Journal: Journal of Hematology & Oncology

Article Title: Directly reprogrammed NK cells driven by BCL11B depletion enhance targeted immunotherapy against pancreatic ductal adenocarcinoma

doi: 10.1186/s13045-025-01730-1

Figure Lengend Snippet: Generation of directly reprogrammed NKs (drNKs) via BCL11B Knockdown. (A) Schematic of the BCL11B one-factor (1 F) reprogramming process. PBMCs were transduced with lentiviruses encoding shRNA against BCL11B in starting cell medium (SCM), followed by sequential incubation in reprogramming medium I (RMI) and reprogramming medium II (RMII) for the indicated durations. (B) Reprogramming efficiency of 1 F-NKs derived from PBMCs using 1 F reprograming medium (1 F RM), compared to four-factor (OSKM-based) reprogramming medium (4 F RM) at day 18 (left). Non-targeting shRNA was used as a control. Fold expansion of CD56 + CD3 − NK cells over time (right). Data are presented as mean ± SD ( n = 4–11). Two-tailed Student’s t-test; * P < 0.001 vs. control. (C) Overview of MSLN-CAR knock-in during 1 F reprogramming. PBMCs were co-transduced with CRISPR-Cas9 targeting BCL11B exon 1 (sgRNA#1/#2) and an adeno-associated virus (AAV) vector carrying the mesothelin-specific CAR (MSLN-CAR), enabling simultaneous BCL11B disruption and site-specific CAR insertion. (D) Schematic of the AAV vector design, comprising anti-MSLN scFv, CD8 hinge, CD8 transmembrane (TM), CD28 intracellular domain (ICD), and CD3ζ signaling domain. (E) Induction of CD56 + MSLN + 1 F-NK cells (MSLN-1 F-NKs) after 18 days of reprogramming with or without MSLN-CAR AAV. Representative flow cytometry plots (upper) and quantification (lower) show successful CAR expression. Data are mean ± SD ( n = 3–5). Two-tailed Student’s t-test; * P < 0.001 vs. sgRNA only. (F) Genomic PCR verification of MSLN-CAR knock-in at the BCL11B locus using primers targeting the pre-left homology arm (LHA) and SFFV promoter in WT-control, sgRNA only, and MSLN + sgRNA. (G) Flow cytometry analysis of CD56 and CD16 surface expression in 1 F-NKs (red), MSLN-1 F-NKs (orange), OSKM 4 F-derived NKs (4 F-NKs; green), iPSC-derived NKs (iPSC-NKs; blue), and PBMC-derived NKs (pNKs, blue). Representative dot plots and histograms are shown. (H-I) Expression of activating receptors (CD16, CD69, and DNAM-I), natural cytotoxicity receptors (NKp30, NKp44, and NKp46), and inhibitory receptors (KIR2DL1, KIR2DL2, and KIR3DL1) in indicated NK cells. (H) Representative histogram plots. (I) Quantitative analysis. Data are mean ± SD ( n = 3–13). Two-tailed Student’s t-test; * P < 0.001 vs. pNKs

Article Snippet: Lentiviral plasmids encoding short hairpin RNAs (shRNAs) targeting human BCL11B or a non-targeting control were obtained from Origene (BCL11B Human shRNA Plasmid Kit, #TL306424).

Techniques: Knockdown, Transduction, shRNA, Incubation, Derivative Assay, Control, Two Tailed Test, Knock-In, CRISPR, Virus, Plasmid Preparation, Disruption, Flow Cytometry, Expressing

Journal: bioRxiv

Article Title: Imp1 acts as a dosage- and stage-dependent temporal rheostat orchestrating radial glial fate transitions and cortical morphogenesis

doi: 10.1101/2025.11.18.688993

Figure Lengend Snippet: (A,D,G) Representative images of control, continuous Imp1 overexpression, and T1 Imp1 conditions showing TEMPO reporters, Cux1/Ctip2 immunostaining and overlays. Boxed regions: Ctip2+ TEMPO neurons (dashed) or double-positive Cux1+/Ctip2+ TEMPO cells (solid). (B,E,H) High magnification images of boxed regions highlight CFP-/RFP-labeled neurons in layers V-VI colocalizing with Ctip2 (outlined arrowheads) or double-positive for both markers (solid arrowheads). (C,F,I) Quantification of marker expression in CFP+ and RFP+ neurons residing in layers V-VI. Following continuous or T1 Imp1 overexpression, neurons in deep layer maintain appropriate deep-layer molecular identities (predominantly Ctip2+), demonstrating that laminar distribution reflects bona fide fate specification changes rather than mislocalization. (C) In control conditions, CFP+: Ctip2 (78.31% ± 13.82%), Cux1 (2.93% ± 2.11%), double-negative (18.76% ± 11.73%). RFP+: Ctip2 (50.35% ± 17.01%), Cux1 (1.85% ± 1.85%), double-negative (46.76% ± 17.20%), double-positive (1.04% ± 1.04%). (F) Following continuous Imp1 overexpression, CFP+: Ctip2+ (71.18% ± 3.06%), Cux1+ (7.38% ± 4.93%), double-negative (16.06% ± 1.93%), double-positive (5.38% ± 1.80%). RFP+: Ctip2+ (58.92% ± 7.18%), Cux1+ (5.84% ± 3.01%), double-negative (5.10% ± 3.12%), double-positive (30.14% ± 11.98%). (I) In T1 Imp1 overexpression, CFP+: Ctip2+ (47.26% ± 4.88%), Cux1+ (14.63% ± 3.76%), double-negative (36.41% ± 5.64%), double-positive (1.68% ± 0.57%). RFP+: Ctip2+ (47.38% ± 13.91%), Cux1+ (9.17% ± 4.68%), double-negative (26.86% ± 5.03%), double-positive (16.59% ± 10.02%). Dashed lines: upper (II-IV), lower cortical layers (V-VI) and subplate zone (SPZ). Scale bars: (A,D,G) 100 µm and (B,E,H) 20 µm. Data show mean±SEM. Statistics: two-tailed unpaired Welch’s t-test (*P < 0.05, **P < 0.01, *** P < 0.001).

Article Snippet: Primary antibodies were applied overnight to amplify TEMPO signals or detect fate markers at 4°C: mouse anti-V5 (1:650, Thermo Fisher R96025) to detect CFP+ cells, rat anti-mCherry (1:500, Thermo Fisher M11217) or chicken anti-mCherry (1:500, AvesLabs MCHERRY AB_2910557) to detect RFP+ cells, rat anti-Ctip2 (1:100, Abcam [25B6] ab18465) to detect Ctip2+ cells, and rabbit anti-Cux1 (1:50, Proteintech 11733-1-AP) to detect Cux1+ cells.

Techniques: Control, Over Expression, Immunostaining, Labeling, Marker, Expressing, Two Tailed Test

Journal: Current biology : CB

Article Title: The RAS GTPase RIT1 compromises mitotic fidelity through spindle assembly checkpoint suppression.

doi: 10.1016/j.cub.2021.06.030

Figure Lengend Snippet: Figure 1. RIT1 interacts directly with the SAC proteins MAD2 and p31comet

Article Snippet: Async., asynchronous growing cells; Noc., cells released from G1/S arrest for 4 h then treated with 100 ng/mL nocodazole for 10 h. (F) Detection of RIT1 S209 phosphorylation on bacterially expressed GST-RIT1 protein incubated with mitotic cell extract (MCE) treated with 1 mM Dinaciclib, 10 mM RO-3306, or DMSO control. (G) Immunoblot of RIT1 S209 phosphorylation on bacterially expressed RIT1 proteins subjected to an in vitro kinase assay with recombinant active CDK1/Cyclin B1. (H) MS quantification of phospho-S209 peptides in RIT1 protein incubated with CDK1/Cyclin B1 as in (G). n = 2, two-sided Student’s t test, data shown as mean, error bars indicate SD, *p % 0.05.

Techniques:

Journal: Current biology : CB

Article Title: The RAS GTPase RIT1 compromises mitotic fidelity through spindle assembly checkpoint suppression.

doi: 10.1016/j.cub.2021.06.030

Figure Lengend Snippet: Figure 2. RIT1 interaction with MAD2 and p31comet is regulated by CDK1 phosphorylation (A) hTERT-RPE1 cell stably expressing mNeonGreen (mNG)-RIT1 and Histone H2B-mCherry undergoing mitosis imaged at 5 min intervals. Anaphase onset set to t = 0 min. Scale bar, 20 mm. (B) Quantification of plasma membrane (PM) to cytoplasmic (Cyto.) ratio of mNG-RIT1 during metaphase (Meta, 5 min) and anaphase (Ana, +5 min) in cells as in (A). Two-sided Student’s paired t test, n = 15, ****p % 0.0001. (C) Immunoblots of subcellular protein fractionation of HeLa cell lysates. Async., asynchronous growing cells; Noc., cells released from G1/S arrest for 4 h then treated with 100 ng/mL nocodazole for 10 h. HRAS included as a PM-bound protein control. (D) Protein pulled down from extracts of HEK293T cells transfected with GST or GST-RIT1 constructs. Immunoblots were probed for endogenous MAD2 and p31comet. (E) HeLa cells stably expressing FLAG-RIT1 released from a G1/S phase arrest and lysed at indicated time points. Immunoprecipitated proteins were probed for RIT1 S209 phosphorylation by immunoblotting. Async., asynchronous growing cells; Noc., cells released from G1/S arrest for 4 h then treated with 100 ng/mL nocodazole for 10 h. (F) Detection of RIT1 S209 phosphorylation on bacterially expressed GST-RIT1 protein incubated with mitotic cell extract (MCE) treated with 1 mM Dinaciclib, 10 mM RO-3306, or DMSO control. (G) Immunoblot of RIT1 S209 phosphorylation on bacterially expressed RIT1 proteins subjected to an in vitro kinase assay with recombinant active CDK1/Cyclin B1. (H) MS quantification of phospho-S209 peptides in RIT1 protein incubated with CDK1/Cyclin B1 as in (G). n = 2, two-sided Student’s t test, data shown as mean, error bars indicate SD, *p % 0.05. See also Figure S2.

Article Snippet: Async., asynchronous growing cells; Noc., cells released from G1/S arrest for 4 h then treated with 100 ng/mL nocodazole for 10 h. (F) Detection of RIT1 S209 phosphorylation on bacterially expressed GST-RIT1 protein incubated with mitotic cell extract (MCE) treated with 1 mM Dinaciclib, 10 mM RO-3306, or DMSO control. (G) Immunoblot of RIT1 S209 phosphorylation on bacterially expressed RIT1 proteins subjected to an in vitro kinase assay with recombinant active CDK1/Cyclin B1. (H) MS quantification of phospho-S209 peptides in RIT1 protein incubated with CDK1/Cyclin B1 as in (G). n = 2, two-sided Student’s t test, data shown as mean, error bars indicate SD, *p % 0.05.

Techniques: Phospho-proteomics, Stable Transfection, Expressing, Clinical Proteomics, Membrane, Western Blot, Fractionation, Control, Transfection, Construct, Immunoprecipitation, Incubation, In Vitro, Kinase Assay, Recombinant

Journal: Current biology : CB

Article Title: The RAS GTPase RIT1 compromises mitotic fidelity through spindle assembly checkpoint suppression.

doi: 10.1016/j.cub.2021.06.030

Figure Lengend Snippet: Figure 3. RIT1 regulates timely anaphase entry and chromosome segregation fidelity (A) Comparison of mitotic transit times between WT and RIT1 knockout (KO) hTERT-RPE1 cells assessed by time-lapse microscopy. Time measured from nuclear envelope breakdown (NEBD). Rev, cells treated with 1 mM Reversine. WT (n = 119), KO (n = 122), KO + Rev (n = 66). (B) Quantification of lagging chromosomes in anaphase hTERT RPE1 cells. Data represent three independent replicates with three WT clones and three KO clones. Error bars indicate SD, ***p % 0.001. Scale bar, 15 mm. (C and D) Duration of mitotic length (NEBD anaphase onset) assessed by time-lapse microscopy in U2-OS cells stably expressing indicated proteins (C) in normal growth conditions (EV [n = 76], M90I [n = 66], and M90I-CAAX [n = 72]) or (D) treated with 15 ng/mL nocodazole (n = 75). Two-sided Student’s t test, error bars indicate SD, **p % 0.01, ***p % 0.001, ****p % 0.0001. (E) Immunoblots of GST-RGL3 RBD pull-down assay with lysates from U2OS cells stably expressing indicated RIT1 constructs. RGL3-RBD protein stained with Coomassie. (F) Duration of mitotic length (NEBD anaphase onset) assessed by time-lapse microscopy in U2-OS cells stably expressing indicated proteins in normal growth conditions: EV (n = 81), M90I (n = 68), M90I/S35N (n = 75), and M90I/Q79L (n = 77). Two-sided Student’s t test, error bars indicate SD, ****p % 0.0001. (G) Comparison of chromosome segregation error rates (lagging and bridging chromosomes) in HCT-116 cells stably expressing indicated constructs. EV, empty vector. Data represent three biologically independent repeats. Two-sided Student’s t test, error bars indicate SD, *p % 0.05, **p % 0.01, ***p % 0.001. (H) Metaphase spread assay compares frequency of aneuploidy, determined by a chromosome count other than the modal number, 45, in HCT-116 cells stably expressing EV (n = 92), S209S (n = 98), S209A (n = 97), or S209D (n = 88). For (A), (C), (D), and (F)–(H), n indicates the number of cells or metaphase spreads counted and data shown are representative of at least two biologically in- dependent experiments. See also Figure S3.

Article Snippet: Async., asynchronous growing cells; Noc., cells released from G1/S arrest for 4 h then treated with 100 ng/mL nocodazole for 10 h. (F) Detection of RIT1 S209 phosphorylation on bacterially expressed GST-RIT1 protein incubated with mitotic cell extract (MCE) treated with 1 mM Dinaciclib, 10 mM RO-3306, or DMSO control. (G) Immunoblot of RIT1 S209 phosphorylation on bacterially expressed RIT1 proteins subjected to an in vitro kinase assay with recombinant active CDK1/Cyclin B1. (H) MS quantification of phospho-S209 peptides in RIT1 protein incubated with CDK1/Cyclin B1 as in (G). n = 2, two-sided Student’s t test, data shown as mean, error bars indicate SD, *p % 0.05.

Techniques: Comparison, Knock-Out, Time-lapse Microscopy, Clone Assay, Stable Transfection, Expressing, Western Blot, Pull Down Assay, Construct, Staining, Plasmid Preparation

Journal: Current biology : CB

Article Title: The RAS GTPase RIT1 compromises mitotic fidelity through spindle assembly checkpoint suppression.

doi: 10.1016/j.cub.2021.06.030

Figure Lengend Snippet: Figure 4. RIT1 inhibits MCC-MAD2 association and promotes degradation of APC/C substrates (A and B) Immunoblots of precipitated proteins from equilibrium competition pull-down assays with 0.2 mM recombinant (A) GST-RIT1 or (B) GST-CDC20 111-138 protein incubated with 0.2 mM His6-MAD2 and titrating amounts of (A) MAD2 binding peptide 1 (MBP1) or a control peptide or (B) full-length RIT1 protein. (C) Pull-down assay with 0.5 mM recombinant GST or GST-MAD2 proteins incubated with 0.5 mM FLAG-RIT1 protein. Precipitated proteins were separated by SDS-PAGE for immunoblot or Coomassie staining. (D) Elution profile of MAD2 protein incubated with or without indicated peptides at 1:10 molar ratio for 1 h at 25C prior to gel filtration. The contents of 12 consecutive 50 mL fractions eluting between 1.2 and 1.8 mL are shown. (E) Immunoblots of anti-CDC20 immunoprecipitated proteins from cells expressing empty vector (EV) or RIT1M90I at indicated time points following nocodazole washout. Immunoprecipitated proteins probed from the same membrane.

Article Snippet: Async., asynchronous growing cells; Noc., cells released from G1/S arrest for 4 h then treated with 100 ng/mL nocodazole for 10 h. (F) Detection of RIT1 S209 phosphorylation on bacterially expressed GST-RIT1 protein incubated with mitotic cell extract (MCE) treated with 1 mM Dinaciclib, 10 mM RO-3306, or DMSO control. (G) Immunoblot of RIT1 S209 phosphorylation on bacterially expressed RIT1 proteins subjected to an in vitro kinase assay with recombinant active CDK1/Cyclin B1. (H) MS quantification of phospho-S209 peptides in RIT1 protein incubated with CDK1/Cyclin B1 as in (G). n = 2, two-sided Student’s t test, data shown as mean, error bars indicate SD, *p % 0.05.

Techniques: Western Blot, Recombinant, Incubation, Binding Assay, Control, Pull Down Assay, SDS Page, Staining, Immunoprecipitation, Expressing, Plasmid Preparation, Membrane

Journal: Brain Pathology

Article Title: Brain heterotopia formation by ciliopathic breakdown of neuroepithelial and blood‐cerebrospinal fluid barriers

doi: 10.1111/bpa.13148

Figure Lengend Snippet: Forebrain heterotopia development upon inhibition of Ift88 or Kif3a in Sox10‐Cre‐lineage cells. (A) To inhibit ciliogenesis in gliogenic RG cells, Kif3a‐flox and Ift88‐flox conditional mice were crossed with Sox10‐Cre mice to obtain conditional primary cilia mutants. Disorganized subpallial ventricular and subventricular zones were visualized via Ki67 and Olig2 staining at P1. In heterozygous control mice, Olig2+ (green) oligogenic progenitors were localized in the subventricular zone away from the Ki67+ (red) ventricular zone. The mutant subpallium shows ventricular Olig2+ cells and dislocated Ki67+ cells (*). (B) Blbp staining showing disruption of the ventricular RG lining in the mutants due to intrusion of Ctip2+ neurons (*). (C) In situ hybridization of Hes5 showing the delaminating RG cells in the mutants (*). (D) Tbr2 staining showing the disorganized subventricular zone and delaminated Tbr2+ intermediate progenitors (*). (E) Sagittal sections showing disruption of the ventricular Blbp+ glial lining, rosette formation in the subpallium (arrows), and heterotopia development in the cortex (*). The left panel is lateral to the right panel (medial). Scale bars = 100 μm

Article Snippet: The primary antibodies used for immunostaining included chicken anti‐GFP (Aves Labs, USA, 1:1000), rabbit anti‐GFP (1:1000, Invitrogen, USA), rat anti‐Pdgfrα (1:300, Millipore) rabbit anti‐ACIII (1:1000, Santa Cruz Biotechnology, USA), rabbit anti‐Blbp (Chemicon, USA, 1:500; Abcam, 1:300), mouse anti‐RC2 (DSHB, USA, 1:200), rat anti‐Ctip2 (Abcam, 1:1000), rabbit anti‐Ki67 (Lab Vision, USA, 1:200), rabbit anti‐Wnt3a (Cell Signaling Technology, USA, 1:1000), rabbit anti‐Pdgf‐A (Abbiotec, USA, 1:1000), rabbit anti‐Tbr2 (Abcam, USA, 1:300), rabbit anti‐Claudin‐5 (United States Biological, USA, 1:300), rabbit anti‐Par3 (Millipore, USA, 1:300), rabbit anti‐Notch1 (Cell Signaling Technology, USA, 1:400), Alexa Fluor 546‐conjugated streptavidin, TRITC‐conjugated phalloidin (Invitrogen, USA, 1:1000), mouse anti‐aTub (Sigma, USA, 1:1000), rabbit anti‐β‐Catenin (Cell Signaling Technology, USA, 1:500), rabbit anti‐Pax6 (Covance, USA, 1:300), rabbit phospho‐Histone H3 (Santa Cruz Biotechnology, USA, 1:1000), rabbit anti‐Pdgfrβ (Cell Signaling Technology, USA, 1:300), biotin‐conjugated 70 kDa dextran (Invitrogen, USA), biotin‐conjugated cadaverine (Invitrogen, USA), mouse anti‐Desmin (DAKO, Denmark, 1:1000), rat anti‐ICAM1 (Abcam, USA, 1:500) and rabbit anti‐Olig2 (Millipore, USA, 1:300).

Techniques: Inhibition, Staining, Mutagenesis, In Situ Hybridization

Journal: Brain Pathology

Article Title: Brain heterotopia formation by ciliopathic breakdown of neuroepithelial and blood‐cerebrospinal fluid barriers

doi: 10.1111/bpa.13148

Figure Lengend Snippet: Kif3a inhibition in CNP‐Cre‐lineage cells did not disrupt the neuroepithelial lining, but Smo inhibition in a Sox10‐Cre mouse line compromised the development of the choroid plexus. (A) Inhibition of Smo expression using Sox10‐Cre (Sox10‐Cre;Smo‐flox/flox) resulted in the expansion of the ventricular zone, as indicated by Ki67 staining (white lines). Olig2+ OPCs were also observed at the apical ventricular surface as well as in the subventricular zone in the mutants (arrows). (A′) The length of the choroid plexus was measured using isolated choroid plexus tissues from the lateral ventricle by spreading tissues on a cover glass at P1. The length of the choroid plexus from Sox10‐Cre;Smo‐flox/flox was normalized with the length of the choroid plexus from Sox10‐Cre;Smo‐flox/+. * p < 0.02 ( n = 3). (B) (a) Mice with OPC‐specific inhibition of primary ciliogenesis (CNP1‐Cre;Kif3a‐flox/flox) showed normal ventricular zones. (b) Mice with OPC‐specific inhibition of primary ciliogenesis showed normal Tbr2+ subventricular zones. (c) Mice with OPC‐specific inhibition of primary ciliogenesis showed normal distributions of Olig2+ OPCs and did not show Ctip2+ heterotopia. (d) Mice with OPC‐specific inhibition of primary ciliogenesis showed normal distributions of Pdgfrα+ OPCs. (B′) The distribution of Olig2+ cells was not significantly different when assessed in bins created from the pia to the lateral ventricle (A‐B‐C‐D). Scale bars = 100 μm except in B‐e (25 μm)

Article Snippet: The primary antibodies used for immunostaining included chicken anti‐GFP (Aves Labs, USA, 1:1000), rabbit anti‐GFP (1:1000, Invitrogen, USA), rat anti‐Pdgfrα (1:300, Millipore) rabbit anti‐ACIII (1:1000, Santa Cruz Biotechnology, USA), rabbit anti‐Blbp (Chemicon, USA, 1:500; Abcam, 1:300), mouse anti‐RC2 (DSHB, USA, 1:200), rat anti‐Ctip2 (Abcam, 1:1000), rabbit anti‐Ki67 (Lab Vision, USA, 1:200), rabbit anti‐Wnt3a (Cell Signaling Technology, USA, 1:1000), rabbit anti‐Pdgf‐A (Abbiotec, USA, 1:1000), rabbit anti‐Tbr2 (Abcam, USA, 1:300), rabbit anti‐Claudin‐5 (United States Biological, USA, 1:300), rabbit anti‐Par3 (Millipore, USA, 1:300), rabbit anti‐Notch1 (Cell Signaling Technology, USA, 1:400), Alexa Fluor 546‐conjugated streptavidin, TRITC‐conjugated phalloidin (Invitrogen, USA, 1:1000), mouse anti‐aTub (Sigma, USA, 1:1000), rabbit anti‐β‐Catenin (Cell Signaling Technology, USA, 1:500), rabbit anti‐Pax6 (Covance, USA, 1:300), rabbit phospho‐Histone H3 (Santa Cruz Biotechnology, USA, 1:1000), rabbit anti‐Pdgfrβ (Cell Signaling Technology, USA, 1:300), biotin‐conjugated 70 kDa dextran (Invitrogen, USA), biotin‐conjugated cadaverine (Invitrogen, USA), mouse anti‐Desmin (DAKO, Denmark, 1:1000), rat anti‐ICAM1 (Abcam, USA, 1:500) and rabbit anti‐Olig2 (Millipore, USA, 1:300).

Techniques: Inhibition, Expressing, Staining, Isolation

Journal: bioRxiv

Article Title: Robotic High-Throughput Biomanufacturing and Functional Differentiation of Human Pluripotent Stem Cells

doi: 10.1101/2020.08.03.235242

Figure Lengend Snippet: (A) Overview of neuronal differentiation strategy compatible with automation. (B) Phase-contrast image showing a typical neuronal culture (day 30; magnification 20x). (C) Neuronal cells develop highly dense network of neurites upon further maturation (day 50; magnification 40x). (D) Immunocytochemical analysis showing cortical neurons expressing TUJ1 and CUX1 (magnification, 20x). (E) Immunocytochemical analysis demonstrating the presence of cortical neurons expressing MAP2 and CTIP2 (magnification, 20x). (F) Majority of cells express vGLUT1, a marker for excitatory neurons (magnification, 20x). (G) Example of neuronal cells showing immunoreactivity for the inhibitory neurotransmitter GABA (magnification, 63x) (H) Robotic MEA platform used for high-throughput electrophysiology and functional cell characterization. (I) Spontaneous activity of hiPSC-derived neurons after 6 weeks of differentiation as measured by MEA. (J) Overlay plot of 10 spikes detected from one channel of a representative MEA recording to demonstrate similarity between spikes detected.

Article Snippet: Primary antibodies used are as follows; OCT4 (Santa Cruz, Sc9801, 1:200), Brachyury (Cell Signaling Technology, 81694, 1:1000), SOX17 (Cell Signaling Technology, 81778, 1:1000), PAX6 (Biolegend, 901301, 1:200), TNNI3 (R&D Systems, MAB8594, 1:100), NKX2.5 (Sigma, SAB1408911, 15 μ g/ml), Flavivirus Group Antigen (Millipore Sigma, MAB10216, 1:200), GABA (Sigma, A2052, 1:1000), vGLUT1 (Sigma, AMAb91041, 1:5000), CUX2 (Abcam, ab130395, 1:500), TUBB3/TUJ1 (Biolegend, 801201, 1:1000), CTIP2 (Novus, NBP2–61702, 1:250), MAP2 (Thermo Fisher Scientific, PA5–17646, 1:500), HNF4a (Santa Cruz, sc-6556, 1:250), AFP (Sigma, A8452, 1:1000), FOXA2 (BD, 561580, 1:200), Albumin (Cedarlane, CL2513A, 1:250) and DAPI (1:1000).

Techniques: Expressing, Marker, High Throughput Screening Assay, Functional Assay, Activity Assay, Derivative Assay

Journal: Journal of Neuroscience

Article Title: Neuronal Basic Helix-Loop-Helix Proteins Neurod2/6 Regulate Cortical Commissure Formation before Midline Interactions

doi: 10.1523/jneurosci.0899-12.2013

Figure Lengend Snippet: Figure3. Projectionneuronsadoptcorticallayer-specificfatesinNeurod2/6doublemutants.A,FluorescentimmunostainingforNestin(blue),Tbr2(green),andCtip2(red)atE13demonstrates normalCPformationandradialmigrationofearly-borndeeperlayerprojectionneurons.B,ImmunostainingforNestin(blue)andBrn2(green)atE16revealsnormalradialmigrationofupperlayer neurons.C,DisturbedsegregationofupperanddeeperlayersatP1asdemonstratedbyimmunostainingforSatb2(green)andCtip2(red).D,CorticaldistributionofSox5-,Ctip2-,andSatb2-positive neuronsincontrols(n5mice)andNeurod2/6doublemutants(n4).Thehistogramsshowabsolutecellnumbersin10equallysizedbinsasdenotedinC.pvalues(two-sidedttest)aredisplayed as *p 0.05, **p 0.01, and ***p 0.001. For regression curves, cells were counted in 50 equally sized bins. E–G, Expression analysis of upper layer-enriched markers by chromogenic immunostaining for Lmo4 (E), Brn2 (F), and in situ hybridization for Cux2 mRNA (G). Coronal (A–C, F, G), horizontal (D), and sagittal (E) paraffin sections (5 m) from control and Neurod2/6 double-mutant brains. 2–6, Cortical layers; MZ, marginal zone.

Article Snippet: Primary antibodies were directed against Brn2 (1:200, polyclonal goat; Santa Cruz Biotechnology), CNPase (1:150, mouse IgG; Sigma), Cntn2 (4D7/TAG1, 1:100, mouse IgM; Developmental Studies Hybridoma Bank), Ctip2 (1:500, rat IgG; Abcam), FNP7 (1:100, mouse IgG; Zymed), Gfap (1:200, polyclonal rabbit; Novocastra), GFP (1:500, polyclonal goat; Rockland), HuC/D (1:250, mouse IgG; Invitrogen), L1cam (1:500, polyclonal rat; Millipore Bioscience Research Reagents), Lmo4 (1:500, rat; J. E. Visvader, University of Sydney, Sydney, Australia), Map2 (1:200, mouse IgG; Millipore Bioscience Research Reagents), Nestin (1:100, mouse IgG; Millipore Bioscience Research Reagents), NeuN (1:200, mouse IgG; Millipore Bioscience Research Reagents), NR1 (1:100, polyclonal rabbit; Abcam), Robo1 (1:2000, polyclonal rabbit; F. Murakami), Satb2 (1:1000; V. Tarabykin), Sox5 (1:200, polyclonal goat; R & D Systems), Tbr2 (1: 200, polyclonal rabbit; Abcam), or Vglut1 (1:400, polyclonal rabbit; Synaptic Systems).

Techniques: Expressing, Immunostaining, In Situ Hybridization, Control, Mutagenesis