assay, Immunofluorescence, Staining, Confocal Microscopy, Fluorescence, Standard Deviation

Journal: iScience

Article Title: Homozygous ALS-linked mutations in TARDBP/TDP-43 lead to hypoactivity and synaptic abnormalities in human iPSC-derived motor neurons

doi: 10.1016/j.isci.2024.109166

Figure Lengend Snippet: TDP-43 MN cultures form a normal axonal network and maintain viability (A and C) Representative images of MNs differentiated for 6 weeks subjected to immunocytochemistry for neuronal markers βIII-tubulin (A) and NF-H (C). Scale bar, 100 μm. (B and D) Quantification of total area and number of branches of βIII-tubulin + axons (B) and NF-H + axons (D). n = 3 independent experiments. Two-way ANOVA. (E) Viability of MN cultures differentiated with and without neurotrophic factors (NF) supplementation over a span of 6 weeks post-plating. n = 4 independent experiments. Two-way ANOVA. (F–G) Immunoblot (F) and quantification (G) of cleaved caspase 3 (CC3) levels. βIII-tubulin was used as loading control. Extractions were performed in MNs harvested after 6 weeks post-plating. n = 4 independent experiments. Ordinary one-way ANOVA. (H) Effect of glutamate treatment (0.1 mM glutamate, 24 h) on viability of MNs differentiated for 4 weeks n = 4 independent experiments. Two-way ANOVA. (I) Effect of ethacrynic acid treatment (50 μM EA, 17 h) on viability of MNs differentiated for 4 weeks. Individual points represent per-well values from 3 independent experiments. Two-way ANOVA. All data shown as mean ± SEM. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001. See also Figures S3 and .

Article Snippet: The following primary antibodies were used: rabbit anti-Nanog (Abcam; Cat#ab21624; 1:200), mouse anti-TRA-1-60 (Stem Cell Tech; Cat#60064; 1:200), mouse anti-SSEA-4 (Santa Cruz; Cat# sc-21704; 1:200), goat anti-Oct-3/4 (Santa Cruz; Cat#sc-8628; 1:500), mouse anti-PAX6 (DSHB; Cat#PAX6; 1:100), rabbit anti-OLIG2 (Millipore; Cat#AB9610; 1:100), rabbit anti-Nestin (Abcam; Cat#ab92391; 1:250), mouse anti-HB9 (DSHB; Cat#81.5C10-c; 1:50), mouse anti-ISL1/2 (DSHB; Cat#39.4D5-c; 1:50), goat anti-ChAT (Millipore; Cat#MAP144P; 1:50), rabbit anti-VAChT (Sigma-Aldrich; Cat#SAB4200559; 1:100), chicken anti-NF-H (EnCor Biotech; Cat#CPCA-NF-H; 1:1000), mouse anti-βIII-tubulin (Millipore; Cat#MAB5564; 1:1000), rabbit anti-TDP-43 (C-terminal) (Proteintech; Cat#12892-1-AP; 1:2000), rabbit anti-TDP-43 (N-terminal) (Proteintech; Cat#10782-2-AP; 1:1000), mouse anti-pTDP-43 (Ser409/410) (Proteintech; Cat#66318-1-lg; 1:500), rabbit anti-Synapsin I (Calbiochem; Cat#574777; 1:500), mouse anti-PSD95 (Millipore; Cat#MABN68; 1:250).

Techniques: Immunocytochemistry, Western Blot

Journal: iScience

Article Title: Homozygous ALS-linked mutations in TARDBP/TDP-43 lead to hypoactivity and synaptic abnormalities in human iPSC-derived motor neurons

doi: 10.1016/j.isci.2024.109166

Figure Lengend Snippet: Subcellular distribution of TDP-43 in MNs (A) Schematics representing the fractionation workflow into nuclear and cytosolic fractions. (B–F) Immunoblot of nuclear and cytosolic fractions (B) and quantification of nuclear (C) and cytosolic (D) TDP-43 levels, and cytosolic C-terminal fragment of 35 kDa (CTF-35) levels (E and F). Histone H3 (nuclear marker) and actin (cytosolic marker) were used as both loading and fractionation controls. n = 6 extractions from 4 independent differentiations. Pooled data from MNs harvested 4- and 6-week post-plating. (G) Representative images of MNs differentiated for 6 weeks subjected to immunocytochemistry for TDP-43 (C-terminal antibody) and NF-H. Scale bar, 50 μm. (H and I) Quantification of TDP-43 distribution using the nuclear/cytosolic ratio of TDP-43 fluorescence signal intensity (H) and the TDP-43/Hoechst correlation coefficient (I). Individual data points represent per-frame mean values from 5 independent experiments. All data shown as mean ± SEM. ∗p < 0.05. Ordinary one-way ANOVA. See also Figure S6 .

Article Snippet: The following primary antibodies were used: rabbit anti-Nanog (Abcam; Cat#ab21624; 1:200), mouse anti-TRA-1-60 (Stem Cell Tech; Cat#60064; 1:200), mouse anti-SSEA-4 (Santa Cruz; Cat# sc-21704; 1:200), goat anti-Oct-3/4 (Santa Cruz; Cat#sc-8628; 1:500), mouse anti-PAX6 (DSHB; Cat#PAX6; 1:100), rabbit anti-OLIG2 (Millipore; Cat#AB9610; 1:100), rabbit anti-Nestin (Abcam; Cat#ab92391; 1:250), mouse anti-HB9 (DSHB; Cat#81.5C10-c; 1:50), mouse anti-ISL1/2 (DSHB; Cat#39.4D5-c; 1:50), goat anti-ChAT (Millipore; Cat#MAP144P; 1:50), rabbit anti-VAChT (Sigma-Aldrich; Cat#SAB4200559; 1:100), chicken anti-NF-H (EnCor Biotech; Cat#CPCA-NF-H; 1:1000), mouse anti-βIII-tubulin (Millipore; Cat#MAB5564; 1:1000), rabbit anti-TDP-43 (C-terminal) (Proteintech; Cat#12892-1-AP; 1:2000), rabbit anti-TDP-43 (N-terminal) (Proteintech; Cat#10782-2-AP; 1:1000), mouse anti-pTDP-43 (Ser409/410) (Proteintech; Cat#66318-1-lg; 1:500), rabbit anti-Synapsin I (Calbiochem; Cat#574777; 1:500), mouse anti-PSD95 (Millipore; Cat#MABN68; 1:250).

Techniques: Fractionation, Western Blot, Marker, Immunocytochemistry, Fluorescence

Journal: iScience

Article Title: Homozygous ALS-linked mutations in TARDBP/TDP-43 lead to hypoactivity and synaptic abnormalities in human iPSC-derived motor neurons

doi: 10.1016/j.isci.2024.109166

Figure Lengend Snippet:

Article Snippet: The following primary antibodies were used: rabbit anti-Nanog (Abcam; Cat#ab21624; 1:200), mouse anti-TRA-1-60 (Stem Cell Tech; Cat#60064; 1:200), mouse anti-SSEA-4 (Santa Cruz; Cat# sc-21704; 1:200), goat anti-Oct-3/4 (Santa Cruz; Cat#sc-8628; 1:500), mouse anti-PAX6 (DSHB; Cat#PAX6; 1:100), rabbit anti-OLIG2 (Millipore; Cat#AB9610; 1:100), rabbit anti-Nestin (Abcam; Cat#ab92391; 1:250), mouse anti-HB9 (DSHB; Cat#81.5C10-c; 1:50), mouse anti-ISL1/2 (DSHB; Cat#39.4D5-c; 1:50), goat anti-ChAT (Millipore; Cat#MAP144P; 1:50), rabbit anti-VAChT (Sigma-Aldrich; Cat#SAB4200559; 1:100), chicken anti-NF-H (EnCor Biotech; Cat#CPCA-NF-H; 1:1000), mouse anti-βIII-tubulin (Millipore; Cat#MAB5564; 1:1000), rabbit anti-TDP-43 (C-terminal) (Proteintech; Cat#12892-1-AP; 1:2000), rabbit anti-TDP-43 (N-terminal) (Proteintech; Cat#10782-2-AP; 1:1000), mouse anti-pTDP-43 (Ser409/410) (Proteintech; Cat#66318-1-lg; 1:500), rabbit anti-Synapsin I (Calbiochem; Cat#574777; 1:500), mouse anti-PSD95 (Millipore; Cat#MABN68; 1:250).

Techniques: Recombinant, Gentle, Cell Viability Assay, DC Protein Assay, Western Blot, Sequencing, Software