Journal: Stem Cell Research & Therapy

Article Title: AKT signaling upregulates BDNF expression in induced neural stem cells that interact with microglia

doi: 10.1186/s13287-025-04489-x

Figure Lengend Snippet: AKT signaling regulates EZH2 phosphorylation, H3K27 trimethylation and BDNF expression in iNSCs. ( A , B ) ChIP assay results showing the levels of EZH2 ( A ) and H3K27me3 ( B ) at Bdnf promoter IV in iNSCs among the iNSC mono-culture, co-culture and co-culture (LY294002; iNSCs pretreated with LY294002) groups at 24 h after co-culture ( n = 6/group; one-way analysis of variance with Tukey’s post hoc test, * P < 0.05, ** P < 0.01, *** P < 0.001). ( C ) Representative immunoblots illustrating the levels of p-AKT, AKT, p-EZH2, EZH2, H3K27me3, H3, BDNF and GAPDH in iNSCs among the three groups at 24 h after co-culture (full-length blots are presented in Additional Fig. ). ( D - G ) Box-whisker plot depicting the relative levels of p-AKT/AKT ( D ), p-EZH2/EZH2 ( E ), H3K27me3/H3 ( F ), and BDNF/GAPDH ( G ) in iNSCs among the three groups at 24 h after co-culture ( n = 3/group; one-way analysis of variance with Tukey’s post hoc test, * P < 0.05, ** P < 0.01, *** P < 0.001). ( H , I ) ChIP assay results showing the levels of EZH2 ( H ) and H3K27me3 ( I ) at Bdnf promoter IV in iNSCs among the iNSC mono-culture, co-culture, co-culture (Control shRNA; iNSCs pretreated with control shRNA), and co-culture (AKT shRNA; iNSCs pretreated with AKT-specific shRNA) groups at 24 h after co-culture ( n = 6/group; one-way analysis of variance with Tukey’s post hoc test, * P < 0.05, *** P < 0.001). ( J ) Representative immunoblots illustrating the levels of p-AKT, AKT, p-EZH2, EZH2, H3K27me3, H3, BDNF and GAPDH in iNSCs among the four groups at 24 h after co-culture (full-length blots are presented in Additional Fig. ). ( K - N ) Box-whisker plot depicting the relative levels of p-AKT/AKT ( K ), p-EZH2/EZH2 ( L ), H3K27me3/H3 ( M ), and BDNF/GAPDH ( N ) in iNSCs among the four groups at 24 h after co-culture ( n = 3/group; one-way analysis of variance with Tukey’s post hoc test, * P < 0.05, ** P < 0.01, *** P < 0.001). Data are presented as mean ± standard deviation. BDNF: brain-derived neurotrophic factor; ChIP: chromatin immunoprecipitation; EZH2: enhancer of zeste homolog 2; GAPDH: glyceraldehyde 3-phosphate dehydrogenase; iNSCs: induced neural stem cells

Article Snippet: In addition, to knock down AKT in iNSCs, we transfected iNSCs with AKT-specific shRNA (sc-29196-V, Santa Cruz Biotechnology, Santa Cruz, CA, USA) or control shRNA (sc-108080, Santa Cruz Biotechnology) according to the shRNA manufacturer’s instructions.

Techniques: Phospho-proteomics, Expressing, Co-Culture Assay, Western Blot, Whisker Assay, Control, shRNA, Standard Deviation, Derivative Assay, Chromatin Immunoprecipitation

Journal: Stem Cell Research & Therapy

Article Title: AKT signaling upregulates BDNF expression in induced neural stem cells that interact with microglia

doi: 10.1186/s13287-025-04489-x

Figure Lengend Snippet: The expression and localization of p-EZH2, H3K27me3 and BDNF in iNSCs. ( A ) Representative staining for the p-EZH2 + (green) and BDNF + (red) depicted p-EZH2 and BDNF levels in iNSCs between the co-culture (Control shRNA; iNSCs pretreated with control shRNA), and co-culture (AKT shRNA; iNSCs pretreated with AKT-specific shRNA) groups at 24 h after co-culture. Nuclei were counterstained with DAPI (blue). Scale bar: 50 μm (5 μm in the magnified images). ( B , C ) Box-whisker plot depicting the relative fluorescence intensity (RFI) values of p-EZH2 ( B ) and BDNF ( C ) in iNSCs between the two groups at 24 h after co-culture ( n = 6/group; student’s t -test, *** P < 0.001). ( D ) Representative staining for the H3K27me3 + (green) and BDNF + (red) depicted H3K27me3 and BDNF levels in iNSCs between the two groups at 24 h after co-culture. Nuclei were counterstained with DAPI (blue). Scale bar: 50 μm (5 μm in the magnified images). ( E , F ) Box-whisker plot depicting the RFI values of H3K27me3 ( E ) and BDNF ( F ) in iNSCs between the two groups at 24 h after co-culture ( n = 6/group; student’s t -test, *** P < 0.001). Data are presented as mean ± standard deviation. BDNF: brain-derived neurotrophic factor; EZH2: enhancer of zeste homolog 2; iNSCs: induced neural stem cells

Article Snippet: In addition, to knock down AKT in iNSCs, we transfected iNSCs with AKT-specific shRNA (sc-29196-V, Santa Cruz Biotechnology, Santa Cruz, CA, USA) or control shRNA (sc-108080, Santa Cruz Biotechnology) according to the shRNA manufacturer’s instructions.

Techniques: Expressing, Staining, Co-Culture Assay, Control, shRNA, Whisker Assay, Fluorescence, Standard Deviation, Derivative Assay

Journal: Stem Cell Research & Therapy

Article Title: AKT signaling upregulates BDNF expression in induced neural stem cells that interact with microglia

doi: 10.1186/s13287-025-04489-x

Figure Lengend Snippet: Co-culture with LPS-activated microglia increases CREB levels at Bdnf promoters I and IV, as well as CREB phosphorylation in iNSCs. ( A , B ) ChIP assay results showing the levels of CREB at Bdnf promoter I ( A ) and IV ( B ) in iNSCs between the iNSC mono-culture and co-culture groups at 24 h after co-culture ( n = 6/group; student’s t -test, *** P < 0.001). ( C ) Representative immunoblots illustrating the levels of p-CREB and CREB in iNSCs between the two groups at 24 h after co-culture (full-length blots are presented in Additional Fig. ). ( D ) Box-whisker plot depicting the relative levels of p-CREB/CREB in iNSCs between the two groups at 24 h after co-culture ( n = 3/group; student’s t -test, *** P < 0.001). ( E , F ) ChIP assay results showing the levels of CREB at Bdnf promoter I ( E ) and IV ( F ) in iNSCs among the iNSC mono-culture, co-culture and co-culture (666 − 15; iNSCs pretreated with 666 − 15) groups at 24 h after co-culture ( n = 6/group; one-way analysis of variance with Tukey’s post hoc test, * P < 0.05, ** P < 0.01, *** P < 0.001). ( G ) Representative immunoblots illustrating the levels of p-CREB, CREB, BDNF and GAPDH in iNSCs among the three groups at 24 h after co-culture (full-length blots are presented in Additional Fig. ). ( H , I ) Box-whisker plot depicting the relative levels of p-CREB/CREB ( H ) and BDNF/GAPDH ( I ) in iNSCs among the three groups at 24 h after co-culture ( n = 3/group; one-way analysis of variance with Tukey’s post hoc test, * P < 0.05, ** P < 0.01, *** P < 0.001). ( J , K ) ChIP assay results showing the levels of CREB at Bdnf promoter I ( J ) and IV ( K ) in iNSCs among the iNSC mono-culture, co-culture, co-culture (Control shRNA; iNSCs pretreated with control shRNA), and co-culture (CREB shRNA; iNSCs pretreated with CREB-specific shRNA) groups at 24 h after co-culture ( n = 6/group; one-way analysis of variance with Tukey’s post hoc test, *** P < 0.001). ( L ) Representative immunoblots illustrating the levels of p-CREB, CREB, BDNF and GAPDH in iNSCs among the four groups at 24 h after co-culture (full-length blots are presented in Additional Fig. ). ( M , N ) Box-whisker plot depicting the relative levels of p-CREB/CREB ( M ) and BDNF/GAPDH ( N ) in iNSCs among the four groups at 24 h after co-culture ( n = 3/group; one-way analysis of variance with Tukey’s post hoc test, *** P < 0.001). Data are presented as mean ± standard deviation. BDNF: brain-derived neurotrophic factor; ChIP: chromatin immunoprecipitation; CREB: cAMP response element binding protein; GAPDH: glyceraldehyde 3-phosphate dehydrogenase; iNSCs: induced neural stem cells

Article Snippet: In addition, to knock down AKT in iNSCs, we transfected iNSCs with AKT-specific shRNA (sc-29196-V, Santa Cruz Biotechnology, Santa Cruz, CA, USA) or control shRNA (sc-108080, Santa Cruz Biotechnology) according to the shRNA manufacturer’s instructions.

Techniques: Co-Culture Assay, Phospho-proteomics, Western Blot, Whisker Assay, Control, shRNA, Standard Deviation, Derivative Assay, Chromatin Immunoprecipitation, Binding Assay

Journal: Stem Cell Research & Therapy

Article Title: AKT signaling upregulates BDNF expression in induced neural stem cells that interact with microglia

doi: 10.1186/s13287-025-04489-x

Figure Lengend Snippet: AKT signaling regulates BDNF expression by recruiting CREB to their promoters I and IV in iNSCs. ( A , B ) ChIP assay results showing the levels of CREB at Bdnf promoter I ( A ) and IV ( B ) in iNSCs among the iNSC mono-culture, co-culture, co-culture (Control shRNA; iNSCs pretreated with control shRNA), and co-culture (AKT shRNA; iNSCs pretreated with AKT-specific shRNA) groups at 24 h after co-culture ( n = 6/group; one-way analysis of variance with Tukey’s post hoc test, * P < 0.05, ** P < 0.01, *** P < 0.001). ( C ) Representative immunoblots illustrating the levels of p-AKT, AKT, p-CREB, CREB, BDNF and GAPDH in iNSCs among the four groups at 24 h after co-culture (full-length blots are presented in Additional Fig. ). ( D - F ) Box-whisker plot depicting the relative levels of p-AKT/AKT ( D ), p-CREB/CREB ( E ), and BDNF/GAPDH ( F ) in iNSCs among the four groups at 24 h after co-culture ( n = 3/group; one-way analysis of variance with Tukey’s post hoc test, ** P < 0.01, *** P < 0.001). ( G ) Representative staining for the p-CREB + (green) and BDNF + (red) depicted p-CREB and BDNF levels in iNSCs between the co-culture (Control shRNA; iNSCs pretreated with control shRNA), and co-culture (AKT shRNA; iNSCs pretreated with AKT-specific shRNA) groups at 24 h after co-culture. Nuclei were counterstained with DAPI (blue). Scale bar: 50 μm (5 μm in the magnified images). ( H , I ) Box-whisker plot depicting the relative fluorescence intensity (RFI) values of p-CREB ( H ) and BDNF ( I ) in iNSCs between the two groups at 24 h after co-culture ( n = 6/group; student’s t -test, *** P < 0.001). ( J ) Representative immunoblots illustrating the levels of BDNF and GAPDH in the injured cortices among the PBS (CHI mice receiving PBS), iNSC (CHI mice receiving iNSCs pretreated with PBS), and iNSC (AKT shRNA; CHI mice receiving iNSCs pretreated with AKT-specific shRNA) groups on day 7 post-CHI (full-length blots are presented in Additional Fig. ). ( K ) Box-whisker plot depicting the relative levels of BDNF/GAPDH in the injured cortices among the three groups on day 7 post-CHI ( n = 3/group; one-way analysis of variance with Tukey’s post hoc test, * P < 0.05, ** P < 0.01, *** P < 0.001). ( L ) Representative staining for the BDNF + (red) depicted BDNF levels in the injured cortices among the three groups on day 7 post-CHI. Nuclei were counterstained with DAPI (blue). Scale bar: 50 μm. ( M ) Box-whisker plot depicting the RFI values of BDNF in the injured cortices among the three groups on day 7 post-CHI ( n = 6/group; one-way analysis of variance with Tukey’s post hoc test, * P < 0.05, *** P < 0.001). Data are presented as mean ± standard deviation. BDNF: brain-derived neurotrophic factor; CHI: closed head injury; ChIP: chromatin immunoprecipitation; CREB: cAMP response element binding protein; GAPDH: glyceraldehyde 3-phosphate dehydrogenase; iNSCs: induced neural stem cells

Article Snippet: In addition, to knock down AKT in iNSCs, we transfected iNSCs with AKT-specific shRNA (sc-29196-V, Santa Cruz Biotechnology, Santa Cruz, CA, USA) or control shRNA (sc-108080, Santa Cruz Biotechnology) according to the shRNA manufacturer’s instructions.

Techniques: Expressing, Co-Culture Assay, Control, shRNA, Western Blot, Whisker Assay, Staining, Fluorescence, Standard Deviation, Derivative Assay, Chromatin Immunoprecipitation, Binding Assay

Journal: Turkish Journal of Medical Sciences

Article Title: Involvement of SWAP-70 in proteolipid protein-induced experimental autoimmune encephalomyelitis

doi: 10.55730/1300-0144.6161

Figure Lengend Snippet: Kinetics of EAE scores for different shRNA treatment arms and control mice after the PLP immunization. The p-value shown in the upper left corner of the panel was obtained by ANOVA. **, p < 0.01; ***, p < 0.001. Vertical bars denote standard deviations.

Article Snippet: To silence the SWAP-70 gene, a lentiviral particle carrying shRNA (sc-153963-V, Santa Cruz Biotechnology, Dallas, TX, USA) was purchased.

Techniques: shRNA, Control

Journal: Turkish Journal of Medical Sciences

Article Title: Involvement of SWAP-70 in proteolipid protein-induced experimental autoimmune encephalomyelitis

doi: 10.55730/1300-0144.6161

Figure Lengend Snippet: Analysis of EAE lesions in spinal cord sections through semiquantification of the intensity of CD8+ T cell, F4/80+ macrophage infiltration, and demyelination areas. a) Ten-micron spinal cord tissue sections of mice belonging to different shRNA treatment arms and control groups were stained for myelin basic protein (MBP), CD8+ T cells, and F4/80+ macrophages. b) Total cell count is shown per mm 2 area, and the expression levels of MBP are shown as integrated density (IntDen) per μm 2 area. Error bars indicate the standard deviations. The p-values obtained by ANOVA are denoted in the upper left corner of the panel. Significantly different by pair-wise comparison at: ***p < 0.001 for comparisons between SWAP-70 shRNA-treatment versus non-PLP immunized and ##p < 0.01, ###p < 0.001 for comparisons between SWAP-70 shRNA-treatment versus saline and scrambled shRNA treatment arms.

Article Snippet: To silence the SWAP-70 gene, a lentiviral particle carrying shRNA (sc-153963-V, Santa Cruz Biotechnology, Dallas, TX, USA) was purchased.

Techniques: shRNA, Control, Staining, Cell Characterization, Expressing, Comparison, Saline

Journal: Arteriosclerosis, Thrombosis, and Vascular Biology

Article Title: TGFβ (Transforming Growth Factor-Beta)–Activated Kinase 1 Regulates Arteriovenous Fistula Maturation

doi: 10.1161/atvbaha.119.313848

Figure Lengend Snippet: Figure 1. TAK1 (transforming growth factor-beta–activated kinase 1) is upregulated in human mature arteriovenous fistulae (AVF). A, Representative human control vein (left) and human matured AVF (right) by Elastin Van Gieson (EVG) staining (n=3 for each group; scale bar=500 μm). Adjacent bar graphs showing wall thickness and vessel dilation by lumen area in the human control vein and matured AVFs (P=0.0002, wall thickness; P=0.0043, lumen area). B, Representative Western blots and adjacent graphical quantification showing increased phospho-TAK1 (p-TAK1; P=0.0076), TAK1 (P=0.065), phospho-JNK (c-Jun N-terminal kinase; p-JNK; P=0.0008), JNK (P=0.0017), p-p38 (P=0.0134), and p38 (P=0.0032; n=3 each). C, Representative immunofluorescence images showing the venous limbs of human mature AVF and control vein (scale bar=25 μm). Adjacent bar graphs show increased expression in endothelial cells (n=3 each; P=0.0091) and smooth muscle cells (n=3; P=0.0015). D, Representative images showing expression of collagen 1 in human mature AVF, compared with control veins without AVF (dash lines showing internal elastic lamina; scale bar=100 μm). Adjacent bar graphs show quantification of collagen 1 in the intima or media in each group (n=3 each; P<0.0001 in intima and P=0.0015 in media). E, Representative images showing expression of fibronectin in human mature AVF, compared with control veins without AVF (scale bar=100 μm). Adjacent bar graphs show quantification of fibronectin in the intima or media in each group (n=3 each; P<0.0001 in the intima). Statistical tests used: Student t test. DAPI indicates 4′,6-diamidino-2-phenylindole.

Article Snippet: 5Z-7-oxozeaenol (OZ; Millipore Sigma; 49610), which is an irreversible active site inhibitor of TAK1; TAK1 shRNA lentiviral particles (sc-36607-V; Santa Cruz, CA), which contain 3 to 5 expression constructs each encoding target-specific shRNA designed to knockdown TAK1 gene expression; control shRNA lentiviral particles-A (sc-108080; Santa Cruz, CA), containing scrambled shRNA; CopGFP control lentiviral particles (sc108084; Santa Cruz, CA); TAK1 lentiviral activation particle (m2; sc-424044-LAC-2; Santa Cruz, CA), which is a CRISPR (clustered regularly interspaced short palindromic repeats)based transcription activation construct packaged into a single lentivirus that upregulates endogenous TAK1; polybrene (sc-134220; Santa Cruz, CA); puromycin dihydrochloride (sc108071; Santa Cruz, CA).

Techniques: Control, Staining, Western Blot, Immunofluorescence, Expressing

Journal: Journal of Lipid Research

Article Title: High-density lipoprotein attenuates lipopolysaccharide-induced IL-1β activation via scavenger receptor class B type 1

doi: 10.1016/j.jlr.2025.100858

Figure Lengend Snippet: SRB1 is required for LPS-stimulated IL-1β activation. A: Inhibition of SRB1 attenuates LPS-mediated IL-1β activation. RAW264.7 cell were incubated with vehicle or LPS (1 μg/ml) with or without HDL (250 μg/ml) with or without BLT-1 (1 μM) for 4h. Cell lysates were harvested and processed for western blotting for detection of pro IL-1β protein expression. The protein level of β-actin was examined as a loading control. B: Protein levels of pro IL-1β were quantitated by densitometric analysis, normalized to β-actin (mean ± SD, n = 3), ∗ P < 0.05 comparison between IL-1β alone and IL-1β+HDL; and comparison between IL-1β+HDL and IL-1β+HDL+BLT-1, ∗∗∗ P < 0.001comparison between IL-1β alone and IL-1β+HDL+BLT-1. C: Supernatants collected from B were processed for ELISA for detection of active IL-1β production using a mouse ELISA kit, ∗ P < 0.05 comparison between IL-1β+HDL and IL-1β+HDL+BLT-1, ∗∗∗ P < 0.001 comparison between IL-1β alone and IL-1β+HDL; and comparison between IL-1β alone and IL-1β+HDL+BLT-1. D: Knockdown of SRB1 attenuates LPS-mediated IL-1β activation. RAW264.7 (scramble) or RAW264.7 (shSRB1) cells were incubated with vehicle of LPS (1 μg/ml) with or without HDL (250 μg/ml) for 4 h. Cell lysates were harvested and processed for western blotting for detection of the protein expression of SRB1, pro IL-1β, and β-actin. E: Supernatants collected from C were processed for ELISA for detection of active IL-1β production using a mouse ELISA kit, ∗∗∗ P < 0.001. F: Knockdown of SRB1 inhibits LPS uptake by RAW264.7. RAW264.7 (scramble) or RAW264.7 (shSRB1) cells were incubated with fluorescently-labeled LPS (1 μg/ml) in the presence or absence of HDL (250 μg/ml) for 4 h, followed by eBioscience™ Fixable Viability Dye eFluor™ 450 staining for 30 min at room temperature. Cells that engulf fluorescently-labeled LPS were captured by flow cytometry. G: Quantitative results of flow cytometry, n = 3, ∗∗∗ P < 0.001. H: Cells were treated as above and then immunocytochemical staining was conducted using fluorescently-labeled LPS (red) and anti-SRB1 antibody (green). Nuclei were counterstained by DAPI (blue). Scar bars, 20 μm.

Article Snippet: For lentiviral infection, RAW 264.7 cells were incubated with shSRB1 (Santa Cruz biotechnology, sc-44753-v) or non-targeting control (Santa Cruz biotechnology, sc-108080) (1 × 10 7 TU/ml) in RPMI supplemented with Polybrene (5 μg/ml) for 48 h. Transfected cells were sorted on day 5 post-infection by puromycin selection (4 μg/ml) followed by Western blotting to verify the gene silencing.

Techniques: Activation Assay, Inhibition, Incubation, Western Blot, Expressing, Control, Comparison, Enzyme-linked Immunosorbent Assay, Knockdown, Labeling, Staining, Flow Cytometry

Journal: Neurobiology of disease

Article Title: The Warburg micro syndrome protein RAB3GAP1 modulates neuronal morphogenesis and interacts with axon elongation end ER-Golgi trafficking factors

doi: 10.1016/j.nbd.2023.106215

Figure Lengend Snippet: (B) Analysis of RAB3GAP1 expression levels in control of RAB3GAP1 knockdown human neurons. (C–F) Localization of TMF1 to Trans and Cis-Golgi compartments, ERGIC, and ER membranes. Control (top panels) or RAB3GAP1-Lentiviral shRNA (RAB3GAP1-LV-shRNA, bottom panel) are shown for each immunostaining. Neurons were stained with Hoescht (blue) to label the nuclei, and were immunostained with antibodies against MAP2 (cyan), TMF1 (red), and either TGN46 or GM130 or LMAN1 or CALNEXIN (all in green) to label the different compartments. All scale bars are 20 μm. (C) Localization of TMF1 to Trans-Golgi compartment. Whole field is shown for all fluorophores and for the overlay image and enlarged view of just TMF1 and TGN46 is shown. (D) Localization of TMF1 to Cis-Golgi compartment. Whole field is shown for all fluorophores and overlay image. Enlarged view of just TMF1 and GM130 is shown. (E) Localization of TMF1 to ERGIC. Whole field is shown for all fluorophores and overlay image. Enlarged view of just TMF1 and LMAN1. (F) Localization of TMF1 to neuronal ER compartment. Whole field is shown for all fluorophores and overlay image. Enlarged view of just TMF1 and Calnexin is shown. (G) Analysis of TMF1 localization neuronal trans-Golgi (TGN46), cis-Golgi (GM130), ERGIC (LMAN1), or ER (Calnexin) membranes by Pearson’s correlation coefficient. All data is shown as Mean ± SEM with individual data points that represent each cell analyzed for control (open blue circles) and for R3G1-LV-shRNA (gray solid circles). Colocalization of TMF1 with TGN46, in control (0.4387 ± 0.0382; n = 26 cells) and R3G1-shRNA (0.2919 ± 0.04067; n = 35 cells) neurons. Colocalization of TMF1 with GM130, in control (0.5576 ± 0.0312; n = 43 cells) and R3G1-shRNA (0.5033 ± 0.02986; n = 42 cells) neurons. Colocalization of TMF1 with LMAN1 in control (0.5327 ± 0.0265; n = 21 cells) and R3G1-shRNA (0.4962 ± 0.0207; n = 23 cells) neurons. Colocalization of TMF1 with Calnexin, in control (0.2998 ± 0.01974; n = 29 cells) and R3G1-M1 (0.1136 ± 0.0.01119; n = 28 cells) neurons. Statistical analysis was conducted using One way ANOVA corrected for multiple comparisons; ** P < 0.005, **** P < 0.0001, ns = not significant. (H) Protein levels of TMF1 by fluorescence intensity. Analysis of the corrected total fluorescence normalized to the average of the control per batch is shown for control (1.00 ± 0.0457, n = 100 cells) and RAB3GAP1-M1 (1.611 ± 0.0687, n = 136 cells). Samples were analyzed after removing outliers using the ROUT method (Q = 1%). Statistical analysis was conducted using unpaired t -test with Welch’s correction, *** P < 0.0001. (I) Nuclear localization of TMF1 in human neurons. The Mean ± SEM are shown with individual data points representing each cell analyzed for control (open blue circles) and for R3G1-LV-shRNA (gray solid circles) neurons. Colocalization of TMF1 with HOESCHT, in control (0.3695 ± 0.0191, n = 30 cells) and R3G1-LV-shRNA (0.492 ± 0.0186 n = 44 cells) neurons. Statistical analysis was conducted using unpaired t-test with Welch’s correction, **** P < 0.0001. (J-M) Localization of DOCK7 to Trans and Cis-Golgi compartments, ERGIC, and ER membranes. Control (top panels) or RAB3GAP1-Lentiviral shRNA (RAB3GAP1-LV-shRNA, bottom panel) are shown for each immunostaining. Neurons were stained with Hoescht (blue) to label the nuclei, and were immunostained with antibodies against MAP2 (cyan), DOCK7 (red), and either TGN46 or GM130 or LMAN1 or CALNEXIN (all in green) to label the different compartments. All scale bars are 20 μm. (J) Localization of DOCK7 to Trans-Golgi compartment. Whole field is shown for all fluorophores and overlay image. Enlarged view of just DOCK7 and TGN46 is shown. (K) Localization of DOCK7 to cis-Golgi compartment. Whole field is shown for all fluorophores and overlay image. Enlarged view of just DOCK7 and GM130 is shown. (L) Localization of DOCK7 to ERGIC. Whole field is shown for all fluorophores and overlay image. Enlarged view of just DOCK7 and LMAN1 is shown. (M) Localization of DOCK7 to ER compartment. Whole field is shown for all fluorophores and overlay image. Enlarged view of just DOCK7 and CALNEXIN is shown. (N) Analysis of DOCK7 localization neuronal trans-golgi (TGN46), cis-Golgi (GM130), ERGIC (LMAN1), or ER (Calnexin) membranes by Pearson’s correlation coefficient. All data is shown as Mean and SEM with individual data points that represent each cell analyzed for control (open blue circles) and for R3G1-LV-shRNA (gray solid circles). Colocalization of DOCK7 with TGN46, in control (0.5489 ± 0.02245; n = 41 cells) and R3G1-LV-shRNA (0.5894 ± 0.02759; n = 28 cells) neurons. Colocalization of DOCK7 with GM130, in control (0.2426 ± 0.01305; n = 27 cells) and R3G1-LV-shRNA (0.2053 ± 0.0.01597; n = 31 cells) neurons. Colocalization of DOCK7 with LMAN1, in control (0.2781 ± 0.01961; n = 28 cells) and R3G1-LV-shRNA (0.4110 ± 0.02697; n = 25 cells) neurons. Colocalization of DOCK7 with CALNEXIN, in control (0.7887 ± 0.0078; n = 38 cells) and R3G1-LV-shRNA (0.5159 ± 0.0219; n = 44 cells) neurons. (O) Protein levels of DOCK7 by fluorescence intensity. Analysis of the corrected total fluorescence normalized to the average of the control per batch is shown for control (0.9303 ± 0.03690, n = 105 cells) and RAB3GAP1-M1 (0.7991 ± 0.04821, n = 89 cells). Samples were analyzed after removing outliers using the ROUT method (Q = 1%). Statistical analysis was conducted using unpaired t -test with Welch’s correction, * P < 0.05. (P) Analysis of cis-Golgi Area. Measurement of the area covered by GM130 staining was conducted in control (open blue circles) and RAB3GAP1-shRNA (gray solid circles) neurons. The Mean ± SEM is shown with individual data points for control (4.070 ± 0.3202, n = 51 cells) and RAB3GAP1-shRNA (3.019 ± 0.1620, n = 51 cells). Statistical analysis conducted by Unpaired t-test with Welch’s correction, ** P < 0.005. (Q) Analysis of cis-Golgi Compaction. Measurement of GM130 staining compaction ratio was conducted in control (open blue circles) and RAB3GAP1-shRNA (gray solid circles) neurons. The Mean ± SEM is shown with individual data points for control (0.2315 ± 0.02603, n = 30 cells) and RAB3GAP1-shRNA (0.2109 ± 0.02316, n = 32 cells). Statistical analysis conducted by Unpaired t -test with Welch’s correction, ns = not significant. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

Article Snippet: Lentiviral shRNA knockdown was achieved by transducing day 35 neurons with two to five million lentiviral particles (2–5 MOI) (Santa Cruz Biotechnology #sc-152633-V) for 48 h, after which cells were selected with puromycin for 24 h, and analyzed at day 38 of neuronal induction.

Techniques: Expressing, Control, Knockdown, shRNA, Immunostaining, Staining, Fluorescence

Journal: Neurobiology of disease

Article Title: The Warburg micro syndrome protein RAB3GAP1 modulates neuronal morphogenesis and interacts with axon elongation end ER-Golgi trafficking factors

doi: 10.1016/j.nbd.2023.106215

Figure Lengend Snippet: (A) Human neurons knockdown for RAB3GAP1 using Lentiviral shRNA. Representative images are shown for control (left panel) and knockdown (right panel) neurons. Yellow arrows show the longest neurite. Scale bars are 20 μm long. (B–D) Analysis of neuronal morphology in control (open blue circles) and RAB3GAP1-LV-shRNA (gray solid circles) shown as Mean ± SEM with individual points representing individual neurons measured in at least 3 independant experiments. (B) Analysis of primary neurite number in control (5.179 ± 0.237; n = 56 cells) and RAB3GAP1-LV-shRNA (4.956 ± 0.2384; n = 68 cells) neurons. (C) Analysis of mean neurite lenght in control (47.78 ± 2.363; n = 56 cells) and RAB3GAP1-LV-shRNA (32.59 ± 1.384; n = 68 cells) neurons. (D) Sholl analysis of the number of intersections away from the cell soma every 10 m are shown as an average per cell in control (n = 43 cells) and RAB3GAP1-LV-shRNA (n = 26 cells). Statistical analysis (B–C) was done using Unpaired t-test with Welch’s correction; **** P < 0.0001 and with two-way ANOVA for sholl analysis (D); **** P < 0.0001 and ** P < 0.005. (E) Schematic representation of experimental design for siRNA knockdown of RAB3GAP1. (F-G) Analysis of RAB3GAP1 levels in human neurons after siRNA knockdown. (F) Representative western blot is shown for two independent experiments. (G) Control (blue open circles) or RAB3GAP1 (gray solid circles) siRNAs effect on RAB3GAP1 protein levels is shown. Mean ± SEM is shown for control siRNA (1.075 ± 0.050) and RAB3GAP1-siRNA (0.3226 ± 0.055), n = 11 independent experiments. Statistical analysis was conducted using an unpaired t-test, **** P < 0.0001. (H) Representative fields of neurons transfected with either control or RAB3GAP1 siRNA are shown. Neurons were stained β-III-tubulin and images are shown in black and white. Yellow arrows indicate the longest neurite. Scale bar is 50 μm. (I) Analysis of neurite length using Sholl analysis. Neurite length analysis between neurons transfected with either control (blue open circles) or RAB3GAP1 (gray solid circles) siRNAs. Individual points for 4 independent experiments are shown as mean ± SEM for control siRNA (57.58 ± 3.12, n = 149 neurites) and RAB3GAP1 siRNA (43.32 ± 1.98, n = 191 neurites). Statistical analysis was conducted using an unpaired t-test with Welch’s correction, ***P < 0.0001. (J) Analysis of the number of intersections away from the cell soma for neurons transfected with either control (blue open circles) or RAB3GAP1 (gray solid circles) siRNAs. The average for 4 independent experiments is shown per intersection point every 10 μm for control siRNA ( n = 59 neurons) and RAB3GAP1 siRNA ( n = 73 neurons). Mean ± SEM are shown for each average data point. Individual student t -tests were conducted for each intersection point: * P < 0.05 and ** P < 0.001. (K–N) Automated analysis of neuronal arborization by high content imager. Analysis was conducted in 3 independent experiments from 10 random fields for neurons transfected with either control (blue open circles; n = 2274 cells) or RAB3GAP1 (gray solid circles; n = 7568 cells) siRNAs. Four independent measurements were obtained for which the mean ± SEM are shown: (K) Mean outgrowth per cell is the average intensity per cell, and is shown for control siRNA (160 ± 12.90) vs. RAB3GAP1 siRNA (129 ± 5.96); (L) mean neurite number in control siRNA (5.81 ± 0.19) vs. RAB3GAP1 siRNA (6.26 ± 0.27); (M) mean number of branching points in control siRNA (20.41 ± 1.95) vs. RAB3GAP1 siRNA (15.03 ± 0.97); and (N) mean arbor straightness in control siRNA (0.87 ± 0.002) vs. RAB3GAP1 siRNA (0.9 ± 0.001). Individual points are the average per cell for each field per individual experiment. Data was first analyzed using identify outliers using a rout value of 0.1% in graph pad. Statistical analysis was conducted with unpaired t-tests, * P < 0.05, *** P < 0.0001. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

Article Snippet: Lentiviral shRNA knockdown was achieved by transducing day 35 neurons with two to five million lentiviral particles (2–5 MOI) (Santa Cruz Biotechnology #sc-152633-V) for 48 h, after which cells were selected with puromycin for 24 h, and analyzed at day 38 of neuronal induction.

Techniques: Knockdown, shRNA, Control, Western Blot, Transfection, Staining