guide cas9 plasmid pspcas9 bb 2a puro  (Addgene inc)

Journal: Redox Biology

Article Title: Nuclear pore complex dysfunction drives TDP-43 pathology in ALS

doi: 10.1016/j.redox.2025.103824

Figure Lengend Snippet: ALS is associated to altered nucleoporin content in spinal cord and brain cortex . A) Left panel shows a representative western-blot image of FG-repeats containing NUPs (reactive to monoclonal antibody MAb414) of lumbar spinal cord homogenates from ALS patients and healthy, age and sex matched controls. Right panel shows the quantified immunoreactivity. B) The left panel shows representative confocal microscopy images from isolated nuclei from spinal cord samples, quantified in the violin plots from the right panel, demonstrating in situ decreased nucleoporin content.C) Quantitative analyses of NUP107, FG-repeats, and NUP93 in isolated nuclei by flow cytometry analyses, showing decreased content of NUP107 in spinal cord samples from ALS patients. D) RNA Seq data in heatmap showing that the mRNAs content of scaffold NUPs and NUP153 (an FG-repeat containing NUP) was decreased in spinal cord from ALS patients.Right panel indicates TPM (transcrits per kilobase million). E) Left panel shows confocal microscopy images with anti-tubulin β III immunoreactivity in nuclei isolated from brain cortex (area 8) from ALS patients and healthy individuals. Middle panel indicates tubulin β III content distribution, with the vertical line situating median values. Right panel illustrates differences in relationship between the content of FG-repeat NPC and tubulin β III in nuclei, with reference to ALS and tubulin β III content. F) Left panel shows representative confocal microscopy images from isolated nuclei from spinal cord samples, quantified plots from the right panel, demonstrating a decreased amount of nuclei from neuronal cells, based on NeuN content. Bars show mean values ± SEM or % in F. ∗,∗∗, and ∗∗∗∗ indicate p < 0.05, p < 0.01 and p < 0.0001 with reference to control values by Student's T test, Chi-Square (F) or Mann-Whitney U test. In E) For controls, linear relationship equation Y = −0.2721∗X + 0.01782 (p < 0.0004) and for ALS, the equation is Y = 0.3195∗X + 0.008008 (p < 0.0001). Scale white bar length in B, E and F are 50 μm long. Ns: non statistically significant differences.

Article Snippet: To investigate the role of specific NUPs, CRISPR/Cas9 constructs targeting NUP107 were designed using guide RNAs against human NUP107 gene (Santa Cruz Biotechnology, Dallas, TX, US, Cat #sc-405252-KO-2).

Techniques: Western Blot, Confocal Microscopy, Isolation, In Situ, Flow Cytometry, RNA Sequencing, Control, MANN-WHITNEY

Journal: Redox Biology

Article Title: Nuclear pore complex dysfunction drives TDP-43 pathology in ALS

doi: 10.1016/j.redox.2025.103824

Figure Lengend Snippet: NUPs are present in motor neurons and glial cells in human lumbar spinal cord, ventral horn. Representative immunohistochemical images of cellular distribution of the scaffolding NUP TPR (A–D) and the FG-repeat NUP107 (E,F). Samples from grey matter are shown in A,B,E and F, while as white matter cells (i.e. glial cells) are shown in C and D. Samples from healthy donors (A,C and E) and ALS patients (B,D and F) are presented. Arrows in A and B indicate non-nuclear staining of TPR, which were more frequent in ALS samples.

Article Snippet: To investigate the role of specific NUPs, CRISPR/Cas9 constructs targeting NUP107 were designed using guide RNAs against human NUP107 gene (Santa Cruz Biotechnology, Dallas, TX, US, Cat #sc-405252-KO-2).

Techniques: Immunohistochemical staining, Scaffolding, Staining

Journal: Redox Biology

Article Title: Nuclear pore complex dysfunction drives TDP-43 pathology in ALS

doi: 10.1016/j.redox.2025.103824

Figure Lengend Snippet: NUP107 silencing alters autophagy and induces TDP-43 pathology in human cells . A) Left panel indicates representative western-blot of different NUPs in CRISPR-mediated NUP-107 silencing of HEK293 cells. Right panel shows densitometry analyses of different experiments. B) Left panel shows representative western-blot of different autophagy and protein-turnover components in the same cells, with right panel indicating densitometric analyses. C) Confocal microscopy of TDP-43 immunoreactivity of HEK293 CRISPR-mediated NUP107 cells, showing altered TDP-43 immunoreactivity, quantified in the lower panel.D) Confocal microscopy of phospho-TDP-43 immunoreactivity of CRISPR-mediated NUP107-silenced HEK293 cells, showing altered TDP-43 immunoreactivity, quantified in lower panel microscopy. E) Western-blot analyses of TDP-43 and phospho-TDP-43 in CRISPR-mediated NUP107-silenced HEK293 cells, quantified in the right panel. F) Higher magnification of confocal microscopy images showing cytosolic aggregates of phospho-TDP-43 (yellow arrows) induced by CRISPR-mediated NUP107-silencing in HEK293 cells, quantified in the left panel (both size and number of phospho-TDP-43 aggregates). G)Left panel, representative western-blot analyses of NUPs and autophagy components in siRNA-mediated silencing of NUP107 in HeLa cells.Right panel shows the quantitative analyses. Bars show mean values ± SEM from N = 3–5 different experiments. ∗,∗∗, ∗∗∗ and ∗∗∗∗ indicate, respectively, p < 0.05, p < 0.01, p < 0.001 and p < 0.0001 significant differences between silenced or non silenced cells by Student's T test or Mann Whitney U test. In B and D, white scale bar lenght is 50 μm long and 30 μm in F. ns: non statistically significant differences.

Article Snippet: To investigate the role of specific NUPs, CRISPR/Cas9 constructs targeting NUP107 were designed using guide RNAs against human NUP107 gene (Santa Cruz Biotechnology, Dallas, TX, US, Cat #sc-405252-KO-2).

Techniques: Western Blot, CRISPR, Confocal Microscopy, Microscopy, MANN-WHITNEY

Journal: Redox Biology

Article Title: Nuclear pore complex dysfunction drives TDP-43 pathology in ALS

doi: 10.1016/j.redox.2025.103824

Figure Lengend Snippet: Loss of TDP-43 induces alteration in nucleoporin homeostasis. A) Representative immunofluorescence analyses showing NUP107 and MAb414 in TDP-43 silenced cells for a HeLa inducible silencing clone. B) Violin plot showing the quantitative analyses of nuclear (i.e. colocalizing with DAPI) content of NUP107 and MAb414 of cells in A. C) Left panel, western-blot analyses of TPR-content in HeLa cells with inducible TDP-43 silencing, with right panel showing quantitative analyses. D) Western-blot analyses of selected NUPs and LC3B in HeLA cells with doxycycline-inducible TDP-43 silencing under autophagy induction by 24 h of nutrient deprivation (Depriv). E) Quantitative analyses of western-blots shown in D. Bars show mean values ± SEM from N = 3–5 different experiments. ∗,∗∗, and ∗∗∗∗ indicate, respectively, p < 0.05, p < 0.01, and p < 0.0001 significant differences between silenced or non silenced cells by Student's T test, Mann Whitney U test or post-hoc LSD after two-way ANOVA. In A white scale bar lenght is 50 μm long. ns: non statistically significant differences.

Article Snippet: To investigate the role of specific NUPs, CRISPR/Cas9 constructs targeting NUP107 were designed using guide RNAs against human NUP107 gene (Santa Cruz Biotechnology, Dallas, TX, US, Cat #sc-405252-KO-2).

Techniques: Immunofluorescence, Western Blot, MANN-WHITNEY