chemical reagents rsl3  (TargetMol)

Journal: Molecular Neurobiology

Article Title: The DNA Demethylase TET1 is a Pivotal Regulator of the miR-124/ISX9-Instructed Conversion of Astrocytes to Induced Neurons

doi: 10.1007/s12035-026-05873-1

Figure Lengend Snippet: TRN analysis identifies core transcriptional regulators with pivotal roles in miR-124 and miR-124/ISX9 instructed reprogramming. A. Representation of the TRN constructed from RNA-seq data obtained from astrocytes at days 1 and 7 of reprogramming, miR-124-reprogrammed astrocytes on day 7 and miR-124 + ISX9-reprogrammed astrocytes on day 7. Each transcriptional regulator is represented by a circle (or a box for the top 20 TFs), where its size is proportional to its regulon size, and its color is indicative of its activity induced by miR-124 and modified by ISX9 ( Orange: transcriptional regulators whose activity is increased by miR-124 and further enhanced by ISX9 addition (miR-124 + ISX9 > miR-124 > astro day 1/day 7)). Magenta : transcriptional regulators whose activity is increased by miR-124 and mildly reduced by ISX9 (miR-124 > miR-124 + ISX9 > astro day 1/day 7). Red: transcriptional regulators whose activity is uniquely increased by the action of ISX9. Blue: transcriptional regulators whose activity is reduced by the action of miR-124 and further reduced by ISX9 (astro day 1/day 7 > miR-124 > miR-124 + ISX9). Turquoise: transcriptional regulators whose activity is reduced by miR-124 and less reduced by ISX9 (astro day 1/day 7 > miR-124 + ISX9 > miR-124). The top 20 transcriptional regulators inferred from the betweenness centrality analysis of the TRN of miR-124 and miR-124 + ISX9 are also portrayed in this network as boxes, and their names are highlighted in bold and black for common regulators, magenta for unique regulators in miR-124 condition and red in miR-124 + ISX9 condition. B. Heatmap presenting the normalized activity of the top 40 transcriptional regulators for miR-124 and miR-124 + ISX9 conditions inferred via betweenness centrality analysis of the TRN (35 regulons are common, whereas the unique 5 regulons for each condition are indicated by green and blue circles for miR-124 and miR-124 + ISX9, respectively)

Article Snippet: In the miR-124 + ISX9-reprogrammed cells, 10 μΜ ISX9 chemical compound (MedChemExpress) was added from days 2–10.

Techniques: Construct, RNA Sequencing, Activity Assay, Modification

Journal: Molecular Neurobiology

Article Title: The DNA Demethylase TET1 is a Pivotal Regulator of the miR-124/ISX9-Instructed Conversion of Astrocytes to Induced Neurons

doi: 10.1007/s12035-026-05873-1

Figure Lengend Snippet: TET1 is important for the neuronal differentiation of miR-124 + ISX9-iNs and controls key regulators of neurogenic reprogramming. A. Coimmunostaining of astrocytes reprogrammed with miR-124 + ISX9 -/+ siTet1 on day 11 with anti-TUJ1 (in gray), anti-SYN1 (in green) and anti-MAP2 (in magenta) antibodies. B. Quantification of the percentage of TUJ1 + reprogrammed cells with miR-124 + ISX9 + siCntl (n = 4 independent experiments) or miR-124 + ISX9 + siTet1 (n = 3 independent experiments) on day 11. C. Presentation of the proportion of differentiated TUJ1 + iNs (green portions of the bars) in the total TUJ1 + population (as quantified in B for each condition and set to 100%) (the gray portions of the bars indicate the proportion of TUJ1 + iNs exhibiting a transitory still not differentiated morphology). D. Quantification of the percentage of TUJ1 + iNs reprogrammed by miR-124 + ISX9 + siCntl (n = 5 independent experiments) or miR-124 + ISX9 + siTet1 (n = 4 independent experiments) that were also positive for SYN1 and MAP2 on day 11. E. Representative images of a miR-124 + ISX9 + siCntl-iN and a miR-124 + ISX9 + siTet1-iN on day 11 processed by the Filament Tracer module in Imaris. Quantification of the maximum primary neurite length per cell ( F ) and average primary neurite length per cell ( G ) in miR-124 + ISX9 + siCntl-iNs (n = 40 cells) and miR-124 + ISX9 + siTet1-iNs (n = 23 cells) via the Filament Tracer module in Imaris. H. Number of Sholl intersections relative to the distance from the soma (in μm) (Sholl intersections’ distribution) for miR-124 + ISX9 + siCntl-iNs (n = 40 cells) and miR-124 + ISX9 + siTet1-iNs (n = 23 cells). I. Quantification of the number of segment (neurite) branch points per cell for miR-124 + ISX9 + siCntl-iNs (n = 40 cells) and miR-124 + ISX9 + siTet1-iNs (n = 23 cells) via Sholl analysis of the Filament Tracer module in Imaris. The cells that were analyzed via the Filament Tracer module in Imaris were collected from 3 independent experiments for each condition. RT‒qPCR analysis of the mRNA levels of the following transcriptional regulators related to neuronal differentiation: Baf53b ( J ), NeuroD1 ( K ) and Myt1l ( L ) in astrocytes reprogrammed with miR-124 + ISX9 -/+ siTet1 on day 7. n = 3 independent experiments *p < 0.05, **p < 0.01, ***p < 0.001

Article Snippet: In the miR-124 + ISX9-reprogrammed cells, 10 μΜ ISX9 chemical compound (MedChemExpress) was added from days 2–10.

Techniques:

Journal: Molecular Neurobiology

Article Title: The DNA Demethylase TET1 is a Pivotal Regulator of the miR-124/ISX9-Instructed Conversion of Astrocytes to Induced Neurons

doi: 10.1007/s12035-026-05873-1

Figure Lengend Snippet: TET1 regulates genes involved in neuron projection morphogenesis and synaptic signaling in differentiating miR-124 + ISX9-iNs. A. Venn diagram representing the overlap between TET1 direct targets identified in two independent TET1-ChIP experiments and genes upregulated in miR-124 + ISX9-iNs compared to astrocytes on day 1 (log 2 (fold change) > 1, padj < 0.01). B. Gene Ontology (GO) Biological Processes (BP) terms enriched for the 1,163 TET1 direct targets upregulated in miR-124 + ISX9-iNs on day 7. The GO terms are ranked by p adjusted values and color coded by the gene ratio (the number of enriched genes divided by the total number of genes in the GO term). Heatmaps showing the expression of genes associated with the GO BP terms “neuron projection morphogenesis” ( C ) and the GO MF terms “SNARE binding, glutamate receptor binding and neurotransmitter binding activity” in astrocytes and iNs ( D ). RT‒qPCR analysis of the mRNA levels of genes related to synaptic activity: Syt4 (n = 3 independent experiments) (E ), Vamp2 (n = 4 independent experiments) ( F ), Celsr3 (n = 4 independent experiments) ( G ) and Kif5a (n = 3 independent experiments) ( H ) in astrocytes reprogrammed with miR-124 + ISX9 -/+ siTet1 on day 7. **p < 0.01, ***p < 0.001

Article Snippet: In the miR-124 + ISX9-reprogrammed cells, 10 μΜ ISX9 chemical compound (MedChemExpress) was added from days 2–10.

Techniques: Expressing, Binding Assay, Activity Assay

Journal: Molecular Neurobiology

Article Title: The DNA Demethylase TET1 is a Pivotal Regulator of the miR-124/ISX9-Instructed Conversion of Astrocytes to Induced Neurons

doi: 10.1007/s12035-026-05873-1

Figure Lengend Snippet: Silencing of Lin28a affects the morphological transition of miR-124 + ISX9-iNs. A. Schematic representation of the protocol used to silence Lin28a during the reprogramming process. B. Estimation of the degree of Lin28a mRNA silencing on day 7 of reprogramming by miR-124 + ISX9 -/+ siLin28a (n = 4 independent experiments) by qRT‒PCR. C. Immunostaining of astrocytes reprogrammed with miR-124 + ISX9 -/+ siLin28a on day 7 with an anti-TUJ1 antibody (in red); the inset areas show representative cell morphologies. D. Quantification of the percentage of TUJ1 + reprogrammed cells by miR-124 + ISX9 -/+ siLin28a on day 7 (n = 4 independent experiments) and on day 11 (n = 5 independent experiments). E. Presentation of the proportion of differentiated TUJ1 + iNs (green portions of the bars) in the total TUJ1 + population (as quantified in D for each condition and set to 100%) (the gray portions of the bars indicate the proportion of TUJ1 + iNs exhibiting a transitory still not differentiated morphology). F. Immunostaining of astrocytes reprogrammed with miR-124 + ISX9 -/+ siLin28a on day 7 with an anti-TUJ1 antibody (in magenta) and an anti-LIN28A antibody (in green); representative cells exhibiting high LIN28A levels and a differentiated morphology (categories 1 or 2) are indicated with arrows, while representative cells with low LIN28A levels and a less differentiated morphology (categories 2 or 3) are indicated with asterisks. Measurement of the total mean fluorescence intensity (f.i.) ( G ), nuclear mean f.i. ( H ) and cytoplasmic mean f.i. ( I ) of LIN28A protein levels on day 7 in control cells treated with sc-miRNA + siCntl and TUJ1 + reprogrammed cells treated with miR-124 + ISX9 -/+ siLin28a (n = 80 cells for each condition from 3 independent experiments) (mean ± SEM, p < 0.0001, two tailed t-test assuming unequal variance). J. Representative images of a miR-124 + ISX9 + siCntrl-iN and a miR-124 + ISX9 + siLin28a-iN on day 11 processed by the Filament Tracer module in Imaris. K. Representation of the number of Sholl intersections relative to the distance from the soma (in μm) (Sholl intersections’ distribution) for miR-124 + ISX9 + siCntrl-iNs (n = 40 cells) and miR-124 + ISX9 + siLin28a-iNs (n = 39 cells). L. Quantification of the number of segment (neurite) branch points per cell for miR-124 + ISX9 + siCntrl-iNs (n = 40 cells) and miR-124 + ISX9 + siLin28a-iNs (n = 39 cells) using Sholl analysis with the Filament Tracer module in Imaris. M. Quantification of the average primary neurite length per cell in miR-124 + ISX9 + siCntrl-iNs (n = 40 cells) and miR-124 + ISX9 + siLin28a-iNs (n = 39 cells) using the Filament Tracer module in Imaris. The cells that were analyzed with the Filament Tracer module in Imaris were collected from 3 independent experiments for each condition. *p < 0.05, **p < 0.01, ***p < 0.001. N. Coimmunostaining of miR-124 + ISX9 + siCntl-iNs and miR-124 + ISX9 + siLin28a-iNs on day 18 with an anti-MAP2 antibody (in magenta) and an anti-SYN1 antibody (in green); the inset areas show higher magnifications of representative cells and their processes stained with SYN1

Article Snippet: In the miR-124 + ISX9-reprogrammed cells, 10 μΜ ISX9 chemical compound (MedChemExpress) was added from days 2–10.

Techniques: Immunostaining, Fluorescence, Control, Two Tailed Test, Staining

Journal: Molecular Neurobiology

Article Title: The DNA Demethylase TET1 is a Pivotal Regulator of the miR-124/ISX9-Instructed Conversion of Astrocytes to Induced Neurons

doi: 10.1007/s12035-026-05873-1

Figure Lengend Snippet: LIN28A and TET1 co-regulate a set of synaptic genes expressed in differentiating miR-124 + ISX9-iNs. A. Venn diagram representing the overlap between LIN28A direct targets derived from a publicly available LIN28A-ChIP experiment and genes upregulated in miR-124 + ISX9-iNs compared to astrocytes on day 1 (log 2 (fold change) > 1, padj < 0.01). B. Gene Ontology (GO) Biological Processes (BP) terms enriched for the 238 LIN28A direct targets upregulated in miR-124 + ISX9-iNs. The GO terms are ranked by p adjusted values and color coded by the gene ratio (the number of enriched genes divided by the total number of genes in the GO term). C. Heatmap showing the expression of genes associated with the GO BP “synaptic signaling”. RT‒qPCR analysis of the mRNA levels of genes related to synaptic activity, Rab3c ( D ), Cacng2 ( E ) and Syn1 ( F ), in astrocytes reprogrammed with miR-124 + ISX9 -/+ siLin28a on day 7. n = 3 independent experiments. RT‒qPCR analysis of the mRNA levels of the genes Rab3c ( G ), Cacng2 ( H ), Vamp2 ( I ) and Syt4 ( J ) in astrocytes reprogrammed with miR-124 + ISX9 + siControl (n = 5 independent experiments for Rab3c and Cacng2 and n = 4 independent experiments for Vamp2 and Syt4 ), miR-124 + ISX9 + siLin28a (n = 3 independent experiments for Rab3c , Cacng2 and Vamp2 and n = 4 independent experiments for Syt4 ), miR-124 + ISX9 + siTet1 (n = 5 independent experiments for Rab3c , n = 4 independent experiments for Cacng2 and Vamp2 and n = 3 independent experiments for Syt4) or miR-124 + ISX9 + siTet1/Lin28a (n = 3 independent experiments) on day 7. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001

Article Snippet: In the miR-124 + ISX9-reprogrammed cells, 10 μΜ ISX9 chemical compound (MedChemExpress) was added from days 2–10.

Techniques: Derivative Assay, Expressing, Activity Assay