Journal: Cell Death Discovery

Article Title: Digital color-coded molecular barcoding reveals dysregulation of common FUS and FMRP targets in soma and neurites of ALS mutant motoneurons

doi: 10.1038/s41420-023-01340-1

Figure Lengend Snippet: A Analysis of AP2B1 and PTEN mRNA levels by real-time qRT-PCR in FMRP RIP samples from FUS WT or FUS P525L iPSC-derived spinal MNs. The housekeeping gene ATP5O is used as negative control. The graph shows the relative enrichment of the mRNAs pulled down by FMRP immunoprecipitation (IP), calculated as the percentage of input, in IP or control IgG samples, after normalization with an artificial spike RNA. The average from three independent differentiation experiments is showed and error bars indicate the standard deviation (Student’s t -test; unpaired; two tails; * p < 0.05). B Luciferase assay in HeLa cells expressing RFP, RFP-FUS WT , or RFP-FUS P525L and transfected with Renilla luciferase reporter constructs containing the 3′UTR of AP2B1 (RLuc- AP2B1 -3′UTR), MAP1B (RLuc- MAP1B -3′UTR) or PTEN (RLuc- PTEN -3′UTR), in combination with plasmids overexpressing FMRP, eGFP or alone (MOCK) (Student’s t -test; paired; two tails; * p < 0.05; ** p < 0.01; *** p < 0.001; **** p < 0.0001).

Article Snippet: Western blot analysis was carried out as described [ ] using anti-AP2B1 (1:2000; 15690-1-AP; Proteintech, Rosemont, IL, USA; RRID:AB_2056351), anti-MAP1B (1:1000; 21633-1-AP; Proteintech; RRID:AB_10793666), anti-PTEN (1:2000; 22034-1-AP; Proteintech; RRID:AB_2878977), anti-GAPDH (1:2000; MAB-10578; Immunological Sciences) primary antibodies and HRP Donkey Anti-Mouse IgG (H + L) (IS20404; Immunological Sciences) and HRP Donkey Anti-Rabbit IgG (H + L) (IS20405; Immunological Sciences) secondary antibodies.

Techniques: Quantitative RT-PCR, Derivative Assay, Negative Control, Immunoprecipitation, Standard Deviation, Luciferase, Expressing, Transfection, Construct

Journal: Frontiers in Cellular Neuroscience

Article Title: Endothelin-1, over-expressed in SOD1 G93A mice, aggravates injury of NSC34-hSOD1G93A cells through complicated molecular mechanism revealed by quantitative proteomics analysis

doi: 10.3389/fncel.2022.1069617

Figure Lengend Snippet: NSC34-hSOD1G93A cells display a series of injured features and ET-A and ET-B receptors are both expressed in the cell model. (A) Immunofluorescence labeling of MAP1B showing different neurites of the three cell groups (NSC34-E, NSC34-hSOD1WT and NSC34-hSOD1G93A). (B) Immunofluorescence staining indicates increased cFOS expression in NSC34-hSOD1G93A cells. (C) Immunofluorescence staining shows hSOD1-positive aggregates in NSC34-hSOD1G93A cells. (D) Quantification of neurite length from multiple fields of view was analyzed by the Sholl analysis. (E , F) Bar graphs showing quantification of cFOS and hSOD1 fluorescence intensities from multiple fields of view. * P < 0.05, a pairwise comparison marked by a horizontal line. Data represent the mean ± SEM, statistical significance was assessed by one-way ANOVA followed by LSD- t test. (G,H) Similar ET-A and ET-B expression are observed in the three cell groups. Bar = 50 μm in (A,B,G,H) . Bar = 20 μm in (C) .

Article Snippet: The sections were then incubated with primary antibodies in blocking buffer overnight at 4 ° C. Primary antibodies include goat anti Iba-1 (Wako, 019-19741, 1:250), mouse anti-GFAP (Millipore, MAB360, 1:400), mouse anti-NeuN (Millipore, MAB377, 1:100), mouse anti-APC (Millipore, OP80, 1:400), rabbit anti-SOD1 (Immunoway, YT4364, 1:100), rabbit anti-cFOS (Abcam, ab222699, 1:100), rabbit anti-MAP1B (Proteintech, 21633-1-AP, 1:100), mouse anti-SMI32 (Biolegend, 801701, 1:100), rabbit anti-GFP (Life tech, G10362, 1:100), rabbit anti-Endothelin-1 (Abbiotec, 250633, 1:100), rabbit anti-ET-A (Bioss, bs-1757R, 1:300) and rabbit anti-ET-B (Abcam, ab117529, 1:500).

Techniques: Immunofluorescence, Labeling, Staining, Expressing, Fluorescence