Journal: bioRxiv

Article Title: Deletion of TRPC6 , an autism risk gene, induces hyperexcitability in cortical neurons derived from human pluripotent stem cells

doi: 10.1101/2022.11.14.516407

Figure Lengend Snippet: ( a ) Schematic of hPSC differentiation into NPCs and mature cortical neurons. The arrows indicate the time point of splitting the cells. ( b ) Immunostaining of control and TRPC6 KO cortical neurons (from two different hPSC clones, C21 and C47) differentiated for 8 weeks. Cortical neuron markers; CTIP2, SATB2, TBR2, and BRN2. MAP2, BTUB; pan-neuronal markers. ( c ) GFAP, a marker for glia. vGLUT1, a marker for glutamatergic excitatory synapses. ( d ) Synaptophysin (Syn), a synaptic vesicle protein. Nuclei are stained with DAPI. ( e ) Validation of TRPC6 protein expression in TRPC6 KO cortical neurons differentiated for 8 weeks. Scale, 100 μm.

Article Snippet: Primary antibodies consisted of SOX2 (Rabbit, 1:200, Invitrogen: MA1-014), FOXG1 (Rabbit, 1:200, Abcam: ab18259), OTX2 (Goat, 1:300, R&D: AF1979), Nestin (Mouse, 1:100, Invitrogen: MA1110, MAP2 (chicken, 1:500, Abcam: ab5392), MAP2 (Mouse, 1:500, Invitrogen: 13-1500), beta tubulin III (Mouse, 1:300, MAB1637), TBR2 (Rabbit,1:300, Cell signaling: 66325), CTIP2 (Rabbit, 1:200, Cell signaling:), BRN2 (Rabbit, 1:200, Cell signaling:12137), GFAP (Chicken, 1:400, Abcam: ab4674), and SATB2 (Rabbit, 1:200, Invitrogen:PA5-83092).

Techniques: Immunostaining, Clone Assay, Marker, Staining, Expressing

Journal: bioRxiv

Article Title: iPSC Motor Neurons with Familial ALS Mutations Capture Gene Expression Changes in Postmortem Sporadic ALS Motor Neurons

doi: 10.1101/2022.10.25.513780

Figure Lengend Snippet: Differentiation of SMNs, cortical neurons, and sensory neurons using inducible transcription factors. (A) Distinct tet-inducible transcription factors contained within PiggyBac constructs were nucleofected into iPSCs to generate different cell types. Timeline summarizes the differentiation protocol. (B) Masking of neurons using BFP2 and TUJ1 staining. Darker, lighter, and gray colors indicate nuclei, cytoplasm, and neurites, respectively. (C) Cyclic immunofluorescence of SMNs for HB9, NEUN, ISL1, TUJ1, and two different CHAT antibodies; quantified in (D). “Combined” indicates the percentage of cells positive for all markers. (E) Cortical neurons stained for BRN2, FOXG1, and TUJ1; quantified (F). (G) Sensory neurons stained for BRN3A, ISL1, and TUJ1; quantified in (H). Scale bars = 100uM; n=8 wells for all groups. Across panels, arrows indicate the same neuron, which is magnified in the inset.

Article Snippet: The primary antibodies used in this study were: anti-Brn2 (1:1000, Cell Signaling 12137S), anti-FoxG1 (1:500, Millipore Sigma MABD79), anti-Tuj1 (1:500, Aves TUJ-0020), anti-Brn3a (1:200, Millipore MAB1585), anti-Isl1 (1:200, Abcam ab203406), anti-GFAP (1:250, abcam ab194324), anti-S100β (1:200, abcam ab52642), and anti-TDP-43 (1:500, proteintech 10782-2-AP).

Techniques: Construct, Staining, Immunofluorescence

Journal: Frontiers in Integrative Neuroscience

Article Title: Single Extracellular Vesicle Analysis Using Flow Cytometry for Neurological Disorder Biomarkers

doi: 10.3389/fnint.2022.879832

Figure Lengend Snippet: NCAM as a neuronal exosome marker in plasma. (A–C) Quantitative analysis of plasma EVs using flow cytometry. Dot plots of fluorescent intensity for plasma EVs stained with PE-CY7-labeled CD63 antibody. EVs were unstained (A) and stained with either isotype control (B) or CD63 antibody (C) . (D) A18945 iPSC-derived neurons cultured for 6 weeks and immunostaining with mature neuronal markers (DAPI, MAP2, NeuN, and merged image). Images were taken using Zeiss LSM confocal microscope at ×63 magnification. Scale bar, 20 μm. (E) Representative images of cortical organoids at day 90 of differentiation. Scale bar, 1 mm. (F) The organoids express cortical layer marker BRN2 (also called POU3F2). The nuclei were stained with DAPI. Scale bar, 100 μm. (G i ) Markers for proliferating neural progenitors (SOX2) and cortical neuron marker CTIP2 (also known as BCL11B) with nuclei DAPI staining (G ii ) . (H) Western blot analysis of NCAM and CD63 from EVs released from iPSCs, iPSC-derived cortical neurons, and iPSC-derived brain organoids. EVs were isolated using SEC from the cell culture media of each sample, and equal EV particle numbers (6 × 10 8 ) were subject to immunoblotting with NCAM and CD63 antibodies. (I–L) Flow cytometry dot plots of fluorescent intensity for plasma EVs double-stained with DiI and NCAM antibody. EV samples were unstained (I) , single stained with DiI (J) , and double-stained with DiI and NCAM antibody in the absence (K) and presence (L) of 0.1% Triton X-100.

Article Snippet: For immunostaining, the freezing medium was washed with PBS, and tissues were permeabilized with 0.3% Triton-X in PBS for 40 min. Tissues were blocked in 3% BSA in PBS containing 0.2% Tween-20 (PBST) for 2 h. Primary antibodies BRN2 (1:200, cell signaling, 12137), CTIP2 (1:200, cell signaling, 12120), and SOX2 (1:300, Invitrogen, MA1-014) were diluted in blocking solution and added to the tissue overnight at 4°C.

Techniques: Marker, Flow Cytometry, Staining, Labeling, Derivative Assay, Cell Culture, Immunostaining, Microscopy, Western Blot, Isolation

Journal: Nature Communications

Article Title: The long noncoding RNA H19 regulates tumor plasticity in neuroendocrine prostate cancer

doi: 10.1038/s41467-021-26901-9

Figure Lengend Snippet: A Nuclear localization of H19 in NCI-H660. y -Axis represents the percent abundance of RNA. Nuclear U1 RNA was used as a control. B WB of NE associated genes (SOX2, CHGA, BRN2, and EZH2), H3K27me3, and H3K4me3 in various AdPC and NEPC cell lines and organoids. C WB of H3K27me3, H3K4me3 in CRPC cell line V16D CRPC and AdPC cell line LNCaP after transient overexpression of H19 . The bar graph shows the relative H19 RNA levels in both the cell lines upon H19 overexpression. 18S was used as an endogenous control. D WB analysis of the levels of EZH2 (Actin as control) and histone H3K27me3 level (Histone H3 as control) in Control (Lv-Scr) and H19 knockdown (Lv-shH19) OWCM-155 NEPC organoids. Numerical values shown under the blot are calculated relative to the control samples. E Relative enrichment of H19 binding to PRC2 complex members EZH2, SUZ12 in LASCPC-01, LNCaP, and V16D CRPC cells with transient overexpression of control (EV) and H19 (H19). F WB analysis of LNCaP cells stably expressing doxycycline (DOX) inducible H19 FL (full-length H19 ) or H19 DEL (5′ deleted H19 fragment) with or without DOX treatment (200 ng/mL, 48 h). Actin was used as a control. Data are mean ± SD ( A , C ), or mean ± SEM ( E ); n = 3 ( A , C , E ) biologically independent replicates. p Values were calculated by unpaired two-tailed Student’s t test.

Article Snippet: Antibodies used: SOX2 (sc-365823, Santa Cruz Biotechnology, 1:400 dilution), H3K27me3 (9733S, Cell Signaling Technology, 1:1000 dilution), EZH2 ((D2C9) XP Rabbit mAb (5246, Cell Signaling Technology, 1:1000 dilution), NSE (sc-21738, Santa Cruz Biotechnology, 1:500 dilution), Synaptophysin (sc-365488, Santa Cruz Biotechnology, 1:500 dilution), CHGA (60893S, Cell Signaling Technology, 1:1000 dilution), BRN2 ((D2C1L) Rabbit mAb 12137, Cell Signaling Technology, 1:1000 dilution), H3 ((D1H2) XP ® Rabbit mAb 4499, Cell Signaling Technology, 1:1000 dilution), Androgen receptor (5153S, Cell Signaling Technology, 1:1000 dilution), H3K4me3 (ab8580, abcam, 1:1000 dilution), P53 (2527 S, Cell Signaling Technology, 1:1000 dilution), Rb (9313S, Cell Signaling Technology, 1:1000 dilution), CK8 (sc–57004, Santa Cruz Biotechnology, 1:300 dilution), PSA (sc-7316, Santa Cruz Biotechnology, 1:250 dilution), HRP conjugated anti-β-actin (Cat. no. A3854, Sigma, 1:10,000 dilution).

Techniques: Over Expression, Binding Assay, Stable Transfection, Expressing, Two Tailed Test

Journal: Nature Communications

Article Title: The long noncoding RNA H19 regulates tumor plasticity in neuroendocrine prostate cancer

doi: 10.1038/s41467-021-26901-9

Figure Lengend Snippet: A Assessment of sensitivity and specificity of H19 in NEPC for use as a diagnostic along with other recently identified NEPC oncogenes (BRN2, SRRM4, and PEG10). Arrows denote patients that are negative for CHGA (black) or CHGA, SYP, and NSE (red) yet positive for H19 . B , C , Kaplan–Meir estimates for the impact of H19 on prostate cancer clinical outcomes. B Survival analysis for H19 in ADT untreated patients and C ADT treated patients for biochemical recurrence (BCR—left panels) and metastasis (MET—right panels). Samples were split by H19 tertile expression to test if either of these subgroups of patients were more likely to develop BCR and MET in the context of treatment. P values were calculated using a Kaplan–Meier estimator statistical test to determine significance for the observed stratification in probabilities across the three subgroups in each panel. D , Illustration of H19 transcription and mechanism of action during PCa, NEtD, and tNEPC. During early stage disease, adenocarcinoma cancer cells are driven by an active AR bound to androgens that prevent the transcription of H19 . As the disease progresses (gray triangle), ADTs suppress AR activity, SOX2 increases, and results in the persistent transcription, expression, and activation of H19 . Once active, H19 operates via dual epigenetic mechanisms; (on the left) binds PRC2 complex members, aiding in altering methylation on H3K4Me3/H3K27Me3 histone marks of PRC2 target genes and (on the right) genome-wide alteration of methylation at CpG sites on DNA. Collectively, these epigenetic changes result in the activation of NE gene expression and deactivation of AR signaling gene expression.

Article Snippet: Antibodies used: SOX2 (sc-365823, Santa Cruz Biotechnology, 1:400 dilution), H3K27me3 (9733S, Cell Signaling Technology, 1:1000 dilution), EZH2 ((D2C9) XP Rabbit mAb (5246, Cell Signaling Technology, 1:1000 dilution), NSE (sc-21738, Santa Cruz Biotechnology, 1:500 dilution), Synaptophysin (sc-365488, Santa Cruz Biotechnology, 1:500 dilution), CHGA (60893S, Cell Signaling Technology, 1:1000 dilution), BRN2 ((D2C1L) Rabbit mAb 12137, Cell Signaling Technology, 1:1000 dilution), H3 ((D1H2) XP ® Rabbit mAb 4499, Cell Signaling Technology, 1:1000 dilution), Androgen receptor (5153S, Cell Signaling Technology, 1:1000 dilution), H3K4me3 (ab8580, abcam, 1:1000 dilution), P53 (2527 S, Cell Signaling Technology, 1:1000 dilution), Rb (9313S, Cell Signaling Technology, 1:1000 dilution), CK8 (sc–57004, Santa Cruz Biotechnology, 1:300 dilution), PSA (sc-7316, Santa Cruz Biotechnology, 1:250 dilution), HRP conjugated anti-β-actin (Cat. no. A3854, Sigma, 1:10,000 dilution).

Techniques: Diagnostic Assay, Expressing, Activity Assay, Activation Assay, Methylation, Genome Wide

Journal: iScience

Article Title: Podoplanin drives dedifferentiation and amoeboid invasion of melanoma

doi: 10.1016/j.isci.2021.102976

Figure Lengend Snippet: Loss of podoplanin restores pigmentation and melanocyte differentiation (A and B) Tumors (A) and cells pellets (B) of PDPN + and PDPN KO B16F10 cell lines. (C) Imaging of pigmentation (brightfield; left) of PDPN + and PDPN KO B16F10 cell lines, labeled with mOrange (red) or CFP (blue) respectively (middle). The scale bar represents 50 microns. (D) Heatmaps showing expression ( Z score) of podoplanin ( PDPN ) and eight dedifferentiation-associated genes in datasets of primary tumor samples of melanoma patients (top; Riker et al. GSE7553 ) and metastatic melanoma cultures (bottom; Mannheim cohort, GSE4843 ; from Hoek et al., 2006 ). For the Mannheim cohort, each number indicates a separate metastatic melanoma culture, and the BRAF and/or NRAS mutation status is indicated for each. (E) mRNA expression of five melanocyte-associated ( Mlana/Mart-1 (p = 0.2045), Tyr (p = 0.9276), Dct (p = 0.9859), Gpnmb (p = 0.1308), Pmel (p = 0.9994); left) and three invasion-associated ( Kit (p > 0.9999), Mitf (∗∗∗∗p < 0.0001), Pou3f2 (encodes Brn2; ∗∗∗∗p < 0.0001; right) genes in PDPN + B16F10 cells. mRNA expression is calculated as fold change of Gapdh expression and normalized to expression in PDPN KO B16F10 cells (set at 1 as indicated by dashed line). Data shown as mean with dots representing n = 3 biological replicates. Two-way ANOVA with Sidak's multiple comparisons; p values indicated above. (F) Western blot analysis of Podoplanin, Melan-A, Tyrosinase and Brn2 (encoded by Pou3f2 gene) in PDPN KO and PDPN + B16F10 cells. Tubulin and histone H3 are used as loading controls.

Article Snippet: Western blots were incubated with rabbit anti-Phospho-Myosin Light Chain 2 (Thr18/Ser19) (ppMLC; 1:750, Cell Signaling Technology, 3674), rabbit anti-Myosin Light Chain 2 (MLC; 1:750, Cell Signaling Technology, 3672), rabbit anti-MelanA (EPR20380, 1:2000, Abcam, ab210546), mouse anti-human Tyrosinase (T311, 1:100, Santa Cruz, sc-20035), rabbit anti-Brn2/POU3F2 (D2CIL, 1:1000, Cell Signaling Technology, 12137), rabbit anti-podoplanin (1:1000, iSpyBio, ABT34), or as loading controls mouse anti-Tubulin (1:20,000, Sigma Aldrich, T7816), mouse anti-GAPDH (1:10,000, clone 6C5, Millipore, MAB374) or mouse anti-Histone H3 (1:2500, Abcam, ab24824), in PBS supplemented with 1% skim milk powder and 0.2% BSA overnight at 4°C, followed by staining with appropriate HRP-conjugated secondary antibodies (Abcam) for 2 hours at RT.

Techniques: Imaging, Labeling, Expressing, Mutagenesis, Western Blot