Journal: Journal of bone and mineral research : the official journal of the American Society for Bone and Mineral Research

Article Title: Neurexin Superfamily Cell Membrane Receptor Contactin-Associated Protein Like-4 (Cntnap4) Is Involved in Neural EGFL-Like 1 (Nell-1)-Responsive Osteogenesis

doi: 10.1002/jbmr.3524

Figure Lengend Snippet: Confirmation of binding affinity between Cntnap4 phages and Nell-1 using a binding dissociation constant ELISA assay. (A) The Basic Local Alignment Search Tool (BLAST) result of the amino acid sequence displayed by the T7 phage constructed with human brain cDNA matched the human Cntnap4 partial protein sequence. Query = the amino acid sequence enclosed by the T7 phage DNA; Sbjct = the matched amino acid sequence Cntnap4. The LamG domains are highlighted in pink. (B) By increasing the number of phages incubated with Nell-1 precoated ELISA plates, the Cntnap4 phage demonstrated significantly higher binding affinity than the control phage. (C) The Cntnap4 phage revealed high binding affinity only to full-length Nell-1 and not to LamG domain-deleted Nell-1. (D) Structures of Nell-1 and Cntnap4 and their potential interaction domains. Nell-1 is a secreted protein composed of 810 amino acids with a molecular weight of ~90 kDa before N-glycosylation and oligomerization. It contains several structural motifs including a laminin G (LamG) domain, a coiled-coil (CC) domain, five cysteine-rich (CR) domains, and six epidermal growth factor (E)-like domains. Cntnap4 is a transmembrane protein of 1310 amino acids consisting of a large extracellular domain, a single membrane-spanning domain, and a short cytoplasmic region at the carboxy-terminus. The extracellular region is composed of a discoidin-like domain (DISC), a fibrinogen-related domain (FreD), two E repeats, and four LamG domains. The cytoplasmic region contains a binding site for PDZ domains. The potential binding domain of Nell-1 and Cntnap4 is highlighted by the blue dashed line. TM= transmembrane. Mean ± SEM of six independent experiments performed in triplicate is shown. *p < 0.05 when compared with control phage.

Article Snippet: Cntnap4 flox/flox -GFP (mCntnap4-CKO-GFP-CKI) mice were provided by Applied StemCell, Inc. (Milpitas, CA, USA) mated with Wnt1-Cre mice (stock #022501; Jackson Laboratory, Bar Harbor, ME, USA) to obtain Cntnap4 flox/ + ; Wnt1-Cre mice ( Supplemental Materials and Methods, subsection 1 ).

Techniques: Binding Assay, Enzyme-linked Immunosorbent Assay, Sequencing, Construct, Incubation, Molecular Weight

Journal: Journal of bone and mineral research : the official journal of the American Society for Bone and Mineral Research

Article Title: Neurexin Superfamily Cell Membrane Receptor Contactin-Associated Protein Like-4 (Cntnap4) Is Involved in Neural EGFL-Like 1 (Nell-1)-Responsive Osteogenesis

doi: 10.1002/jbmr.3524

Figure Lengend Snippet: Cntnap4 and Nell-1 colocalization in osteogenic-committed cells. (A) Of the eight types of tested cell lines, MC3T3-E1 pre-osteoblasts expressed the highest levels of Cntnap4. (B) Of the four types of tested primary cells, NMCC exhibited the highest expression levels of Cntnap4. NMCC = newborn mouse calvarial cells. mRC = mouse rib chondrocytes. hBMSC = human bone marrow stem cells. hARC = human articular chondrocytes. Mean ± SEM of six independent experiments performed in triplicate is shown (A, B). In addition, Nell-1 significantly increased the levels of Cntnap4 in both MC3T3-E1 pre-osteoblasts (C) and NMCC (D). On the contrary, expression of Cntnap2, which was markedly lower than that of Cntnap4, was not responsive to Nell-1 simulation. Mean ± SEM of three independent experiments performed in duplicate is shown (C, D). *p < 0.05 when compared with the group without Nell-1 treatment; #p < 0.05 when compared with the Cntnap4 expression. Moreover, CLSM revealed the colocalization of Nell-1 and Cntnap4 in MC3T3-E1 pre-osteoblasts after 30 minutes of incubation with exogenous recombinant human Nell-1 (E). Colocalization was predominantly found on the plasma membrane. The direct Nell-1/Cntnap4 interaction was validated by Duolink PLA. Similar Nell-1 and Cntnap4 colocalization and protein interactions were also observed in the plasma membrane of NMCC with 30 minutes of Nell-1 treatment (F). Scale bar = 50 μm.

Article Snippet: Cntnap4 flox/flox -GFP (mCntnap4-CKO-GFP-CKI) mice were provided by Applied StemCell, Inc. (Milpitas, CA, USA) mated with Wnt1-Cre mice (stock #022501; Jackson Laboratory, Bar Harbor, ME, USA) to obtain Cntnap4 flox/ + ; Wnt1-Cre mice ( Supplemental Materials and Methods, subsection 1 ).

Techniques: Expressing, Incubation, Recombinant

Journal: Journal of bone and mineral research : the official journal of the American Society for Bone and Mineral Research

Article Title: Neurexin Superfamily Cell Membrane Receptor Contactin-Associated Protein Like-4 (Cntnap4) Is Involved in Neural EGFL-Like 1 (Nell-1)-Responsive Osteogenesis

doi: 10.1002/jbmr.3524

Figure Lengend Snippet: Cntnap4 and Nell-1 colocalization in mouse calvarial bone marrow cavities. (A, B) Calvarial bones of 60-day-old mice showed high-intensity double staining of Nell-1 and Cntnap4 inside the bone marrow cavities. White arrows = marrow cavity cells with both Nell-1/Cntnap4 colocalization staining and PLA signaling; yellow arrow = bone lining cells with Nell-1/Cntnap4 colocalization staining. Nell-1 was detected in the calcified bone of 60-day-old mouse calvaria (C) but not in the surrounding muscle (D). However, unlike the marrow cavity where Nell-1 and Cntnap4 colocalize, Cntnap4 was barely detected in the calcified areas of mouse calvarial bone and the surrounding muscle. Scale bar = 500 μm (black), 100 μm (blue), and 20 μm (white).

Article Snippet: Cntnap4 flox/flox -GFP (mCntnap4-CKO-GFP-CKI) mice were provided by Applied StemCell, Inc. (Milpitas, CA, USA) mated with Wnt1-Cre mice (stock #022501; Jackson Laboratory, Bar Harbor, ME, USA) to obtain Cntnap4 flox/ + ; Wnt1-Cre mice ( Supplemental Materials and Methods, subsection 1 ).

Techniques: Double Staining, Staining

Journal: Journal of bone and mineral research : the official journal of the American Society for Bone and Mineral Research

Article Title: Neurexin Superfamily Cell Membrane Receptor Contactin-Associated Protein Like-4 (Cntnap4) Is Involved in Neural EGFL-Like 1 (Nell-1)-Responsive Osteogenesis

doi: 10.1002/jbmr.3524

Figure Lengend Snippet: Physical interaction between Nell-1 and Cntnap4. Pull-down assays were performed with MC3T3-E1 pre-osteoblasts (A) and NMCC (B). Increased Cntnap4 was detected when beads were coated with His-tagged Nell-1. Co-immunoprecipitation assay with MC3T3-E1 pre-osteoblasts (C) and NMCC (D) demonstrated an increase in Cntnap4 when cells were incubated with Nell-1. To confirm specificity, no Cntnap4 was detected when the agarose beads were not coated with anti-Nell-1 antibody. (E) SPR assay was performed to assess the binding affinity and dynamic relationship between pentameric Nell-1 (Nell-1(5)) and the immobilized Cntnap4extra, which demonstrated classical ligand-receptor binding. Red dashed lines present the kinetic projection performed by Scrubber 2.0 (BioLogic Software Pty Ltd., Campbell, Australia).

Article Snippet: Cntnap4 flox/flox -GFP (mCntnap4-CKO-GFP-CKI) mice were provided by Applied StemCell, Inc. (Milpitas, CA, USA) mated with Wnt1-Cre mice (stock #022501; Jackson Laboratory, Bar Harbor, ME, USA) to obtain Cntnap4 flox/ + ; Wnt1-Cre mice ( Supplemental Materials and Methods, subsection 1 ).

Techniques: Co-Immunoprecipitation Assay, Incubation, SPR Assay, Binding Assay, Software

Journal: Journal of bone and mineral research : the official journal of the American Society for Bone and Mineral Research

Article Title: Neurexin Superfamily Cell Membrane Receptor Contactin-Associated Protein Like-4 (Cntnap4) Is Involved in Neural EGFL-Like 1 (Nell-1)-Responsive Osteogenesis

doi: 10.1002/jbmr.3524

Figure Lengend Snippet: Cntnap4 is indispensable for Nell-1 osteogenic bioactivity in vitro. (A) ALP staining on day 9 and Alizarin red staining on day 14 revealed increased staining in the Nell-1 and BMP2 groups of control shRNA-transfected MC3T3-E1 cells. In Cntnap4-KD MC3T3-E1 cells, high staining intensities of ALP and Alizarin red were only present in the BMP2 group. (B) A time-dependent, steady increase in Ocn and Opn staining was observed in both PBS and recombinant human Nell-1-treated control MC3T3-E1 cells. At each time point, the Nell-1-treated group demonstrated increased staining intensity when compared with the PBS-treated group. In Cntnap4-KD MC3T3-E1 cells, neither PBS nor Nell-1 treatment resulted in detectable positive staining of Ocn or Opn. (C) In Control MC3T3-E1 cells, Alp, Collagen Iα1, and Collagen Iα2 reached peak expression levels 9 days after stimulation, whereas Ocn, Opn, and Bsp displayed time-dependent patterns of increased expression. Notably, higher expression levels were detected for each gene in the Nell-1 treatment group when compared with the PBS group. However, Cntnap4-KD MC3T3-E1 cells did not exhibit significant changes in any osteogenic markers nor any differences between the PBS group and the Nell-1 group. Mean ± SEM of six independent experiments performed in triplicate is shown. *p < 0.05 when compared with the Control + PBS group. Scale bar = 100 μm.

Article Snippet: Cntnap4 flox/flox -GFP (mCntnap4-CKO-GFP-CKI) mice were provided by Applied StemCell, Inc. (Milpitas, CA, USA) mated with Wnt1-Cre mice (stock #022501; Jackson Laboratory, Bar Harbor, ME, USA) to obtain Cntnap4 flox/ + ; Wnt1-Cre mice ( Supplemental Materials and Methods, subsection 1 ).

Techniques: In Vitro, Staining, shRNA, Transfection, Recombinant, Expressing

Journal: Journal of bone and mineral research : the official journal of the American Society for Bone and Mineral Research

Article Title: Neurexin Superfamily Cell Membrane Receptor Contactin-Associated Protein Like-4 (Cntnap4) Is Involved in Neural EGFL-Like 1 (Nell-1)-Responsive Osteogenesis

doi: 10.1002/jbmr.3524

Figure Lengend Snippet: Cntnap4-KD blocks the osteogenic effects of Nell-1 ex vivo. (A) Mineral deposition in the mouse calvarial explants was revealed by Alizarin Complexone during the culture period. Lentiviral overexpression of Nell-1 increased the density of Alizarin Complexone; however, when Nell-1 was overexpressed in Cntnap4-KD samples, the Alizarin Complexone staining was comparable to the control (without Cntnap4-KD or Nell-1 overexpression). (B) Quantification of the maximal width of the frontal and parietal bone overlapping area. Nell-1 overexpression alone increased the overlapping area, whereas Cntnap4-KD alone slightly reduced the overlapping area. When the Cntnap4-KD samples were treated with Nell-1 lentiviral overexpression, the maximal width of the overlapping area remained unchanged (similar to that of the control group). (C) Quantification of the unclosed anterior fontanel area. The calvarial explants in the Nell-1 overexpression group demonstrated completely closed fontanels, whereas anterior fontanels in the control group remained open. Cntnap4-KD alone slightly inhibited the closure of the anterior fontanels. The Cntnap4-KD + Nell-1 overexpression group showed a largely open fontanel area. The edge of each calvarial bone is outlined by a white dotted line; yellow arrows represent the maximal width of the frontal and parietal bone overlapping area (in the coronal suture). P=parietal; F=frontal. Eight calvaria explants were used for each group. For B and C, the means were used as center values. The Mann–Whitney test was used for statistical analysis. p < 0.05, p < 0.01. Scale bar = 100 μm.

Article Snippet: Cntnap4 flox/flox -GFP (mCntnap4-CKO-GFP-CKI) mice were provided by Applied StemCell, Inc. (Milpitas, CA, USA) mated with Wnt1-Cre mice (stock #022501; Jackson Laboratory, Bar Harbor, ME, USA) to obtain Cntnap4 flox/ + ; Wnt1-Cre mice ( Supplemental Materials and Methods, subsection 1 ).

Techniques: Ex Vivo, Over Expression, Staining, MANN-WHITNEY

Journal: Journal of bone and mineral research : the official journal of the American Society for Bone and Mineral Research

Article Title: Neurexin Superfamily Cell Membrane Receptor Contactin-Associated Protein Like-4 (Cntnap4) Is Involved in Neural EGFL-Like 1 (Nell-1)-Responsive Osteogenesis

doi: 10.1002/jbmr.3524

Figure Lengend Snippet: Wnt1-Cre-mediated Cntnap4-knockout (Cntnap4flox/+;Wnt1-Cre) neonatal mice demonstrate decreased craniofacial development. Representative superior view of gross appearance (A), skeletal staining (B), and 3D micro-CT reconstruction (C) images of the craniofacial skeleton of neonatal Cntnap4flox/+;Wnt1-Cre mouse and its wild-type littermate demonstrate the significant difference in the cranial bone formation and sutural patency. In comparison with its wild-type littermate, the neonatal Cntnap4flox/+;Wnt1-Cre mouse has significantly less bone in the anterior fontanel of calvaria (red asterisk). In addition, defective mineralization and bone formation are also observed in the coronal suture (red arrows) of the Cntnap4flox/+;Wnt1-Cre mouse skull, which can also be appreciated from the lateral view (D) and 3D micro-CT reconstructions (E). Six pairs of neonatal Cntnap4flox/+;Wnt1-Cre mice and their wild-type littermates were compared. F = frontal bone; P = parietal bone; I = interparietal bone; N = nasal bone. Scale bar = 1 mm.

Article Snippet: Cntnap4 flox/flox -GFP (mCntnap4-CKO-GFP-CKI) mice were provided by Applied StemCell, Inc. (Milpitas, CA, USA) mated with Wnt1-Cre mice (stock #022501; Jackson Laboratory, Bar Harbor, ME, USA) to obtain Cntnap4 flox/ + ; Wnt1-Cre mice ( Supplemental Materials and Methods, subsection 1 ).

Techniques: Knock-Out, Staining, Micro-CT

Journal: Journal of bone and mineral research : the official journal of the American Society for Bone and Mineral Research

Article Title: Neurexin Superfamily Cell Membrane Receptor Contactin-Associated Protein Like-4 (Cntnap4) Is Involved in Neural EGFL-Like 1 (Nell-1)-Responsive Osteogenesis

doi: 10.1002/jbmr.3524

Figure Lengend Snippet: Cntnap4 is indispensable for the Nell-1-responsive activation of MAPK and Wnt signaling pathways in vitro. (A) Activation of MAPK signaling in Control and Cntnap4-KD MC3T3-E1 pre-osteoblasts stimulated with Nell-1. In Control MC3T3-E1 cells, significantly higher levels of pERK and pJNK were detected 10 minutes and 30 minutes after Nell-1 stimulation, respectively. There was no change detected in the phosphorylation level of P38. In Cntnap4-KD MC3T3-E1 cells, Nell-1 stimulation did not alter the expression levels of pERK and pJNK. Expression of Wnt signaling molecules in the whole cell lysate (B) and cell nuclear lysate (C) of Control and Cntnap4-KD MC3T3-E1 cells treated with Nell-1. In Control MC3T3-E1 cells, Nell-1 significantly increased the expression levels of Axin2 and active β-catenin, whereas no effect was observed on these markers in Cntnap4-KD cells treated with Nell-1. The charts demonstrate mean relative band intensity (normalized to control MC3T3-E1 at 0 minutes) ± SEM for three individual experiments. p < 0.05 when compared with the Control group at 0 minutes, #p < 0.05 when compared with the Cntnap4-KD group at 0 minutes.

Article Snippet: Cntnap4 flox/flox -GFP (mCntnap4-CKO-GFP-CKI) mice were provided by Applied StemCell, Inc. (Milpitas, CA, USA) mated with Wnt1-Cre mice (stock #022501; Jackson Laboratory, Bar Harbor, ME, USA) to obtain Cntnap4 flox/ + ; Wnt1-Cre mice ( Supplemental Materials and Methods, subsection 1 ).

Techniques: Activation Assay, In Vitro, Expressing

Journal: Journal of bone and mineral research : the official journal of the American Society for Bone and Mineral Research

Article Title: Neurexin Superfamily Cell Membrane Receptor Contactin-Associated Protein Like-4 (Cntnap4) Is Involved in Neural EGFL-Like 1 (Nell-1)-Responsive Osteogenesis

doi: 10.1002/jbmr.3524

Figure Lengend Snippet: Schematic diagram of Nell-1 signaling pathways during osteogenesis. As a secreted molecule, Nell-1 initiates cellular signaling through binding to its specific receptor, Cntnap4, on the cell surface. The MAPK and Wnt signaling pathways play critical roles in Nell-1-mediated osteogenesis. Nell-1 preferentially activates ERK and JNK in MAPK signaling and also promotes the phosphorylation/activation of Runx2, which stimulates the expression of Nell-1 and Ocn by directly binding to the OSE2 region of their promoters. In addition, Nell-1 promotes the expression of Axin2 and active β-catenin and increases the nuclear translocation of active β-catenin.

Article Snippet: Cntnap4 flox/flox -GFP (mCntnap4-CKO-GFP-CKI) mice were provided by Applied StemCell, Inc. (Milpitas, CA, USA) mated with Wnt1-Cre mice (stock #022501; Jackson Laboratory, Bar Harbor, ME, USA) to obtain Cntnap4 flox/ + ; Wnt1-Cre mice ( Supplemental Materials and Methods, subsection 1 ).

Techniques: Binding Assay, Activation Assay, Expressing, Translocation Assay

Journal: PLOS One

Article Title: Targeted knockdown of DNA methyltransferase 3a (DNMT3a) unlocks dedifferentiation and neurogenic potential in mouse retinal Müller glia

doi: 10.1371/journal.pone.0337891

Figure Lengend Snippet: (A–C) Time-course analysis of mRNA expression levels of progenitor markers Ascl1, Nestin and Lin28 , normalized to Gapdh , measured by qRT-PCR. NMDA and DNMT3a KD alone transiently upregulated these markers, with a more pronounced and sustained effect observed in the DNMT3a KD NMDA group. Data represent mean ± SEM of three independent experiments; *p < 0.05 vs. CTRL at the corresponding point (two-way ANOVA with Tukey’s post hoc test). (D) Quantification of Lin28 fluorescence intensity in arbitrary units (A.U.) from immunocytochemistry images shown in (E). Significant increases in Lin28 expression were observed in NMDA, DNMT3a KD and DNMT3a KD NMDA groups compared to CTRL (*p < 0.05 vs. CTRL; #p < 0.05 vs. NMDA; one-way ANOVA with Tukey’s post hoc test). (E) Representative phase contrast and immunofluorescence images showing DAPI (nuclei, blue) and Lin28 (green) staining at 48 h post-treatment. CTRL: control, NMDA: exposure to NMDA 100 µM, MOCK: electroporation without plasmid, dCas9: transfection of empty plasmid, CRISPRi: CRISPR interference-mediated Dnmt3a knockdown, CRISPRi NMDA: Dnmt3a knockdown with simultaneous exposure to NMDA 100 µM. Phase contrast images scale bar = 100 μm, 10X; Immunofluorescence images scale bar = 20 µm, 40X.

Article Snippet: After that the samples were incubated with following primary antibodies: mouse monoclonal anti-βlll tubulin antibody (Santa Cruz sc-80005) (1:300); rabbit polyclonal anti-GS antibody (Abcam ab73593) (1:300); mouse monoclonal anti-nestin antibody (Santa Cruz sc-23927) (1:200); rabbit polyclonal anti-Lin28 (Abcam ab46020) (1:200) diluted in 1:3 blocking solution at 4°C overnight.

Techniques: Expressing, Quantitative RT-PCR, Fluorescence, Immunocytochemistry, Immunofluorescence, Staining, Control, Electroporation, Plasmid Preparation, Transfection, CRISPR, Knockdown

Journal: Biology Open

Article Title: Cullin-3 and its adaptor protein ANKFY1 determine the surface level of integrin β1 in endothelial cells

doi: 10.1242/bio.029579

Figure Lengend Snippet: ANKFY1 is a BTBP associating with CUL3 to regulate cellular distribution of integrin β1, cell spreading on the BM, and angiogenesis. (A) Western blots of cell lysates of HUVECs at 72 h post-transfection of siRNAs. (B) Confocal images of intracellular integrin β1 and α2. HUVECs were fixed after 72 h transfection of siRNAs. Magnifications of the squared areas are shown on the right. Representative colocalized integrin β1 and α2 are indicated by arrows. (C) Confocal images of the cell surface integrin β1. HUVECs were fixed after 72 h transfection of siRNA and stained for integrin β1 by Alexa488-conjugated TS2/16 without membrane permeabilization. (D) Quantitation of C; 50 of cells from three independent experiments were analyzed. Data show the mean±s.e.m. *** P <0.001. (E) Confocal images of HUVECs spreading on the BM. HUVECs treated with control siRNA and ANKFY1 siRNA #1, #2 were seeded on the BM and incubated for 1 h. Bars; 20 µm. (F) Areas of the cells in E; >70 cells from three independent experiments were analyzed. Data show the mean±s.e.m. *** P <0.001. (G) Confocal images of tube formation. HUVECs seeded on collagen I gel were treated with control, CUL3 or ANKFY1 siRNA and packed on collagen I followed by VEGF stimulation for 66 h. Scale bars: 20 µm in B, C and E; 200 µm in G.

Article Snippet: The following antibodies were purchased from the manufacturers as indicated: goat anti-integrin β1 antibody (N-20, dilution 1:1000 for western blotting; Santa Cruz Biotechnology), mouse anti-integrin β1 antibody (P5D2, dilution 1:1000; R&D Systems), mouse anti-integrin β1 antibody (TS2/16, dilution 1:500 for western blotting and immunofluorescence; BioLegend, San Diego, USA), mouse anti-CUL3 antibody (CUL3-9, dilution 1:1000; Sigma-Aldrich), Alexa488-conjugated mouse anti-integrin β1 antibody (TS2/16, dilution 1:200; BioLegend), rabbit anti-integrin α2 antibody (EPR17338, dilution 1:6000 for western blotting, dilution 1:1000 for immunofluorescence; Abcam), mouse anti-ANKFY1 antibody (B-6, dilution 1:100 for western blotting, dilution 1:50 for immunofluorescence; Santa Cruz Biotechnology), mouse anti-paxillin antibody (349, dilution 1:200; BD Bioscience), mouse anti-vinculin antibody (hVIN-1, dilution 1:1000; Sigma-Aldrich), rabbit anti-EGFR antibody (D38B1, dilution 1:1000; Cell Signaling Technology), mouse anti-Calnexin antibody (ab2798, dilution 1:1000; Abcam), mouse anti-GAPDH antibody (5A12, dilution 1:6000; Wako, Tokyo, Japan), rabbit anti-HA antibody (Y-11, dilution 1:1000 for western blotting and immunofluorescence, dilution 1:100 for immunoprecipitation; Santa Cruz Biotechnology), mouse anti-Myc antibody (9E10, dilution 1:1000; Santa Cruz Biotechnology), mouse anti-FLAG antibody (M2, dilution 1:1000; Sigma-Aldrich), rabbit anti-Alexa488 antibody (A-11094, dilution 1:200; Thermo Fisher Scientific), HRP-conjugated anti-mouse IgG antibody (W4021, dilution 1:2000; Promega, Madison, USA), HRP-conjugated anti-rabbit IgG antibody (W4011, dilution 1:2000; Promega), HRP-conjugated anti-goat IgG antibody (V805A, dilution 1:2000; Promega), goat Alexa488-conjugated anti-mouse IgG antibody (A11001, dilution 1:2000; Molecular Probes, Eugene, USA), donkey Alexa488-conjugated anti-rat IgG antibody (A21208, dilution 1:2000; Molecular Probes), goat Alexa568-conjugated anti-mouse IgG antibody (A11004, dilution 1:2000; Molecular Probes), and goat Cy3-conjugated anti-rabbit IgG antibody (111-165-144, dilution 1:2000; Jackson ImmunoResearch Laboratories).

Techniques: Western Blot, Transfection, Staining, Membrane, Quantitation Assay, Control, Incubation

Journal: Biology Open

Article Title: Cullin-3 and its adaptor protein ANKFY1 determine the surface level of integrin β1 in endothelial cells

doi: 10.1242/bio.029579

Figure Lengend Snippet: Interaction of ANKFY1 and CUL3. (A) FLAG-CUL3, ANKFY1-HA, HA-ANKFY1, Myc-Nedd8 and mock plasmid (pcDNA3.1) were expressed in HEK293T cells for 48 h. ANKFY1 tagged at its N terminus or C terminus with HA was expressed to validate the effects of the location of the tag on its interaction with CUL3. The lysates were then immunoprecipitated with anti-HA antibody. Total cell lysates (input) and immunoprecipitates (IP) were separated by SDS-PAGE and then blotted for CUL3 and HA. The asterisks indicate neddylated CUL3. IgG heavy and light chains are shown in the blot with anti-Myc antibody. (B) FLAG-CUL3, ANKFY1-HA and Myc-Nedd8 were expressed in HEK293T cells for 48 h. The lysates were then immunoprecipitated with anti-HA antibody. Total cell lysates (input) and IP were separated by SDS-PAGE, and then blotted for CUL3 and HA. Before cell lysis, HUVECs were treated with 1 µM MLN-4924 for 20 h. The asterisks indicate neddylated CUL3. IgG heavy and light chains are shown in the blot with anti-Myc antibody.

Article Snippet: The following antibodies were purchased from the manufacturers as indicated: goat anti-integrin β1 antibody (N-20, dilution 1:1000 for western blotting; Santa Cruz Biotechnology), mouse anti-integrin β1 antibody (P5D2, dilution 1:1000; R&D Systems), mouse anti-integrin β1 antibody (TS2/16, dilution 1:500 for western blotting and immunofluorescence; BioLegend, San Diego, USA), mouse anti-CUL3 antibody (CUL3-9, dilution 1:1000; Sigma-Aldrich), Alexa488-conjugated mouse anti-integrin β1 antibody (TS2/16, dilution 1:200; BioLegend), rabbit anti-integrin α2 antibody (EPR17338, dilution 1:6000 for western blotting, dilution 1:1000 for immunofluorescence; Abcam), mouse anti-ANKFY1 antibody (B-6, dilution 1:100 for western blotting, dilution 1:50 for immunofluorescence; Santa Cruz Biotechnology), mouse anti-paxillin antibody (349, dilution 1:200; BD Bioscience), mouse anti-vinculin antibody (hVIN-1, dilution 1:1000; Sigma-Aldrich), rabbit anti-EGFR antibody (D38B1, dilution 1:1000; Cell Signaling Technology), mouse anti-Calnexin antibody (ab2798, dilution 1:1000; Abcam), mouse anti-GAPDH antibody (5A12, dilution 1:6000; Wako, Tokyo, Japan), rabbit anti-HA antibody (Y-11, dilution 1:1000 for western blotting and immunofluorescence, dilution 1:100 for immunoprecipitation; Santa Cruz Biotechnology), mouse anti-Myc antibody (9E10, dilution 1:1000; Santa Cruz Biotechnology), mouse anti-FLAG antibody (M2, dilution 1:1000; Sigma-Aldrich), rabbit anti-Alexa488 antibody (A-11094, dilution 1:200; Thermo Fisher Scientific), HRP-conjugated anti-mouse IgG antibody (W4021, dilution 1:2000; Promega, Madison, USA), HRP-conjugated anti-rabbit IgG antibody (W4011, dilution 1:2000; Promega), HRP-conjugated anti-goat IgG antibody (V805A, dilution 1:2000; Promega), goat Alexa488-conjugated anti-mouse IgG antibody (A11001, dilution 1:2000; Molecular Probes, Eugene, USA), donkey Alexa488-conjugated anti-rat IgG antibody (A21208, dilution 1:2000; Molecular Probes), goat Alexa568-conjugated anti-mouse IgG antibody (A11004, dilution 1:2000; Molecular Probes), and goat Cy3-conjugated anti-rabbit IgG antibody (111-165-144, dilution 1:2000; Jackson ImmunoResearch Laboratories).

Techniques: Plasmid Preparation, Immunoprecipitation, SDS Page, Lysis

Journal: Biology Open

Article Title: Cullin-3 and its adaptor protein ANKFY1 determine the surface level of integrin β1 in endothelial cells

doi: 10.1242/bio.029579

Figure Lengend Snippet: CUL3 determines the endosomal localization of ANKFY1. (A) Confocal images of HUVECs treated with control siRNA and CUL3 siRNA #1 for 72 h. CUL3-knockdown of HUVECs infected with siRNA of resistant-FLAG-CUL3 (WT or K712R)-carrying lentivirus for 48 h. Cells were fixed, permeabilized and stained for ANKFY1. Magnifications of the squared areas are shown in the lower panels. Scale bars: 20 µm. (B) Quantitation of A. The fluorescence intensities of ANKFY1 signals in 50 cells from three independent experiments were analyzed. Data show the mean±s.e.m. ** P <0.01; * P <0.05. (C) Western blots of HUVEC lysates treated with control siRNA and CUL3 siRNA #1 for 72 h.

Article Snippet: The following antibodies were purchased from the manufacturers as indicated: goat anti-integrin β1 antibody (N-20, dilution 1:1000 for western blotting; Santa Cruz Biotechnology), mouse anti-integrin β1 antibody (P5D2, dilution 1:1000; R&D Systems), mouse anti-integrin β1 antibody (TS2/16, dilution 1:500 for western blotting and immunofluorescence; BioLegend, San Diego, USA), mouse anti-CUL3 antibody (CUL3-9, dilution 1:1000; Sigma-Aldrich), Alexa488-conjugated mouse anti-integrin β1 antibody (TS2/16, dilution 1:200; BioLegend), rabbit anti-integrin α2 antibody (EPR17338, dilution 1:6000 for western blotting, dilution 1:1000 for immunofluorescence; Abcam), mouse anti-ANKFY1 antibody (B-6, dilution 1:100 for western blotting, dilution 1:50 for immunofluorescence; Santa Cruz Biotechnology), mouse anti-paxillin antibody (349, dilution 1:200; BD Bioscience), mouse anti-vinculin antibody (hVIN-1, dilution 1:1000; Sigma-Aldrich), rabbit anti-EGFR antibody (D38B1, dilution 1:1000; Cell Signaling Technology), mouse anti-Calnexin antibody (ab2798, dilution 1:1000; Abcam), mouse anti-GAPDH antibody (5A12, dilution 1:6000; Wako, Tokyo, Japan), rabbit anti-HA antibody (Y-11, dilution 1:1000 for western blotting and immunofluorescence, dilution 1:100 for immunoprecipitation; Santa Cruz Biotechnology), mouse anti-Myc antibody (9E10, dilution 1:1000; Santa Cruz Biotechnology), mouse anti-FLAG antibody (M2, dilution 1:1000; Sigma-Aldrich), rabbit anti-Alexa488 antibody (A-11094, dilution 1:200; Thermo Fisher Scientific), HRP-conjugated anti-mouse IgG antibody (W4021, dilution 1:2000; Promega, Madison, USA), HRP-conjugated anti-rabbit IgG antibody (W4011, dilution 1:2000; Promega), HRP-conjugated anti-goat IgG antibody (V805A, dilution 1:2000; Promega), goat Alexa488-conjugated anti-mouse IgG antibody (A11001, dilution 1:2000; Molecular Probes, Eugene, USA), donkey Alexa488-conjugated anti-rat IgG antibody (A21208, dilution 1:2000; Molecular Probes), goat Alexa568-conjugated anti-mouse IgG antibody (A11004, dilution 1:2000; Molecular Probes), and goat Cy3-conjugated anti-rabbit IgG antibody (111-165-144, dilution 1:2000; Jackson ImmunoResearch Laboratories).

Techniques: Control, Knockdown, Infection, Staining, Quantitation Assay, Fluorescence, Western Blot

Journal: bioRxiv

Article Title: The RNA helicase UPF1 associates with mRNAs co-transcriptionally and is required for the release of mRNAs from transcription sites

doi: 10.1101/395863

Figure Lengend Snippet: ( A ) Fluorescence imaging of tetramethylrhodamine conjugated Wheat Germ Agglutinin (WGA, red) and UPF1-GFP (green) in salivary gland cells. Lower panels are magnified view of boxed area in upper panels. ( B ) Imaging of WGA (red) in wild type (upper panel) and FkhGAL4>UPF1-RNAi (lower panel) salivary gland cells. Yellow arrow indicates nuclear envelope. Cells were counter-stained with DAPI (blue).

Article Snippet: These antibodies were used for immunostaining: mouse anti-UPF1 (described in this paper 7B12, typically diluted 1:100), mouse IgM anti-Ser2 Pol II (H5, Covance AB_10143905, 1:500), mouse anti-hnRNPA1 (Hrb87F, P11, 1:50) , mouse anti-GFP (B-2, Santa Cruz, SC-9996, 1:200), Tetramethylrhodamine Conjugate Wheat Germ Agglutinin (Thermo Fisher, W7024, 10µg/mL).

Techniques: Fluorescence, Imaging, Staining

Journal: bioRxiv

Article Title: The RNA helicase UPF1 associates with mRNAs co-transcriptionally and is required for the release of mRNAs from transcription sites

doi: 10.1101/395863

Figure Lengend Snippet: ( A ) Fluorescence immunolocalization of UPF1 using the 7B12 monoclonal antibody (Cy3, red) on polytene chromosomes (blue) of wild type (I, II) and UPF1-RNAi (III, IV) salivary glands. ( B ) Western blotting probed with 7B12 mab for protein extracts of 3 rd instar larval salivary gland from FkhGAL4>UPF1-GFP (lane I), wild type (lane II) and FkhGAL4>UPF1-RNAi (lane III). Ponceau staining of the same blot showing equal protein loading. ( C ) Immunolocalization of UPF1-GFP (FITC, green, I, III) on polytene chromosomes of FkhGAL4>UPF1-GFP salivary glands, detected using anti-GFP antibody. Chromosomes were counter stained with DAPI (blue, II, III). Line profiles in IV show both signal intensities along the white line traced on the chromosome arm in III. Note that UPF1 signal peaks at chromatin-decondensed regions characterised by low DAPI signal.

Article Snippet: These antibodies were used for immunostaining: mouse anti-UPF1 (described in this paper 7B12, typically diluted 1:100), mouse IgM anti-Ser2 Pol II (H5, Covance AB_10143905, 1:500), mouse anti-hnRNPA1 (Hrb87F, P11, 1:50) , mouse anti-GFP (B-2, Santa Cruz, SC-9996, 1:200), Tetramethylrhodamine Conjugate Wheat Germ Agglutinin (Thermo Fisher, W7024, 10µg/mL).

Techniques: Fluorescence, Western Blot, Staining

Journal: bioRxiv

Article Title: The RNA helicase UPF1 associates with mRNAs co-transcriptionally and is required for the release of mRNAs from transcription sites

doi: 10.1101/395863

Figure Lengend Snippet: ( A ) UPF1 (red) and Ser2 Pol II (pink) ChIP-seq enrichment profiles at RpL23A (on left) and RpS12 (on right) gene loci. ( B ) Real-time PCR quantification of average ChIP signal of either endogenous UPF1 (red) or GFP (as negative control, grey) at the RpL23 and RpS12 genes in salivary glands expressing GFP. The locations of the primer pairs used are indicated by the black boxes (P) shown below the genes schematics in A. ( C ) Real-time PCR quantification of average UPF1 association in control (red) or UPF1-RNAi (blue) S2 cells. ( D ) Real-time PCR quantification of average UPF1 association at three distinct regions of RpL23 in S2 cells (same primers pairs as in ) with (blue) or without (red) RNase A treatment. ( E ) Ser2 Pol II immunoprecipitation of S2 cell nuclear extracts using anti-Ser2 Pol II antibody (ab5095) and detection (same blot) of Ser2 Pol II, UPF1, eIF4AIII and hnRNPA1, in control (lanes 2-3) or RNase treated samples (lanes 4-5). IP refers to immunoprecipitated fractions, Ub to unbound fractions.

Article Snippet: These antibodies were used for immunostaining: mouse anti-UPF1 (described in this paper 7B12, typically diluted 1:100), mouse IgM anti-Ser2 Pol II (H5, Covance AB_10143905, 1:500), mouse anti-hnRNPA1 (Hrb87F, P11, 1:50) , mouse anti-GFP (B-2, Santa Cruz, SC-9996, 1:200), Tetramethylrhodamine Conjugate Wheat Germ Agglutinin (Thermo Fisher, W7024, 10µg/mL).

Techniques: ChIP-sequencing, Real-time Polymerase Chain Reaction, Negative Control, Expressing, Immunoprecipitation

Journal: Cancers

Article Title: Extracellular Vesicle Transfer from Endothelial Cells Drives VE-Cadherin Expression in Breast Cancer Cells, Thereby Causing Heterotypic Cell Contacts

doi: 10.3390/cancers12082138

Figure Lengend Snippet: Cancer–endothelial cell interaction induces expression of endothelial cell (EC) markers in breast cancer cells. ( A ) Immunofluorescent staining for vascular endothelial cadherin (VE-cadherin) (red) in MCF7-green fluorescent protein (GFP) cells (green), which were added onto a human umbilical vein endothelial cell (HUVEC) monolayer for 24, 48, and 72 h. The protein ( B ) (full western blot figures and mRNA ( C ) levels of VE-cadherin were analyzed in GFP-labeled cancer cells, MCF7-GFP and MDA-MB-231-GFP, which were isolated by fluorescence-activated cell sorting (FACS) after co-culturing with HUVECs for different time points (24, 48, and 72 h). Values are presented as means ± SD ( n = 3) (* p ≤ 0.05; ** p ≤ 0.01). ( D ) The mRNA levels of vascular endothelial growth factor receptor I (VEGFRI), and VEGFRII were determined by qPCR in the isolated MCF7-GFP and MDA-MB-231-GFP cells. Values are presented as means ± SD of the fold changes as compared to the monocultured tumor cells (TCs) ( n = 3) (* p ≤ 0.05; ** p ≤ 0.01) ( E ) The soluble VE-cadherin ectodomains, soluble VE (sVE)-cadherin, shedded by HUVECs into the cell supernatant, were detected in cancer cell lysates with the BV9 antibody by Western blot. sVE-cadherin was not stable in cancer cells and was lost within 24 h (lane R) after the removal of the HUVEC-conditioned medium (full western blot figure. As a positive control, lysates of cancer cells co-cultured with HUVECs were used (two left lanes). ( F ) Immunofluorescence labeling of VE-cadherin in MCF7 cells treated with HUVEC medium for 48 h showed increased VE-cadherin-positive signal in the nucleus. ( G ) The positive VE-cadherin staining in the nucleus was biometrically quantified by ImageJ. For the calculation of VE-cadherin-positive signal in the nucleus, we evaluated n = 141 Ctrl cells (gray bar), and n = 130 MCF7-cells treated with HUVEC supernatant (black bar) for 24 h. Means values ± SD are shown (** p ≤ 0.01). ( H ) MCF7 cells transfected with the VE-cadherin-tdTomato reporter gene were treated with HUVEC culture supernatant, co-cultured with HUVECs (positive control), or monocultured (negative control). The activity of VE-cadherin promoter was quantified by staining the cells with a primary antibody against tdTomato and secondary antibody against tdTomato conjugated with Alexa Fluor 488 (green). Western blots of ( B , E ) are shown , ( G ) is shown in , ( C ) is shown in .

Article Snippet: Membranes were incubated with the following primary antibodies: goat anti-VE-cadherin (C-19) (1:100; Santa Cruz, Dallas, TX, USA), mouse anti-VE-cadherin (BV9) (1:100; Santa Cruz), mouse anti-Cadherin 11 (1:50; Invitrogen), mouse anti-E-cadherin (1:100; BD Biosciences), or mouse anti-vimentin (1:50; BD Biosciences), followed by incubation with HRP-conjugated secondary IgG (1:1000; Dako, Hamburg, Germany).

Techniques: Expressing, Staining, Western Blot, Labeling, Isolation, Fluorescence, FACS, Positive Control, Cell Culture, Immunofluorescence, Transfection, Negative Control, Activity Assay

Journal: Nature Communications

Article Title: A drug-free cardiovascular stent functionalized with tailored collagen supports in-situ healing of vascular tissues

doi: 10.1038/s41467-024-44902-2

Figure Lengend Snippet: a Representative rhodamine-conjugated phalloidin (TRITC-Phalloidin, red) and 4′,6-diamidino-2-phenylindole (DAPI, blue) fluorescence images of HUVECs showing the (rhCol III/PDA-PEI) n coatings loaded with different amounts of rhCol III all encouraged HUVECs compared with control PLA group. Scale bars, 200 μm. Quantification of ( b , c ) cell number and ( d , e ) cell viability of HUVECs cultured on uncoated and (rhCol III/PDA-PEI) n -coated PLA ( n = 1, 2, and 4) sheets after 1, 3, 5, and 7 days of culture ( n = 5 independent samples). f Volcano plot showing differentially expressed genes in the (rhCol III/PDA-PEI) 2 group compared to the PLA control group. Downregulated and upregulated genes are colored blue and red, respectively, at significantly differentially expressed thresholds │log 2 FC│ > 1.2 and p < 0.05. g Gene Ontology (GO) analysis of differentially expressed genes in PLA versus (rhCol III/PDA-PEI) 2 . h Heatmap of the differentially expressed genes in the PLA and (rhCol III/PDA-PEI) 2 groups. i Circular visualization of the results of gene-annotation enrichment analysis. j Quantification of the expression of representative genes in PLA versus (rhCol III/PDA-PEI) 2 , validated by qRT-PCR arrays. The value in the PLA group was normalized to that in the (rhCol III/PDA-PEI) 2 group ( n = 3 independent samples). k Representative immunofluorescence staining of CCL5 (green), GATA3 (green), XBP1 (green), and CEACAM6 (green) of HUVECs on 3 days of culture. Scale bar, 50 µm. l Schematic diagram of the potential three signaling pathways involved in the regulation of HUVEC behavior induced by the (rhCol III/PDA-PEI) 2 coating, including PI3K/AKT, mTOR, and MAPK. Two-way ANOVA with Tukey’s multiple comparisons was used for the comparisons in ( b )–( e ) and ( j ). Two-sided Student’s t test with multiple testing corrections was used in ( f ) and ( g ). The data are presented as the mean ± SD ( p values < 0.05 were considered statistically significant).

Article Snippet: The primary antibodies used in this study included mouse monoclonal XBP1 (Cat. No.: sc-8015, Clone: F-4, Santa Cruz Biotechnology, USA, 1:50), mouse polyclonal CCL5 (Cat. No.: sc-365826, Clone: A-4, Santa Cruz Biotechnology, USA, 1:50), mouse monoclonal CEACAM6 (Cat. No.: sc-59899, Clone: 9A6, Santa Cruz Biotechnology, USA, 1:50), rabbit monoclonal GATA3 (Cat. No.: ab199428, Clone: EPR16651, Abcam, USA, 1:500), rabbit polyclonal F4/80 (Cat. No.: 29414-1-AP, Proteintech, China, 1:100), rabbit monoclonal CD68 (Cat. No.: ab283654, Clone: EPR23917-164, Abcam, USA, 1:100), rabbit polyclonal CD86 (Cat. No.: bs-1035R, Biosynthesis Biotechnology co., ltd, USA, 1:200), rabbit monoclonal CD206 (Cat. No.: 24595, Clone: E6T5J, Cell Signaling Technology, USA, 1:200), mouse monoclonal α-SMA (Cat. No.: ab7817, Clone: 1A4, Abcam, USA, 1:200), and rabbit monoclonal MMP2 (Cat. No.: 10373-2-AP, Clone: SB13a, Proteintech, USA, 1:200), mouse monoclonal CD31 (Cat. No.: ab9498, Clone: JC/70A, Abcam, USA, 1:200), and rabbit polyclonal eNOS (Cat. No.: ab5589, Abcam, USA, 1:100).

Techniques: Fluorescence, Control, Cell Culture, Expressing, Quantitative RT-PCR, Immunofluorescence, Staining, Protein-Protein interactions

Journal: Nature Neuroscience

Article Title: Neuronal activity rapidly reprograms dendritic translation via eIF4G2:uORF binding

doi: 10.1038/s41593-024-01615-5

Figure Lengend Snippet: a , Immunofluorescence (IF) images of primary cortical neurons immunostained for glial fibrillary acidic protein (GFAP) and oligodendrocyte transcription factor 2 (OLIG2) simultaneously with PSD95 to show that the cultures are devoid of glial cells or oligodendrocytes, respectively. DAPI for nuclei; PSD95 for excitatory neurons. Magnification, ×40. Scale bars, 50 μm. b , TurboID-PSD95 was cloned without (top row) and with (bottom row) its 5′ and 3′ UTRs and lentivirally expressed in primary cortical neurons. White dashed boxes are zoomed in areas in black&white images. DAPI for nuclei; MAP2 for dendrites; Flag for each TurboID. % dendritically localized TurboID-PSD95 is quantified by co-localization with MAP2 signal in ImageJ. 3 different areas of images per replicate ( n = 3). Magnification, ×20. Scale bars, 50 μm. Significance was derived from biological replicates, showing the center line at mean. c , IF images of TurboID-PSD95-transduced neurons immunostained for DAPI (blue, for nuclei), PSD95 (red, for endogenous PSD95) and TurboID-PSD95 (cyan, detected by Flag). Magnification, ×60. Scale bar, 50 μm. d , IF images show the expression of a presynaptic marker, Synaptophysin (cyan), and TurboID-PSD95 (red, detected by Flag antibody) in primary cortical neurons transduced with TurboID-PSD95. DAPI (blue) marker for nuclei. Three zoomed in regions are marked by the white boxes. Magnification, ×60. Scale bar, 10 μm. e , IF images show TurboID expression and biotinylation in primary cortical neurons transduced with TurboID-PSD95 or Pan-TurboID after 30 minutes of biotin incubation. DAPI (blue, nuclei); MAP2 (green, dendrites); Flag (red, TurboID); and Streptavidin (cyan, biotinylated proteins). Magnification, ×20. Scale bars, 50 μm. f , Western blots stained for Flag and β-Actin from Pan-TurboID and TurboID-PSD95-transduced neurons in the absence (−) or presence (+) of exogenous biotin shown to indicate the relative expression levels of TurboID proteins. Quantifications of TurboID protein levels normalized to β-Actin are shown on the right ( n = 3); relative levels are not significant by two-tailed, paired Student’s t -test. g , Western blots stained for streptavidin signal in inputs (‘in’) and streptavidin pulldowns (‘pd’) from Pan-TurboID or TurboID-PSD95-transduced neurons in the absence (−) or presence (+) of exogenous biotin. h , Streptavidin pulldowns shown for dendritic (SHANK3, GKAP, NLGN1 and HOMER1) and negative control (GAPDH) proteins from TurboID-PSD95-transduced neurons in the absence (−) or presence (+) of exogenous biotin. Flag signal indicates self-biotinylation of each construct. Loaded on the gel are 10% (by volume) of input and 50% (by volume) of pulldowns. Percent isolated by TurboID-PSD95 in each condition is calculated by dividing the signal in the pulldown lane by that of the input lane, after each is adjusted to total, and quantifications are shown as bar graphs ( n = 3). P values: Flag = 0.58, SHANK3 = 0.0061, GKAP = 0.018, NLGN1 = 0.00052, HOMER1 = 0.021, GAPDH = 0.42. i , Streptavidin pulldowns shown for dendritic (BAIAP2 and DLGAP3) and nuclear (TBR1, H4 and H2AX) proteins from Pan-TurboID and TurboID-PSD95-transduced neurons in the presence (+) of exogenous biotin. Loaded on the gel are 10% (by volume) of input and 50% (by volume) of pulldowns. Percent isolated by each TurboID is calculated as in (h) ( n = 3). P values: BAIAP2 = 0.0052, DLGAP3 = 0.0035, TBR1 = 0.0063, H4 = 0.018, H2AX = 0.0037. j , Phosphorylation of EEF2, eIF2α, ERK1/2 and IRE1 and total levels of ATF4 and CHOP are shown in resting (rest), activated (DHPG, Dep) and stressed (Sodium arsenite (NaAsO 2 )) cells by using phospho-specific and total antibodies. The amount of phosphorylated or total protein is shown in the bar graphs, calculated by dividing the phosphorylated signal to total and β-Actin for the phosphorylated proteins and by dividing the total to β-Actin for ATF4 and CHOP ( n = 3). Significance was calculated with respect to rest. P values: P-EEF2 (DHPG = 0.0088, Dep = 0.0023, NaAsO 2 = 0.039), P-eIF2α (DHPG = 0.018, Dep = 0.0034, NaAsO 2 = 0.028), P-ERK1/2 (DHPG = 0.015, Dep = 0.0067, NaAsO 2 = 0.00084), P-IRE1 (DHPG = 0.06, Dep = 0.37, NaAsO 2 = 0.0027), ATF4 (DHPG = 0.038, Dep = 0.42, NaAsO 2 = 0.016), CHOP (DHPG = 0.044, Dep = 0.18, NaAsO 2 = 0.024). k , Quantitative PCR (qPCR) results shown for immediate early genes, Arc , Fos and Jun . The fold changes for each gene are calculated by first normalizing to the house-keeping gene β-Actin in each condition, then dividing the value of each condition by that of the resting state ( n = 3). l , Dendritic spine size in resting and KCl-depolarized neurons are measured using the Keyence microscope. Red squares are examples of spines that are counted ( n = 3, 12 spines from each biological replicate are counted as technical replicates). Significance was derived from the biological replicates using the two-tailed, unpaired Student’s t -test. Box plots show the min and max, with the center line at median. Magnification, ×100. Scale bars, 5 μm. m , Fluo-4-AM staining in resting, KCl-depolarized and DHPG-depolarized cells. Fluo4-AM was loaded in resting cells and measurements were taken at indicated time points after Fluo4-AM removal. In depolarized cells, the dye was loaded during silencing. After silencing, fluorescence was measured during stimulus at 10, 30 and 60-minute time points for the KCl treatment and at 10-minute for the DHPG-induced activation. Fluorescence was also measured 60 minutes after the stimulus removal (60′post KCl and 60′post DHPG). Circles represent data from 2 biological and 3 technical replicates. Below: Examples of Fluo4-AM fluorescence are shown in resting, 10-minute KCl-treated and 10-minute DHPG-treated neurons. Fluo4-AM loading (45 minutes) was performed during the last 45 minutes of the silencing step prior to stimulus addition for the KCl and DHPG treatment and simultaneously for the resting neurons. Imaging was performed 10 minutes after the stimulus was added. Scale bars, 50 μm. (b,f,h-k,m) Data are mean ± s.d. Significance was calculated using the two-tailed, paired Student’s t -test. P values: ns (not significant) >0.05; * <0.05; ** <0.01; *** <0.001; **** <0.0001. n indicates the number of biologically independent samples.

Article Snippet: Puromycin (1:3,000, mouse, Kerafast, EQ0001, RRID: AB_2620162), Flag (1:3,000, mouse, Sigma-Aldrich, F1804, RRID: AB_262044), β-Actin antibody (1:2,500, mouse, Sigma-Aldrich, A1978, RRID: AB_476692), RPL10A (1:1,000, rabbit, Abcam, ab174318), MAP2 (1:2,500, guinea pig, Synaptic Systems, 188004, RRID: AB_2138181), GFAP (1:500, rabbit, Abcam, ab7260, RRID: AB_305808), OLIG2 (1:500, rabbit, Proteintech, 13999-1-AP, RRID: AB_2157541), PSD95 (1:500, mouse, Millipore, MABN68, RRID: AB_10807979), Synaptophysin (1:300, mouse, Abcam, ab8049, RRID: AB_2198854), SHANK3 (1:500, mouse, Novus, NBP1-47610, RRID: AB_10010567), GKAP (1:500, rabbit, Novus, NBP1-76911, RRID: AB_11017331), NLGN1 (1:200, mouse, Novus, NBP2-42192), HOMER1 (1:1,000, rabbit, Proteintech, 12433-1-AP, RRID: AB_2295573), GAPDH (1:5,000, mouse, Thermo Fisher Scientific, AM4300, RRID: AB_2536381), BAIAP2 (1:500, rabbit, Proteintech, 11087-2-AP, RRID: AB_2063075), DLGAP3 (1:500, rabbit, Proteintech, 55056-1-AP, RRID: AB_10858793), TBR1 (1:500, rabbit, Proteintech, 20932-1-AP, RRID: AB_10695502), H4 (1:1,000, mouse, Abcam, ab31830, RRID: AB_1209246), H2A.X (1:1,000, rabbit, Proteintech, 10856-1-AP, RRID: AB_2114985), EEF2 (1:1,000, rabbit, Cell Signaling Technology, 2332, RRID:AB_10693546), P-EEF2 (1:1,000, rabbit, Cell Signaling Technology, 2331, RRID: AB_10015204), eIF2α (1:1,000, rabbit, Cell Signaling Technology, 9722, RRID: AB_2230924), P-eIF2α (1:1,000, rabbit, Cell Signaling Technology, 3398, RRID: AB_2096481), p42/44 MAPK (1:1,000, rabbit, Cell Signaling Technology, 4695, RRID: AB_390779), P-p42/44 MAPK (1:1,000, rabbit, Cell Signaling Technology, 9101, RRID: AB_331646), P-IRE1 (1:500, rabbit, Novus, NB100-2323SS, RRID: AB_10145203), IRE1 (1:500, rabbit, Novus, NB100-2324SS, RRID: AB_10000972), CHOP (1:1,000, mouse, Cell Signaling, 2895T, RRID: AB_2089254), ATF4 (1:1,000, rabbit, Cell Signaling Technology, 11815S, RRID: AB_2616025), MPHOSPH (1:300, rabbit, Biorbyt, orb100446), KCNJ9 (1:300, rabbit, LSBio, LS-C352416), KCNJ9 (1:300, mouse, Antibodies Incorporated, 75-445, RRID: AB_2686912), eIF4G2 (1:1,000, rabbit, Cell Signaling Technology, RRID: AB_10622189 and rabbit, Cell Signaling Technology, RRID: AB_2261993), NSUN3 (1:250, rabbit, LSBio, LS-C163024), MTF1 (1:300, rabbit, Novus, NBP1-86380, RRID: AB_11011361), ZFP64 (1:300, rabbit, Proteintech, 17187-1-AP, RRID: AB_2218826) and KATNBL1 (1:250, rabbit, Proteintech, 24795-1-AP, RRID: AB_2879730).

Techniques: Immunofluorescence, Clone Assay, Derivative Assay, Expressing, Marker, Transduction, Incubation, Western Blot, Staining, Two Tailed Test, Negative Control, Construct, Isolation, Real-time Polymerase Chain Reaction, Microscopy, Fluorescence, Activation Assay, Imaging