Journal: Frontiers in Molecular Neuroscience

Article Title: Cohesive Regulation of Neural Progenitor Development by microRNA miR-26, Its Host Gene Ctdsp and Target Gene Emx2 in the Mouse Embryonic Cerebral Cortex

doi: 10.3389/fnmol.2018.00044

Figure Lengend Snippet: Ctdsp2 increases neural progenitor (NP) proliferation. (A–C) Overexpression of Ctdsp2 , but not the control construct pCAGIG, increased the proportion of cells expressing both proliferative marker bromodeoxyuridine + (BrdU + )/green fluorescence protein + (GFP + ) and radial glial cell (RGC) marker Pax6 + /GFP + , but not intermediate progenitor (IP) marker Tbr2 + /GFP + , in GFP-positive cells. (D–F) shRNA-mediated knockdown (sh Ctdsp2 ) of Ctdsp2 decreased the proportion of both BrdU + /GFP + cells and Pax6 + /GFP + cells, but not Tbr2 + /GFP + cells in GFP-positive cells, compared to the control construct pSilencer. Values represent mean ± SEM. n = 9 sections from at least three brains. ** P < 0.01; ns, not significant. ANOVA with post hoc test was used. Scale bar = 50 μm.

Article Snippet: Primary antibodies against the following antigens were used: BrdU (1:50, Developmental Studies Hybridoma Bank at University of Iowa (DSHB)), Ki67 (1:500, Abcam), Pax6 (1:500, Covance), Pax6 (1:15 DSHB), Tbr2 (1:500, Abcam), GFP (1:1000, Abcam, chicken), and GFP (1:1000, Rockland, rabbit).

Techniques: Over Expression, Control, Construct, Expressing, Marker, Fluorescence, shRNA, Knockdown

Journal: Frontiers in Molecular Neuroscience

Article Title: Cohesive Regulation of Neural Progenitor Development by microRNA miR-26, Its Host Gene Ctdsp and Target Gene Emx2 in the Mouse Embryonic Cerebral Cortex

doi: 10.3389/fnmol.2018.00044

Figure Lengend Snippet: miR-26 promotes NP proliferation. (A,D,G) Overexpression of miR-26a, but not the control construct pCAGIG, proportion of cells expressing both proliferative marker BrdU + /GFP + and RGC marker Pax6 + /GFP + , but not IP marker Tbr2 + /GFP + , in GFP-positive cells. (B,E,H) miRNA sponge-mediated knockdown (miR-26-SP), but not the mutated sponge (miR-26-SPmut), decreased proportion of cells expressing both proliferative marker BrdU + /GFP + and RGC marker Pax6 + /GFP + in GFP-positive cells. (C,F,I) Ratio of BrdU + /GFP + , Pax6 + /GFP + or Tbr2 + /GFP + cells vs. GFP + cells in the electroporated cortex. Values represent mean ± SEM. n = 9 sections from at least three brains. * P < 0.05; ** P < 0.01; ns, not significant. ANOVA with post hoc test was used. Scale bar = 50 μm.

Article Snippet: Primary antibodies against the following antigens were used: BrdU (1:50, Developmental Studies Hybridoma Bank at University of Iowa (DSHB)), Ki67 (1:500, Abcam), Pax6 (1:500, Covance), Pax6 (1:15 DSHB), Tbr2 (1:500, Abcam), GFP (1:1000, Abcam, chicken), and GFP (1:1000, Rockland, rabbit).

Techniques: Over Expression, Control, Construct, Expressing, Marker, Knockdown

Journal: Frontiers in Molecular Neuroscience

Article Title: Cohesive Regulation of Neural Progenitor Development by microRNA miR-26, Its Host Gene Ctdsp and Target Gene Emx2 in the Mouse Embryonic Cerebral Cortex

doi: 10.3389/fnmol.2018.00044

Figure Lengend Snippet: Emx2 is functionally inhibited by miR-26 in regulating NP proliferation. (A–F) Overexpression of Emx2 , but not the control construct pCAGIG, decreased the proportion of cells expressing both proliferative marker BrdU + /GFP + and RGC marker Pax6 + /GFP + , but not IP marker Tbr2 + /GFP + , in GFP-positive cells. short hairpin RNA (shRNA)-mediated knockdown (sh Emx2 ) of Emx2 increased the proportion of both BrdU + /GFP + cells and Pax6 + /GFP + cells, but not Tbr2 + /GFP + cells in GFP-positive cells, compared to the control construct pSilencer. (G–J) Emx2 expression suppressed the proportion of cells expressing both proliferative marker BrdU + /GFP + and RGC marker Pax6 + /GFP + in GFP-positive cells. Co-expressing Emx2 with miR-26, but not miR-26-mut, dramatically reversed the suppression. (K,L) Emx2 expression did not alter the proportion of cells expressing IP marker Tbr2 + /GFP + , in GFP-positive cells. Values represent mean ± SEM. n = 9 sections from at least three brains. ** P < 0.01; *** P < 0.001; ns, not significant. ANOVA with post hoc test was used. Scale bar = 50 μm.

Article Snippet: Primary antibodies against the following antigens were used: BrdU (1:50, Developmental Studies Hybridoma Bank at University of Iowa (DSHB)), Ki67 (1:500, Abcam), Pax6 (1:500, Covance), Pax6 (1:15 DSHB), Tbr2 (1:500, Abcam), GFP (1:1000, Abcam, chicken), and GFP (1:1000, Rockland, rabbit).

Techniques: Over Expression, Control, Construct, Expressing, Marker, shRNA, Knockdown

Journal: Cell stem cell

Article Title: Human embryonic stem cell-derived motor neurons are sensitive to the toxic effect of glial cells carrying an ALS-causing mutation.

doi: 10.1016/j.stem.2008.09.017

Figure Lengend Snippet: Figure 1. Differentiation of Human ESCs into Motor Neurons (A) Diagram outlining the protocol used to differen- tiate human ESCs into motor neurons. Undifferen- tiated human ESC colonies are dissociated in col- lagenase and grown as EBs for the first 14 days in EB media, then are induced to a rostrocaudal identity with retinoic acid (RA) and Shh for another 14 days. Finally, EBs are matured in the presence of GDNF for 14 more days. At this point the EBs can either be plated whole or dissociated with papain and then plated. (B) Immunohistochemistry was performed to de- tect neuronal markers PAX 6, NKX 6.1, ISL1/2, and HB9 in EB sections at 14, 28, and 42 days after collagenase treatment of undifferentiated human ESCs. (C) Percent of sectioned EBs (n = 20) staining pos- itive for PAX6, NKX6.1, ISL1/2, or HB9 at day 0, day 14, day 28, and day 42 of differentiation. (D) Percent of cells per sectioned EB (mean ± SD; n = 3; cells counted in sections randomly selected from different EBs) staining positive for PAX6, NKX6.1, ISL1/2, or HB9 at day 0, day 14, day 28, and day 42 of differentiation.

Article Snippet: After blocking, the cells were incubated overnight at 4 C with primary antibodies: mouse anti-b-tubulin III (Covance); rabbit anti-b-tubulin III (SIGMA); Pax6, Nkx6.1, Nkx2.2, Isl 1, Hb9 (DSHB); Chx10, Lhx2 (Santa Cruz Technologies); ChAT (Chemicon); and rabbit anti-GFP conjugated Alexa Fluor 488 (Molecular Probes) in the blocking solution.

Techniques: Immunohistochemistry, Staining

Journal: Cell stem cell

Article Title: Human embryonic stem cell-derived motor neurons are sensitive to the toxic effect of glial cells carrying an ALS-causing mutation.

doi: 10.1016/j.stem.2008.09.017

Figure Lengend Snippet: Figure 3. Characterization of the Hb9::GFP Human ESC Line (A) DNA construct used for the electroporation of human ESCs. (B) GFP expression during different stages of the differentiation from human ESCs into motor neurons. (C–F) Expression of neuronal markers HB9 (C), PAX6 (D), ISL1/2 (E), choline acetyltransferase (ChAT) (F) in motor neurons derived from Hb9::GFP human ESCs.

Article Snippet: After blocking, the cells were incubated overnight at 4 C with primary antibodies: mouse anti-b-tubulin III (Covance); rabbit anti-b-tubulin III (SIGMA); Pax6, Nkx6.1, Nkx2.2, Isl 1, Hb9 (DSHB); Chx10, Lhx2 (Santa Cruz Technologies); ChAT (Chemicon); and rabbit anti-GFP conjugated Alexa Fluor 488 (Molecular Probes) in the blocking solution.

Techniques: Construct, Electroporation, Expressing, Derivative Assay

Journal:

Article Title: A binding site for Pax proteins regulates expression of the gene for the neural cell adhesion molecule in the embryonic spinal cord

doi:

Figure Lengend Snippet: Mutations in PBS-1 and PBS-2 decrease N-CAM promoter activity in a N2A cell line stably expressing Pax-6. Cells were transfected with 0.5 μg of CMVβ and 2 μg of either a CAT gene construct driven by the −414/−15 N-CAM, a similar construct (designated −414/−15 M12) with mutations in PBS-1 and PBS-2, or the promoterless CAT gene (pCATbasic). CAT assays were performed in duplicate. The numbers refer to the relative levels of CAT activity produced by each construct.

Article Snippet: For promoter activation assays, a N2A neuroblastoma cell line permanently expressing Pax-6 was transfected with reporter gene constructs prepared in the pCATbasic plasmid (Promega).

Techniques: Activity Assay, Stable Transfection, Expressing, Transfection, Construct, Produced

Journal: EMBO Molecular Medicine

Article Title: Paired box 6 gene delivery preserves beta cells and improves islet transplantation efficacy

doi: 10.15252/emmm.202317928

Figure Lengend Snippet: A EndoC‐βH1 cells with PAX6 knockdown were subjected to cell proliferation measurement indexed by BrdU labeling after 72‐h treatment with insulin (100 nM), Exendin‐4 (10 nM), GIP (10 nM), or IGF1 (50 ng/ml) ( n = 8–10). B–D (B) Cell apoptosis ( n = 8), (C) GSIS ( n = 5), and (D) insulin content ( n = 10–11) were measured in control and PAX6 knockdown cells. E Protein expression of insulin and incretin signaling components in cells with PAX6 knockdown ( n = 4). F Phosphorylated and total Akt abundance in cells with PAX6 knockdown were measured after 15‐min insulin (100 nM) stimulation ( n = 4). G, H Phosphorylated and total CREB abundance in cells with PAX6 knockdown were measured after 15‐min (G) Exendin‐4 (10 nM) or (H) GIP (10 nM) stimulation ( n = 4). I, J cAMP concentration was measured in cells with PAX6 knockdown after 15‐min (I) Exendin‐4 (10 nM) or (J) GIP (10 nM) stimulation ( n = 6–7). Data information: Each n represents an independent biological replicate (A–J). Mann–Whitney test (A). Unpaired Student's t ‐test (B–E, I, J). Two‐way ANOVA (F–H). Data are means ± SEM. ns, nonsignificant. Source data are available online for this figure.

Article Snippet: PAX6‐overexpressing construct was generated by subcloning the coding sequence expressing human PAX6 (OriGene, Rockville, MD, USA) into the lentiviral vector (Addgene, Cambridge, MA, USA).

Techniques: Knockdown, Labeling, Control, Expressing, Concentration Assay, MANN-WHITNEY

Journal: EMBO Molecular Medicine

Article Title: Paired box 6 gene delivery preserves beta cells and improves islet transplantation efficacy

doi: 10.15252/emmm.202317928

Figure Lengend Snippet: A EndoC‐βH1 cells with PAX6 overexpression were subjected to cell proliferation measurement indexed by BrdU labeling after 72‐h exposure to normal or HGPA condition with insulin (100 nM), Exendin‐4 (10 nM), GIP (10 nM), or IGF1 (50 ng/ml) ( n = 8). B–E After 72‐h exposure to normal or HGPA condition, (B) cell apoptosis ( n = 8), (C) GSIS ( n = 5), (D) insulin content ( n = 8), and (E) protein expression of insulin and incretin signaling components ( n = 4) were measured in cells with PAX6 overexpression. F Phosphorylated and total Akt abundance were measured in cells with PAX6 overexpression after 15‐min insulin (100 nM) stimulation ( n = 4). G, H Phosphorylated and total CREB abundance were measured in cells with PAX6 overexpression after 15‐min (G) Exendin‐4 (10 nM) or (H) GIP (10 nM) stimulation ( n = 4). Data information: Each n represents an independent biological replicate (A–H). Unpaired Student's t ‐test (A). One‐way ANOVA (B–E). Two‐way ANOVA (F–H). Data are means ± SEM. ns, nonsignificant. Source data are available online for this figure.

Article Snippet: PAX6‐overexpressing construct was generated by subcloning the coding sequence expressing human PAX6 (OriGene, Rockville, MD, USA) into the lentiviral vector (Addgene, Cambridge, MA, USA).

Techniques: Over Expression, Labeling, Expressing

Journal: EMBO Molecular Medicine

Article Title: Paired box 6 gene delivery preserves beta cells and improves islet transplantation efficacy

doi: 10.15252/emmm.202317928

Figure Lengend Snippet: A, B (A) Fasting blood glucose and (B) glucose tolerance of control or STZ‐treated mice with AAV injection ( n = 10–12). C Glucose profiles calculated as AUC ( n = 10–12). D–G (D) Serum insulin, (E) pancreatic insulin content, (F) serum glucagon, and (G) pancreatic glucagon content of control or STZ‐treated mice with AAV injection ( n = 10–12). H–J Representative immunostaining and quantification showing (H) alpha‐to‐beta cell ratio, (I) insulin/Ki67 and (J) insulin/TUNEL signals in pancreatic islets of control or STZ‐treated mice with AAV injection ( n = 6). Scale bar = 20 μm. Data information: Each n represents the measurement of a sample from distinct mice (A–J). Unpaired Student's t ‐test and Mann–Whitney test (A, B). STZ + AAV‐PAX6 versus STZ + AAV‐Ctrl. One‐way ANOVA (C–J). Data are means ± SEM. AUC, area under the curve. Source data are available online for this figure.

Article Snippet: PAX6‐overexpressing construct was generated by subcloning the coding sequence expressing human PAX6 (OriGene, Rockville, MD, USA) into the lentiviral vector (Addgene, Cambridge, MA, USA).

Techniques: Control, Injection, Immunostaining, TUNEL Assay, MANN-WHITNEY

Journal: EMBO Molecular Medicine

Article Title: Paired box 6 gene delivery preserves beta cells and improves islet transplantation efficacy

doi: 10.15252/emmm.202317928

Figure Lengend Snippet: A, B (A) Fasting blood glucose and (B) serum insulin of db /+ or db/db mice at the indicated time points after AAV injection ( n = 10). C IPGTT of db /+ or db/db mice with AAV injection ( n = 10). D Glucose profiles calculated as AUC ( n = 10). E Serum insulin during IPGTT expressed as percent of basal ( n = 10). F–H (F) Serum glucagon, (G) islet insulin content, and (H) islet apoptosis of db /+ or db/db mice with AAV injection ( n = 8–10). I, J Representative immunostaining and quantification showing (I) insulin/Ki67 and (J) insulin/TUNEL signals in pancreatic islets of db /+ or db/db mice with AAV injection ( n = 6). Scale bar = 20 μm. K Protein expression of insulin and incretin signaling components in pancreatic islets of db /+ or db/db mice with AAV injection ( n = 4). Data information: Each n represents the measurement of a sample from distinct mice (A–K). Unpaired Student's t ‐test and Mann–Whitney test (A–C). db/db + AAV‐PAX6 versus db/db + AAV‐Ctrl. Unpaired Student's t ‐test (E). db/db + AAV‐PAX6 versus db/db + AAV‐Ctrl. One‐way ANOVA (D, F–K). Data are means ± SEM. AUC, area under the curve; ns, nonsignificant. Source data are available online for this figure.

Article Snippet: PAX6‐overexpressing construct was generated by subcloning the coding sequence expressing human PAX6 (OriGene, Rockville, MD, USA) into the lentiviral vector (Addgene, Cambridge, MA, USA).

Techniques: Injection, Immunostaining, TUNEL Assay, Expressing, MANN-WHITNEY

Journal: EMBO Molecular Medicine

Article Title: Paired box 6 gene delivery preserves beta cells and improves islet transplantation efficacy

doi: 10.15252/emmm.202317928

Figure Lengend Snippet: A Top KEGG pathways enriched by DEGs in human islets with PAX6 overexpression compared with the control group. Up‐ or down‐regulated genes enriched pathways were represented in red and blue colors, respectively. B–D GSEA enrichment graphs of pancreatic secretion pathway, acute inflammatory pathway, and MAPK signaling pathway. E, F Heatmaps of DEGs showing (E) the increased expression of INS, INSR, and IRS2 genes and (F) the decreased expression of disallowed genes in human islets with PAX6 overexpression. G Venn diagram showing the top overlapping KEGG pathways between the PAX6‐bound genes (GSE87530) (Swisa et al , ) and the DEG list of our PAX6‐overexpressing islets (adjusted P ‐value < 0.05; cutoff log 2 FC > 0.5). H–J (H) Fasting blood glucose, (I) glucose tolerance, and (J) glucose profiles calculated as AUC of control or STZ‐treated mice with normal human islet transplantation ( n = 4–9). K Serum human insulin ( n = 4–9). L Representative immunostaining showing insulin/glucagon signal and ELISA measurement of human insulin content in human islet grafts ( n = 4–6). M Representative immunostaining and quantification showing insulin/TUNEL signal in human islet grafts ( n = 5). Scale bar = 100 μm. Data information: Each n represents the measurement of a sample from distinct mice (H–M). Hypergeometric test was applied for the pathway enrichment analysis and the P ‐values were adjusted by Benjamini–Hochberg correction method (A). Gene list ranked by their log 2 fold changes were used for GSEA based on the Kolmogorov–Smirnov test. False discovery rate < 0.05 was set as significance threshold (B–D). One‐way ANOVA (H–L). Unpaired Student's t ‐test (M). Data are means ± SEM. DEGs, differentially expressed genes; AUC, area under the curve; ns, nonsignificant. Source data are available online for this figure.

Article Snippet: PAX6‐overexpressing construct was generated by subcloning the coding sequence expressing human PAX6 (OriGene, Rockville, MD, USA) into the lentiviral vector (Addgene, Cambridge, MA, USA).

Techniques: Over Expression, Control, Expressing, Transplantation Assay, Immunostaining, Enzyme-linked Immunosorbent Assay, TUNEL Assay

Journal: Scientific reports

Article Title: Pax6 regulates neuronal migration and cell proliferation via interacting with Wnt3a during cortical development.

doi: 10.1038/s41598-025-88662-5

Figure Lengend Snippet: Fig. 2. Neurons were detained in the intermediate zone of cortex after Pax6 overexpression. Three days after the transfection of pCAGGS-EGFP (Control), and pCAGGS-Pax6-EGFP (Pax6 overexpression) constructs into E15.5 embryo cortex, brain samples (E15.5 + 3d) were coronally sectioned for subsequent examinations (A). Immunostainings against Pax6 were performed to track Pax6-overexpressing cells: DAPI (Blue in B and C), GFP (Green in D and E), Pax6 (Red in F and G), and Merge (H and I). (J) Statistical graph of percentage of GFP+ cells in different cortical regions of the control and Pax6 overexpression group. (K) Pie chart of the proportion of GFP+/Pax6+ cells in Pax6-overexpressing cortex. L) Statistical graph of percentage of GFP+ or GFP+/Pax6+ cells in different cortical regions of the control and Pax6 overexpression group. (M) Schematic diagrams of neuronal migration in Pax6-overexpressing cortex at E15.5 + 3d. GFP+/Pax6+ cells are pointed by white arrowheads in (G and I). Scale bar = 50 μm. Abbreviations: OE, overexpression; VZ, ventricular zone; SVZ, Subventricular zone; IZ, intermediate zone; CP, cortical plate; MZ, marginal zone. n = 3. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.

Article Snippet: The relative gray values for Pax6 and Wnt3a were calculated by normalizing the gray value of each protein’s band to that of its corresponding loading control (GAPDH, 1:5000, 60004-1-lg, proteintech, China). n = 3 for each group.

Techniques: Over Expression, Transfection, Control, Construct, Migration

Journal: Scientific reports

Article Title: Pax6 regulates neuronal migration and cell proliferation via interacting with Wnt3a during cortical development.

doi: 10.1038/s41598-025-88662-5

Figure Lengend Snippet: Fig. 3. Pax6 overexpression temporally disrupted neuronal migration during corticogenesis. pCAGGS- EGFP (Control) or pCAGGS-Pax6-EGFP (Pax6 overexpression) constructs were transfected into E13.5 embryo cortex, and brain samples were collected respectively at 1 day (E13.5 + 1d, A-H), 3 days (E13.5 + 3d, I-P), 5 days (E13.5 + 5d, Q-X), and 7 days (E13.5 + 7d, Y-f) after electroporation for immunostainings against Pax6. Immunostaining shows DAPI (Blue in A, E, I, M, Q, U, Y, and c), GFP (Green in B, F, J, N, R, V, Z, and d), Pax6 (Red in C, G, K, O, S, W, a, and e), and Merge (D, H, L, P, T, X, b, and f). White arrowheads highlight GFP+/Pax6+ cells in Pax6 overexpression group. (g-j) Statistical graph of percentage of GFP+ or GFP+/Pax6+ cells in different cortical regions of the control and Pax6 overexpression at 1 day (g), 3 days (h), 5 days (i) and 7 days (j) after electroporation. (k) Schematic diagrams of neuronal migration in Pax6 overexpressing cortex at E13.5 + 1d, E13.5 + 3d, E13.5 + 5d and E13.5 + 7d. Scale bar = 50 μm. Abbreviations: OE, overexpression; VZ/ SVZ, ventricular zone/subventricular zone; IZ, intermediate zone; CP, cortical plate; MZ, marginal zone; ns, no significance. n = 3. *p < 0.05, ***p < 0.001, ****p < 0.0001.

Article Snippet: The relative gray values for Pax6 and Wnt3a were calculated by normalizing the gray value of each protein’s band to that of its corresponding loading control (GAPDH, 1:5000, 60004-1-lg, proteintech, China). n = 3 for each group.

Techniques: Over Expression, Migration, Control, Construct, Transfection, Electroporation, Immunostaining

Journal: Scientific reports

Article Title: Pax6 regulates neuronal migration and cell proliferation via interacting with Wnt3a during cortical development.

doi: 10.1038/s41598-025-88662-5

Figure Lengend Snippet: Fig. 4. Axonal outgrowth was affected by Pax6 overexpression. Immunostaining against Pax6 was performed on E15.5 + 3d brain sections (A-H): DAPI (Blue in A and B), GFP (Green in C and D), Pax6 (Red in E and F), and Merge (G and H). (I) Statistical graph of GFP+ and GFP+/Pax6+ cells with vertical or lateral migratory morphology in the IZ of control and Pax6 overexpression group, respectively. (J and K) are the amplification of the framed area in G and H. White arrowheads in J and K indicate GFP+ and GFP+/Pax6+ cells exhibiting distinct morphologies in the IZ of control and Pax6 overexpression group, respectively. (L) Statistical graph of neuronal cells of different morphologies in the IZ of the control and Pax6 overexpression group. GFP-SH-SY5Y and Pax6 OE-SH-SY5Y cell lines were generated by lentiviral transduction, followed by immunostaining against Pax6: DAPI in blue, GFP in green, and Pax6 in red (M and N). White arrowheads in M and N highlight GFP-SH-SY5Y and Pax6 OE-SH-SY5Y cells with different shapes, respectively. (O) Statistical graph of SH-SY5Y cells of different shapes in the control and Pax6 overexpression group. Scale bars are 50 μm in A-H, M, and N; 20 μm in J and K. Abbreviations: OE, overexpression; VZ, ventricular zone; SVZ, Subventricular zone; IZ, intermediate zone; CP, cortical plate; MZ, marginal zone. n = 3. *p < 0.05, **p < 0.01.

Article Snippet: The relative gray values for Pax6 and Wnt3a were calculated by normalizing the gray value of each protein’s band to that of its corresponding loading control (GAPDH, 1:5000, 60004-1-lg, proteintech, China). n = 3 for each group.

Techniques: Over Expression, Immunostaining, Control, Amplification, Generated, Transduction

Journal: Scientific reports

Article Title: Pax6 regulates neuronal migration and cell proliferation via interacting with Wnt3a during cortical development.

doi: 10.1038/s41598-025-88662-5

Figure Lengend Snippet: Fig. 6. Pax6 overexpression induced the expression of Wnt3a. (A) Table of 5 Pax6 potential binding sites within the regulatory sequence of Wnt3a. Immunostaining against Wnt3a was conducted on coronal sections of Pax6-overexpressing brain (B-I): DAPI (Blue in B and F), GFP (Green in C and G), Wnt3a (Red in D and H), and Merge (E and I). J and K are the amplification of the framed area in E and I, respectively. (L) Quantitative PCR was performed to detect the alteration of Pax6 and Wnt3a at the mRNA level in Pax6- overexpressing N2a cells. The level of Pax6/Wnt3a protein in Pax6-overexpressing N2a cells (M) and their medium (O) was examined by Western blot. (N and P) are the statistical graph of (M and O). Scale bars are 50 μm in (B-I), 25 μm in (J and K). Abbreviations: Con, control; OE, overexpression; VZ, ventricular zone; SVZ, Subventricular zone; IZ, intermediate zone; CP, cortical plate; MZ, marginal zone. n = 3 in B-I and M-P; n = 4 in L. *p < 0.05, **p < 0.01, ****p < 0.0001.

Article Snippet: The relative gray values for Pax6 and Wnt3a were calculated by normalizing the gray value of each protein’s band to that of its corresponding loading control (GAPDH, 1:5000, 60004-1-lg, proteintech, China). n = 3 for each group.

Techniques: Over Expression, Expressing, Binding Assay, Sequencing, Immunostaining, Amplification, Real-time Polymerase Chain Reaction, Western Blot, Control