rabbit anti pard3  (Proteintech)

Journal: bioRxiv

Article Title: NDR1/2 kinases regulate cell polarization and cell motility through Cdc42 GTPase and Pard3 signaling in mammalian cells

doi: 10.1101/2025.10.30.685405

Figure Lengend Snippet: A-C Fibroblasts were fixed 10 h after wound scratch and subjected to immunofluorescence analysis. Pard3 was visualized with Alexa Fluor 647 (A). Junctional organization was quantified by lacunarity (B) and average puncta size (C)., ****P < 0. 0001, ***P < 0.001, ** P < 0.01, One-Way ANOVA followed by Bonferroni’s test vs. Scr-shRNA; n = 4. (D-E) Pard3 protein expression levels were analyzed by western blot, ***P < 0.001, ** P < 0.01, One-Way ANOVA followed by Bonferroni’s test vs. Scr-shRNA; n = 5. (F) Co-localization of Pard3 and the junctional protein ZO-1 was assessed by co-immunofluorescence staining. Pard3 was visualized with Alexa Fluor 647 (red), ZO-1 with Alexa Fluor 488 (green), and nuclei with DAPI(Blue). (G) Schematic of the pLV-Pard3-GFP lentiviral construct driven by the Ubc promoter. Construct integrity was confirmed by sequencing and western blot analysis. (H) Fibroblasts with shRNA-mediated knockdown of NDR1/2 were infected with Pard3- GFP–expressing lentivirus and enriched by FACS. Pard3 localization was visualized by GFP in both live-cell and fixed-cell imaging. The white dashed line indicates the scratch wound edge. Scale bars in (A, F, H): 20 µm.

Article Snippet: After blocking, the slides were incubated with primary antibodies overnight 4°C with antibodies including mouse anti-Vinculin (1: 300, Sigma, V9131), rabbit anti-GM130 (1:1000, CST, 12480T), rabbit anti- Pard3(1:400, Proteintech, 11085-1-AP), mouse anti-Zo-1 (1:500, Proteintech, 66452-1-Ig).

Techniques: Immunofluorescence, shRNA, Expressing, Western Blot, Staining, Construct, Sequencing, Knockdown, Infection, Imaging

Journal: bioRxiv

Article Title: NDR1/2 kinases regulate cell polarization and cell motility through Cdc42 GTPase and Pard3 signaling in mammalian cells

doi: 10.1101/2025.10.30.685405

Figure Lengend Snippet: (A) Schematic of Pard3 (top) highlighting the consensus NDR kinase phosphorylation motif (H.R..[S/T]); The N- terminal region of Pard3 (tagged with Myc at the N-terminus and 6×His at the C- terminus) contains the consensus phosphorylation site at Ser144. Wild-type (WT) and S144A mutant constructs were cloned into the pET22b backbone for inducible expression in E. coli DE3 cells (bottom). (B) Fibroblasts stably expressing lentivirus encoding GFP alone were subjected to wound-healing assays as controls; ****P < 0.0001; Two-Way ANOVA followed by Tukey’s post hoc tests, n = 40. (C-F) Validation of Pard3 phosphorylation at Ser144 by in vitro kinase assays. NDR1 and NDR2 (WT or kinase-dead [KD], , K118A for NDR1 and K119A for NDR2) were purified from HEK293T cells and incubated with purified WT N-Pard3 or S144A-mutant N-Pard3 protein. Reactions were performed in the presence of ATP-γ-S, and thiophosphorylation was detected by western blot using an anti–thiophosphate ester antibody, ***P < 0.001, **P < 0.01; One-Way ANOVA followed by Dunnett’s post hoc tests, n = 3. (G-H) Rescue wound-healing assays were performed to evaluate the effect of exogenous Pard3 or the Pard3-S144A mutant on fibroblast migration following NDR1/2 knockdown over 20 h, ****P < 0.0001, ***P < 0.001, *P < 0.05; Two-Way ANOVA followed by Tukey’s post hoc tests, n = 40-46.

Article Snippet: After blocking, the slides were incubated with primary antibodies overnight 4°C with antibodies including mouse anti-Vinculin (1: 300, Sigma, V9131), rabbit anti-GM130 (1:1000, CST, 12480T), rabbit anti- Pard3(1:400, Proteintech, 11085-1-AP), mouse anti-Zo-1 (1:500, Proteintech, 66452-1-Ig).

Techniques: Phospho-proteomics, Mutagenesis, Construct, Clone Assay, Expressing, Stable Transfection, Biomarker Discovery, In Vitro, Purification, Incubation, Western Blot, Migration, Knockdown

Journal: Frontiers in Cell and Developmental Biology

Article Title: Nox1-based NADPH oxidase regulates the Par protein complex activity to control cell polarization

doi: 10.3389/fcell.2023.1231489

Figure Lengend Snippet: Antibodies used in this manuscript.

Article Snippet: Rabbit polyclonal anti-Par3 antibodies were used for immunoprecipitation (Proteintech) and immunofluorescence (Millipore).

Techniques: Control

Journal: Frontiers in Cell and Developmental Biology

Article Title: Nox1-based NADPH oxidase regulates the Par protein complex activity to control cell polarization

doi: 10.3389/fcell.2023.1231489

Figure Lengend Snippet: Nox1 controls Par3 activity. (A) Rac activity was determined using GLISA assay in WT and Nox1 y/− cells after PDGF-BB stimulation for the indicated times. Differences between genotypes was analyzed with one-way ANOVA (* p < 0.05, ** p < 0.01, n = 4). (B) WT and Nox1 y/- MEFs, were seeded on collagen-I-coated coverslips, serum starved for 16 h, and stimulated with 10 ng/mL PDGF-BB for 30 min. Cells were fixed and incubated with GST-Rac1G15A and then immunostained for GST and Tiam. Tiam activation depended on Nox1 and was tracked by colocalization of GST, and Tiam signals at the membrane of lamellipodia and protrusions. Look up table (LUT) panels correspond to the magnification of the lamellipodia area shown on merge images (white boxes). Scale bar = 10 μm (C) Quantification of colocalization between Tiam and GST-Rac1G15A signal. The graph shows Pearson’s R values at the lamellipodium area for WT and protrusion area for Nox1 y/- cells. Differences were evaluated with an unpaired t-test (**** p < 0.001, n = 3, and 5-7 cells per condition in each independent experiment). (D) MEF cells were serum starved for 16 h and stimulated with 10 ng/mL of PDGF-BB for 3 min. Phosphorylated proteins were isolated by affinity chromatography and analyzed by immunoblot using an antibody against Par3. Cortactin and actin were used as controls. Graph shows densitometric analysis of observed levels of phosphoproteins for the 180, 150, and 100 KDa bands corresponding to different Par3 isoforms from three independent experiments. Statistical significance was evaluated with a two-way ANOVA (* p < 0.05, n = 3). (E) Cells were serum starved for 16 h and stimulated for 15 and 30 min with 10 ng/mL PDGF-BB. Cell lysates were analyzed for immunoblot for pThr410/403-PKCζ/λ corresponding to the activation loop of atypical PKCs. Graphs show the densitometric analysis of four independent experiments. Statistical significance was evaluated with a two-way ANOVA (* p < 0.02, p **<0.003, n = 4).

Article Snippet: Rabbit polyclonal anti-Par3 antibodies were used for immunoprecipitation (Proteintech) and immunofluorescence (Millipore).

Techniques: Activity Assay, Incubation, Activation Assay, Membrane, Isolation, Affinity Chromatography, Western Blot

Journal: Frontiers in Cell and Developmental Biology

Article Title: Nox1-based NADPH oxidase regulates the Par protein complex activity to control cell polarization

doi: 10.3389/fcell.2023.1231489

Figure Lengend Snippet: Nox1 regulates polarity through PP2A phosphatase activity. (A) Cells were serum starved for 16 h and stimulated for 15 and 30 min with 10 ng/mL PDGF-BB. Cell lysates were analyzed for immunoblot for the i nactivating-phosphorylation of PP2A-CA using an antibody against pTyr307. Graphs show the densitometric analysis of 4 independent experiments. Data were analyzed with a two-way ANOVA (* p < 0.05, ** p < 0.01, **** p < 0.0001, n = 4). (B) Cells were co-transfected with siGlo and siRNA Control (CTRL) or siRNA against PP2A-CA (sequence #4 or #7). Representative images of morphology observed for WT and Nox1 y/- cells. Arrows show cells with polar shapes (single lamellipodium), and stars show cells with multiple lamellipodia. Scale bar = 10 μm (C) . Efficiency of knockdown using the siRNA Control (CTRL) or siRNA against Par3 (sequence #4 or #7). (D) Quantification of cells that showed a polarized shape, including a single lamellipodium. Statistical differences were determined with two-way ANOVA from three independent experiments (n = 3). Each group was compared (n = 3, 200 cells, **** p < 0.001). (E) Quantification of cells with single lamellipodium (black bars), multiple lamellipodia (white bars), or no lamellipodia (grey bars). Data were analyzed with a one-way ANOVA within each phenotype. Each group was compared with the control sample in basal (WT) (n = 3, 200 cells; *** p < 0.001).

Article Snippet: Rabbit polyclonal anti-Par3 antibodies were used for immunoprecipitation (Proteintech) and immunofluorescence (Millipore).

Techniques: Activity Assay, Western Blot, Phospho-proteomics, Transfection, Control, Sequencing, Knockdown

Journal: Frontiers in Cell and Developmental Biology

Article Title: Nox1-based NADPH oxidase regulates the Par protein complex activity to control cell polarization

doi: 10.3389/fcell.2023.1231489

Figure Lengend Snippet: PP2A controls Par3 localization. WT and Nox1 y/- cells were co-transfected with siGlo and siRNA Control (CTRL) or siRNA against PP2A-CA (sequence #4 or #7: siPP2A#4 and siPP2A#7), serum starved for 16 h and stimulated with 10 ng/mL PDGF-BB for 30 min. Cells were fixed and immunostained for Par3 (green) and nucleus (DAPI, blue). Representative images of Par3 localization observed for WT and Nox1 y/- cells. Scale bar = 10 μm.

Article Snippet: Rabbit polyclonal anti-Par3 antibodies were used for immunoprecipitation (Proteintech) and immunofluorescence (Millipore).

Techniques: Transfection, Control, Sequencing

Journal: Frontiers in Cell and Developmental Biology

Article Title: Nox1-based NADPH oxidase regulates the Par protein complex activity to control cell polarization

doi: 10.3389/fcell.2023.1231489

Figure Lengend Snippet: Okadaic acid affects cell polarity, the number of lamellipodia, and Par3 localization in lamellipodia. Cells were seeded on Collagen I-coated coverslips, allowed to attach, and serum starved for 16 h. Then, they were incubated with 1 μM of Okadaic acid (OKA) for 30 min and stimulated with 10 ng/mL of PDGF-BB for an additional 30 min. Cells were fixed and stained for (A) Cortactin (red), F-actin (phalloidin, green), and nucleus (DAPI, blue). The lower panel corresponds to the magnification of the above pictures, showing F-actin distribution in single lamellipodia and protrusion in WT and NOX1 y/- cells with and without OKA. Scale bar = 10 μm. (B) Quantification of the percentage of polarized cells and (C) Number of lamellipodia after OKA treatment. Percentage of polarized cells (B) and number of lamellipodia (C) were calculated from images of 8–10 random field of view per condition (807 cells total from three independent experiments). Data were analyzed with one-way ANOVA (* p < 0.05, ** p < 0.01, ns: no significant). (D) Cells were treated as before and stained with an antibody raised against Par3 (green) and DAPI for nucleus (blue). Representative pictures show the distribution of Par3 along the lamellipodia of WT and Nox1 y/- cells in basal and OKA-treated cells. Scale bar = 10 μm.

Article Snippet: Rabbit polyclonal anti-Par3 antibodies were used for immunoprecipitation (Proteintech) and immunofluorescence (Millipore).

Techniques: Incubation, Staining

Journal: Frontiers in Cell and Developmental Biology

Article Title: Nox1-based NADPH oxidase regulates the Par protein complex activity to control cell polarization

doi: 10.3389/fcell.2023.1231489

Figure Lengend Snippet: Nox1 regulates the activity and localization of the Par3/aPKC/Tiam polarity complex by regulating PP2A activity. In WT cells, PDGF stimulation induces the polarized formation of a single lamellipodium via the assembly of Par3/aPKC/Tiam and the activation of Rac at the leading edge. At the rear end of the cell, Par6 interacts with aPKC and Par3 is not binding Tiam. Our current working model propose that the lack of Nox1 (Nox1 y/- ) inhibits the activity of the phosphatase PP2A, inducing the aberrant phosphorylation and activation of the Par3/aPKC/Tiam polarity complex. Par3, PP2A, Nox1-HA, and active Tiam amass at multiple membrane locations forming multiple lamellipodia-like structures probably by Rac activation.

Article Snippet: Rabbit polyclonal anti-Par3 antibodies were used for immunoprecipitation (Proteintech) and immunofluorescence (Millipore).

Techniques: Activity Assay, Activation Assay, Binding Assay, Phospho-proteomics, Membrane

Journal: Journal of Cellular and Molecular Medicine

Article Title: PARD3 gene variation as candidate cause of nonsyndromic cleft palate only

doi: 10.1111/jcmm.17452

Figure Lengend Snippet: PARD3 variant found in a pedigree with NSCP. (A) The pedigree with nonsyndromic cleft palate only. Unfilled and shaded shapes denote healthy and affected individuals, respectively. Squares represent males, and circles represent females. The arrow indicates the proband. (B) Clinical image of two affected individuals (III3 and III6) from the pedigree, which show the cleft of the soft palate. (C) The heterozygous single nucleotide insertion variant in PARD3 was verified by Sanger sequencing. The variant was named according to GenBank: NM_019619.4, NP_062565.2. (D) Protein sequence alignment of PARD3 orthologs was performed by Multiple Sequence Alignment (MUSCLE). The p. E338Gfs*26 variant and the associated new residue sequence are shown above and indicated in red. (E) A schematic diagram of functional domains in the PARD3 (GenBank: NP_062565.2) protein. N= amino terminus; C= carboxy terminus

Article Snippet: The rabbit polyclonal antibody against PARD3 (catalog number 11085‐1‐AP) was purchased from Proteintech.

Techniques: Variant Assay, Sequencing, Residue, Functional Assay

Journal: Journal of Cellular and Molecular Medicine

Article Title: PARD3 gene variation as candidate cause of nonsyndromic cleft palate only

doi: 10.1111/jcmm.17452

Figure Lengend Snippet: Zebrafish with pard3aa and pard3ab disruption displayed ethmoid plate patterning defects. (A) Schematic diagram of the binding and cleavage site of CRISPR/Cas9 in the coding sequence of the zebrafish PARD3 orthologs ( pard3aa , pard3ab ). Red = gRNA target. (B) Comparison of craniofacial structures of F0 CRISPR/Cas9‐mediated mutant zebrafish and Cas9 control zebrafish. The neurocranium was dissected at 3 dpf. Compared with the larvae in the control group, the CRISPR mutant larvae had a mild dysplasia phenotype, with subtle indentations at the upper edge of the ethmoid plate or hypoplastic at the median ethmoid (arrowheads). me = median ethmoid; le = lateral ethmoid. The statistical analysis of abnormal developmental palate was performed (bars indicate the means ± SEM. Student’s t test, * p < 0.05). Scale bar = 200 μm

Article Snippet: The rabbit polyclonal antibody against PARD3 (catalog number 11085‐1‐AP) was purchased from Proteintech.

Techniques: Disruption, Binding Assay, CRISPR, Sequencing, Comparison, Mutagenesis, Control

Journal: Journal of Cellular and Molecular Medicine

Article Title: PARD3 gene variation as candidate cause of nonsyndromic cleft palate only

doi: 10.1111/jcmm.17452

Figure Lengend Snippet: Expression of the patient‐derived truncated PARD3 (c.1012dupG) variant induced hypoplastic ethmoid plate development in zebrafish. (A) Representative images of the control group and the larvae with patient‐derived truncating variant mRNA injection are depicted. Compared with the development of the ethmoid plate in control group larvae, the larvae injected with PARD3‐c.1012dupG mRNA showed ethmoid plate dysplasia, with the median ethmoid plate having a certain degree of absence and failing to form a smooth upper edge of the ethmoid plate. Scale bar = 200 μm. (B) Quantification of hypoplastic development of the ethmoid plate between the experimental groups and the control group (bars indicate the means ± SEM. Student’s t test was used to analyse the data. ** p < 0.01)

Article Snippet: The rabbit polyclonal antibody against PARD3 (catalog number 11085‐1‐AP) was purchased from Proteintech.

Techniques: Expressing, Derivative Assay, Variant Assay, Control, Injection

Journal: Journal of Cellular and Molecular Medicine

Article Title: PARD3 gene variation as candidate cause of nonsyndromic cleft palate only

doi: 10.1111/jcmm.17452

Figure Lengend Snippet: Truncated PARD3‐c.1012dupG variant changed the localization of the wild‐type full‐length PARD3 protein. (A) MCF‐10A cells stably expressing Flag‐tagged PARD3‐c.1012dupG or full‐length PARD3 were developed by lentiviral infection and puromycin selection and formed apical lumens after 10 days of 3D culture in Matrigel. The cysts were stained for Flag‐tagged PARD3 (wild‐type or mutant) (green) and the basolateral membrane marker β‐catenin (red). The truncated PARD3(c.1012dupG) was mainly localized to the basal compartment, while the full‐length PARD3 was mainly localized to the lateral and apical areas. The arrow points to the presence of truncated PARD3 at the basal region. Arrowheads point to the apical region. Scale bar = 10 μm. (B) Mass spectrometry analysis of PARD3‐c.1012dupG products identified endogenous full‐length PARD3 as the candidate interacting protein. Plasmids expressing SBP‐His 8 ‐tagged PARD3‐c.1012dupG or empty vector were stably transfected into HEK‐293T cells, and the cells were harvested and lysed 72 h after selection with hygromycin B. Peptides derived from the trypsin digestion of mutant PARD3 pull down complex were analysed by LC–MS/MS. Herein, we used PARD3 338‐1273 to refer to the C‐terminal signal of endogenous PARD3 bound by the PARD3‐c.1012dupG protein. The number of peptide hits for the C‐terminal signal of endogenous PARD3 (PARD3 338‐1273 ) is shown as a pie chart and table. (C) Endogenous PARD3 interacted with Flag‐tagged PARD3‐p. E338Gfs*26. Flag‐tagged PARD3‐p. E338Gfs*26 was immunoprecipitated from the cell lysate of HEK‐293T cells stably expressing Flag‐tagged PARD3‐p. E338Gfs*26, and the coimmunoprecipitation product was analysed by anti‐PARD3 (C‐terminal immunogen) and anti‐Flag immunoblotting. (D) Substantial proportion of endogenous PARD3 colocalized with Flag‐tagged PARD3‐c.1012dupG mainly at the basement membrane in 3D‐cultured MCF‐10A cells. MCF‐10A cells stably expressing Flag‐tagged PARD3‐c.1012dupG or empty vector were grown in Matrigel, and endogenous PARD3 was analysed with immunofluorescent staining using C‐terminal PARD3 antibody (green). Mutant PARD3 was visualized by Flag antibody (red), and nuclei were stained with DAPI (blue). Scale bar = 7.5 μm

Article Snippet: The rabbit polyclonal antibody against PARD3 (catalog number 11085‐1‐AP) was purchased from Proteintech.

Techniques: Variant Assay, Stable Transfection, Expressing, Infection, Selection, Staining, Mutagenesis, Membrane, Marker, Mass Spectrometry, Plasmid Preparation, Transfection, Derivative Assay, Liquid Chromatography with Mass Spectroscopy, Immunoprecipitation, Western Blot, Cell Culture

Journal: Journal of Cellular and Molecular Medicine

Article Title: PARD3 gene variation as candidate cause of nonsyndromic cleft palate only

doi: 10.1111/jcmm.17452

Figure Lengend Snippet: Variants of PARD3 in sporadic cases with NSCP

Article Snippet: The rabbit polyclonal antibody against PARD3 (catalog number 11085‐1‐AP) was purchased from Proteintech.

Techniques: Mutagenesis

Journal: Journal of Cellular and Molecular Medicine

Article Title: PARD3 gene variation as candidate cause of nonsyndromic cleft palate only

doi: 10.1111/jcmm.17452

Figure Lengend Snippet: Functional validation of the N‐terminal truncating variant c.397C>T identified in a sporadic case with NSCP. (A) Representative images of the control group and zebrafish larvae injected with PARD3‐c.397C>T variant mRNA are depicted. Scale bar = 200 μm. (B) Statistical analysis for (A). Bars indicate the means ± SEM. Student’s t test was used to analyse the data. *** p < 0.001. (C) MCF‐10A cells stably expressing PARD3‐c.397C>T were polarized and formed apical lumens after 10 days of 3D culture in Matrigel. The cysts were stained for Flag‐tagged PARD3‐c.397C>T (green) and the basolateral membrane marker β‐catenin (red). PARD3‐c.397C>T localized to the basal compartment rather than the apical region. The arrowhead points to the presence of truncated PARD3 at the basal region. The asterisk indicates the apical area. Scale bar = 25 μm. (D) Endogenous PARD3 interacted with Flag‐tagged PARD3‐p.R133*. Flag‐tagged PARD3‐p. R133* was immunoprecipitated from cell lysates of HEK‐293T cells stably expressing Flag‐tagged PARD3‐p. R133*, and the coimmunoprecipitation product was analysed by anti‐PARD3 (C‐terminal immunogen) and anti‐Flag immunoblotting. (E) 3D culture of MCF‐10A cells was performed, and MCF‐10A cells stably expressing Flag‐tagged PARD3‐c. 397C>T showed that a substantial proportion of endogenous PARD3 colocalized with PARD3‐c.397C>T, which was mainly at the basement membrane. Endogenous PARD3 was analysed with immunofluorescent staining using a C‐terminal PARD3 antibody (green), PARD3‐c.397C>T was visualized with a Flag antibody (red), and nuclei were stained with DAPI (blue). Scale bar = 7.5 μm

Article Snippet: The rabbit polyclonal antibody against PARD3 (catalog number 11085‐1‐AP) was purchased from Proteintech.

Techniques: Functional Assay, Biomarker Discovery, Variant Assay, Control, Injection, Stable Transfection, Expressing, Staining, Membrane, Marker, Immunoprecipitation, Western Blot

Journal: Brain

Article Title: Junctional instability in neuroepithelium and network hyperexcitability in a focal cortical dysplasia human model

doi: 10.1093/brain/awab479

Figure Lengend Snippet: Molecular characterization of FCD cortical organoids. ( A ) Relative expression (RT-qPCR) of ZO-1 in 19-day-old-organoid from four independent batches of organoids per subject ( n = 16 control and n = 16 patients). Each subject comprised two different clones of iPSCs, with three technical replicates per sample, expression levels were normalized to GAPDH; P = 0.0387. ( B ) RT-qPCR of PARD3 in 90-day-old-organoids from four independent batches of organoids per subject ( n = 16 controls and n = 16 patients). Each subject comprised two different clones of iPSCs, with three technical replicates per sample, expression levels were normalized to GAPDH; P = 0.0008. ( C ) Quantification and western blot of ultrafractionated actin samples (F-actin and G- actin) from 90-day-old organoids; four independent batches of organoids per subject ( n = 12 controls and n = 12 patients). Each subject comprised two different clones of iPSCs. The expression levels were normalized to β-tubulin III; P = 0.0312. ( D ) RT-qPCR of RHOA in 90-day-old-organoids from four independent batches of organoids per subject ( n = 16 controls and n = 16 patients). Each subject was composed of two different clones of iPSCs, with three technical replicates per sample, expression levels were normalized to GAPDH; P = 0.0110. ( E ) Quantification and western blot of RHOA protein from 90 day-old-organoids; four independent batches of organoids per subject ( n = 11 controls and n = 10 patients; without F1 organoids). Each subject comprised two different clones of iPSCs. The expression levels were normalized to β-tubulin III; P = 0.0241. ( F ) RT-qPCR of RHOA from fresh-frozen brain tissue of patients with FCD type II ( n = 15 controls and n = 15; ), with three technical replicates per sample. The expression levels were normalized to ACTB; P = 0.0214. ( G ) Quantification and western blot of RHOA protein from fresh-frozen brain tissue of patients with FCD type II ( n = 7) and controls ( n = 9). The expression levels were normalized to β-tubulin III; P = 0.0167. ( H ) RT-qPCR of PARD3 from fresh-frozen brain tissue of patients with FCD type II ( n = 15) and controls ( n = 15), with three technical replicates per sample. The expression levels were normalized to ACTB; P = 0.0199. ( I ) Quantification and western blot of SYNAPSIN I protein from 90-day-old-organoids; four independent batches of organoids per subject ( n = 13 controls and n = 16 patients). Each subject comprised two different clones of iPSCs. The expression levels were normalized to β-tubulin III; P = 0.0098. ( J – L ) Gene expression analysis based on Nanostring neuropathology panel from 90-day-old organoids, FCD versus control, ( n = 4 controls and n = 4 patients; Patients F1, F2, F3 and F4). ( J ) Volcano plot evidencing the differentially expressed genes (purple dots) found when FCD organoids and controls organoids were compared. ( K ) Box plot evidencing the differential expression of MMP2 when comparing patients and controls; P = 0.00476 and a fold change of −4.45. J and K were obtained from the ROSALIND™ platform. ( L ) Quantification and western blotting of MMP2 from 90-day-old organoids; four independent batches of organoids per subject ( n = 12 control and n = 11 patients). Each subject comprised two different clones of iPSCs. The expression levels were normalized to β-tubulin III; P = 0.0489. The results are presented as the mean ± SEM. A one-sample t -test was used to assess statistical significance; * P < 0.05, ** P < 0.001, **** P < 0.0001. Controls: WT83 clone 1◑, clone 2◐; 4C clone 1◨, clone 2 ◧; 969 clone 1◮, clone 2 ◭, 121 clone 1 ◆ clone 2 ◇; patients: F1 clone 1●, clone 2 ○; F2 clone 1■, clone 2 □, F3 clone 1▲, clone 2 △, F4 clone 1▼, clone 2▽.

Article Snippet: Primary antibodies used were: rat anti-CTIP2 (Abcam; ab18465; 1:500); rabbit anti-SATB2 (Abcam; ab34735; 1:200); chicken anti-MAP2 (Abcam; ab5392; 1:1000); rabbit anti-SOX2 (Abcam; ab97959; 1:1000); rabbit anti-CUX1 (CUTL1 or CASP, Abcam; ab54583; 1:200); rabbit anti-vesicular glutamate transporter 1 (VGLUT1; Synaptic Systems; 135311; 1:500); rabbit anti-cleaved caspase 3 (CC3 Cell Signaling; 9664S; 1:500); mouse anti-RHOA (Abcam; ab54835;1:100); mouse anti-postsynaptic density protein 95 (PSD-95; NeuroMab, UC Davis, 1:1000); rabbit anti-synapsin I (Sigma-Aldrich; AB1543P, 1:1000); mouse anti-zona occludens 1 (ZO-1;Thermo Fisher Scientific; ZO1-1A12; 1:500); rabbit anti-partitioning defective 3 (PARD3; Thermo Fisher Scientific; 11085-1-AP; 1:300); mouse anti-N-Cadherin (BD; Clone 32; 1:150); rabbit anti-NeuN (Abcam; ab128886; 1:1000); mouse anti-NESTIN (Abcam; ab22035; 1:200); and rabbit anti-Ki67 (Abcam; ab15580; 1:100).

Techniques: Expressing, Quantitative RT-PCR, Clone Assay, Western Blot