shrna control  (Addgene inc)

Journal: Acta Neuropathologica

Article Title: TDP-43 impairs glycolysis by sequestering hexokinase 1 in amyotrophic lateral sclerosis

doi: 10.1007/s00401-026-02996-6

Figure Lengend Snippet: Pathological TDP-43 elicits glycolytic impairment in cells. a , b Agilent seahorse glycolytic stress test was performed on n = 3 independent biological repeats. Glycolysis and Glycolytic Capacity were measured according to manufacturer’s protocol. ECAR values were normalized to total protein then plotted against time. Individual glycolysis and glycolytic capacity values were normalized to total protein level then to individual control from each biological repeat. a Stable HEK293 cells were overexpressed with either YFP (control), TDP-43 WT or TDP-43 ΔNLS . Data were analyzed by one-way ANOVA followed by post hoc Tukey’s test. b TDP-43 G298S patient iPSC and control iPSC were differentiated into motor neurons with our established protocol. On day 26 following differentiation, cells were treated with MG-132 (1 μM) for 24 h. Data were analyzed by Student’s t-test. c YFP (control), TDP-43 WT or TDP-43 ΔNLS stable cells were transiently transfected with various glycolysis shRNAs. Cell viability was measured via MTT assay. Decreased cell viability in all TDP-43 WT and TDP-43 ΔNLS with various glycolysis shRNA (n = 3). Data were analyzed by one-way ANOVA followed by post hoc Tukey’s test. All data are mean ± SE

Article Snippet: The following constructs pcDNA3.2-YFP (addgene #84910), pcDNA3.2-TDP-43 WT -YFP (human) (addgene #84911), pcDNA3.2-TDP-43 ΔNLS -YFP (human) (addgene #84912), pLD-puro-Cc-TARDBP-A315T_VA Plasmid (human TDP-43 A315T) (addgene #141329), pLD-puro-Cc-TARDBP-WT_VA Plasmid (human TDP-43 WT) (addgene #141327), and shRNA control (addgene: #8453) were purchased from addgene.

Techniques: Control, Transfection, MTT Assay, shRNA

Journal: Stem Cell Reports

Article Title: ASCL1 promotes nuclear shrinkage in transdifferentiation by suppressing NUP37

doi: 10.1016/j.stemcr.2026.102823

Figure Lengend Snippet: Reduction of nuclear size during the direct conversion of human fibroblasts to neurons (A) Schematic for the transdifferentiation of human fibroblasts to iNs by lentiviruses expressing ASCL1, miR124-9-9 ∗ -BclxL, and p53 shRNA (AMp, uppercase for overexpression and lowercase for knockdown). –FBS, serum withdrawal to synchronize cell cycle at the G1/S checkpoint. Scale bar, 100 μm. (B) Phase contrast images of MRC5 cells under conversion at the indicated time points. Scale bar, 100 μm. Insets, super-resolution images of DAPI-stained nuclei. Scale bar, 10 μm. (C) Nuclear volume quantification for MRC5 cells throughout reprogramming. n = 50 frames from 3 independent experiments for each time point, unpaired t test vs. day −2. ∗ p < 0.01. (D) Quantification of nuclear area for MRC5 cells throughout reprogramming. n = 50 frames from 3 independent experiments for each time point, unpaired t test vs. day −2, ∗ p < 0.01. (E) Quantification of nuclear area at the indicated time points as MRC5, AG22056 newborn foreskin fibroblasts, or GM09918 (78 years) skin fibroblasts were being converted to iNs. n = 50 frames from 3 independent experiments for each time point, unpaired t test vs. day −2, ∗ p < 0.01. (F) The average area of MRC5, AG22056, or GM09918 cells as fibroblasts at day −2 (Fib) or TUJ1 + or MAP2 + iNs. ns, no significance. n = 50 frames from three independent experiments. (G) iPSC-derived cortical neurons were co-stained for MAP2 and DAPI at days 30, 40, and 80 of differentiation. Scale bar, 50 μm. Inset, super-resolution images of neuronal nuclei; scale bar, 10 μm. (H) Quantification of nuclear area of the indicated samples. ∗ p < 0.01, vs. the preceding bar (or D30 for iPSC-derived neurons), n = 50 frames from 3 independent experiments.

Article Snippet: We purchased the following plasmids from Addgene: pLKO.1/ p53 shRNA (#19119), pLKO.1/scrambled shRNA (#1864), pMD2.G (#12259), psPAX2 (#12260), pTight-9-124-Bclx (miR9/9 ∗ -124, #60857), pRL-SV40P (#27163), and pGL3 enhancer vector (#212938).

Techniques: Expressing, shRNA, Over Expression, Knockdown, Staining, Derivative Assay

Journal: Stem Cell Reports

Article Title: ASCL1 promotes nuclear shrinkage in transdifferentiation by suppressing NUP37

doi: 10.1016/j.stemcr.2026.102823

Figure Lengend Snippet: NUP37 knockdown significantly enhanced AMp-mediated transdifferentiation and nuclear shrinkage (A) Western blot of NUP37 in MRC5 cells transduced without (−) or with the indicated reprogramming factors. (B–D) MRC5 human fibroblasts reprogrammed with ASCL1, MIR124-9-9 ∗ -BclxL, and p53 shRNA (AMp) (B), AMp and NUP37 shRNA (AMpu) (C), or AMp and NUP37 overexpression (AMpU) (D) were co-stained as indicated on day 14. Scale bar, 100 μm. (E–G) Reprogramming efficiency (E) as measured by the percentages of TUJ1 + or MAP2 + cells among all DAPI + cells, reprogramming yield of MAP2 + cells per frame (F), and the number of DAPI + cells per frame (G) at day 14. # and ∗ , p < 0.05, n = 15 (3 experiments, 5 frames each), vs. AMp for the indicated cell type, unpaired t test. (H) Nuclear area of MAP2 + neurons for each condition. ∗ p < 0.001, n = 50 frames from 3 independent experiments, vs. AMp, unpaired t test. (I–P) MRC5 cells reprogrammed with AMp (I–L) or AMpu (M–P) were co-stained as indicated at different time points. Scale bar, 100 μm. (Q‒S) (Q) Reprogramming efficiency of MAP2 + -generated neurons per DAPI + nuclei. (R) Yield of MAP2 + neurons. (S) Number of DAPI + cells per frame. ∗ p < 0.01, n = 15 (3 experiments, 5 frames each), vs. AMp at the same time point, unpaired t test. (T) RT-qPCR measurement of mature neuronal markers in AMp- or AMpu-induced neurons at D14. ∗ p < 0.05, n = 6 (3 experiments, duplicate for each), vs. AMp, unpaired t test.

Article Snippet: We purchased the following plasmids from Addgene: pLKO.1/ p53 shRNA (#19119), pLKO.1/scrambled shRNA (#1864), pMD2.G (#12259), psPAX2 (#12260), pTight-9-124-Bclx (miR9/9 ∗ -124, #60857), pRL-SV40P (#27163), and pGL3 enhancer vector (#212938).

Techniques: Knockdown, Western Blot, shRNA, Over Expression, Staining, Generated, Quantitative RT-PCR

Journal: Stem Cell Reports

Article Title: ASCL1 promotes nuclear shrinkage in transdifferentiation by suppressing NUP37

doi: 10.1016/j.stemcr.2026.102823

Figure Lengend Snippet: Cooperation of ASCL1 and NUP37 shRNA in reprogramming and nuclear shrinkage (A–P) MRC5 human fibroblasts were reprogrammed without or with the indicated combinations of ASCL1 (A), miR124-9-9 ∗ -BclxL (M), p53 shRNA (p) and NUP37 shRNA (u), and co-stained as indicated at day 14. Bar, 100 μm. (Q–S) Reprogramming efficiency (Q) as measured by the percentages of TUJ1 + or MAP2 + cells among all DAPI + cells, reprogramming yield of MAP2 + cells per frame (R), and the number of DAPI + cells per frame (S) at day 14. # and ∗ , p < 0.001, n = 15 (3 experiments, 5 frames each), vs. the corresponding condition without u for the indicated cell type, unpaired t test. $ p < 0.001, n = 15 (3 experiments, 5 frames each), vs. no virus (−V), unpaired t test. (T) Nuclear area for each condition. ∗ p < 0.05, n = 50 frames from 3 independent experiments, vs. the corresponding condition without u; unpaired t test. $ p < 0.005, n = 50 frames from 3 independent experiments, vs. no virus (−V), unpaired t test.

Article Snippet: We purchased the following plasmids from Addgene: pLKO.1/ p53 shRNA (#19119), pLKO.1/scrambled shRNA (#1864), pMD2.G (#12259), psPAX2 (#12260), pTight-9-124-Bclx (miR9/9 ∗ -124, #60857), pRL-SV40P (#27163), and pGL3 enhancer vector (#212938).

Techniques: shRNA, Staining, Virus