goat anti clusterin  (R&D Systems)

Journal: Cancer Research Communications

Article Title: Age-Related Increases in IGFBP2 Increase Melanoma Cell Invasion and Lipid Synthesis

doi: 10.1158/2767-9764.CRC-23-0176

Figure Lengend Snippet: Aged fibroblasts secrete high levels of IGFBP2. A, Re-analysis of proteomics on CM from young and aged dermal fibroblasts, published in Kaur and colleagues , showing differentially expressed proteins between the two groups, in which red denotes increased expression in aged as compared with young, and blue denotes decreased expression. B, IGFBP2 ELISA analysis in young and aged human dermal fibroblast CM ( P = 0.0055). C, IGFBP2 staining in human melanoma skin reconstructs with young or aged donor-derived dermal fibroblasts. D, IGFBP2 staining in primary tumor tissue from young and aged C57BL6 mice. E, RPPA analysis of young and aged YUMM1.7 mouse tumor lysate for IGFBP2 expression ( P = 0.002). F, Pathway analysis of RPPA analysis of young and aged YUMM1.7 mouse tumor lysate. G, P-AKT (Ser473) staining of tumors in young and aged mice. H, Oil Red O staining of tumors in young and aged mice.

Article Snippet: Mice were i.p. injected with neutralizing Igfbp2 antibody (AF797 from R&D systems) or an IgG control (AB-105-C from R&D systems) at a concentration of 1 mg/kg every day until tumors reached 1,500 mm 3 .

Techniques: Expressing, Enzyme-linked Immunosorbent Assay, Staining, Derivative Assay

Journal: Cancer Research Communications

Article Title: Age-Related Increases in IGFBP2 Increase Melanoma Cell Invasion and Lipid Synthesis

doi: 10.1158/2767-9764.CRC-23-0176

Figure Lengend Snippet: IGFBP2 induces fatty acid synthesis in melanoma cells. A, Western blot analysis of Igfbp2, phospho-Akt, total Akt and Hsp90 (loading control) from young and aged tumor lysate. Quantification of immunoblotting of Igfbp2 and phospho-Akt relative to Hsp90 loading control. B, Correlation analysis of IGFBP2 protein expression and p-AKT S473 expression from RPPA of melanoma PDX samples. C, Correlation analysis of IGFBP2 protein expression and p-AKT T308 expression from RPPA of melanoma PDX samples. D, Western blot analysis of melanoma cells (1205Lu) treated with recombinant IGFBP2 (100 ng/mL) at different times. Cells were probed for IGFBP2, phospho-AKT, total AKT, and HSP90 (loading control). E, Western blot analysis of IGFBP2 knockdown in aged fibroblasts. F, BODIPY (505/515) staining of human melanoma cells (1205Lu) cultured with aged fibroblast CM from fibroblasts transduced with empty vector (PLKO.1) or shRNA constructs (shIGFBP2). Quantification of BODIPY. G, BODIPY (505/515) staining of melanoma cells treated with CM from young fibroblasts treated with recombinant IGFBP2 (50 and 100 ng/mL). Quantification of BODIPY. H, RT-PCR of IGFBP2 expression in melanoma cells after treatment with young or aged CM. I, Western blot analysis of IGFBP2, P-AKT, total AKT, and HSP90 in melanoma cells transfected with either an empty vector control (CTRL) or IGFBP2 (IGFBP2 OE). J, BODIPY (493/503) staining of melanoma cells after transfection with either control or IGFBP2 constructs with quantification. GraphPad Prism 8 was used for plotting graphs and statistical analysis. Unpaired t test was performed.

Article Snippet: Mice were i.p. injected with neutralizing Igfbp2 antibody (AF797 from R&D systems) or an IgG control (AB-105-C from R&D systems) at a concentration of 1 mg/kg every day until tumors reached 1,500 mm 3 .

Techniques: Western Blot, Control, Expressing, Recombinant, Knockdown, Staining, Cell Culture, Transduction, Plasmid Preparation, shRNA, Construct, Reverse Transcription Polymerase Chain Reaction, Transfection

Journal: Cancer Research Communications

Article Title: Age-Related Increases in IGFBP2 Increase Melanoma Cell Invasion and Lipid Synthesis

doi: 10.1158/2767-9764.CRC-23-0176

Figure Lengend Snippet: IGFBP2 increases melanoma cell migration and invasion. A, Wound healing assay of human melanoma cells (1205Lu, WM164) cultured with young or aged CM in the presence or absence of a neutralizing IGFBP2 antibody (5 mg/mL) or recombinant IGFBP2 (150 ng/mL). B, Matrigel invasion assay of melanoma cells (1205Lu, WM164) cultured with young and aged CM in the presence or absence of a neutralizing IGFBP2 antibody or recombinant IGFBP2. C, Melanoma cells (1205Lu) grown in 3D spheroids cultured with aged CM and young CM in the presence or absence of recombinant IGFBP2 (150 ng/mL) for 48 hours. GraphPad Prism 8 was used for plotting graphs and statistical analysis. Unpaired t test was performed.

Article Snippet: Mice were i.p. injected with neutralizing Igfbp2 antibody (AF797 from R&D systems) or an IgG control (AB-105-C from R&D systems) at a concentration of 1 mg/kg every day until tumors reached 1,500 mm 3 .

Techniques: Migration, Wound Healing Assay, Cell Culture, Recombinant, Invasion Assay

Journal: Cancer Research Communications

Article Title: Age-Related Increases in IGFBP2 Increase Melanoma Cell Invasion and Lipid Synthesis

doi: 10.1158/2767-9764.CRC-23-0176

Figure Lengend Snippet: Igfbp2 increases melanoma tumor growth and metastasis in vivo . A, mCherry-tagged YUMM1.7 murine melanoma cells were grown in young (8 weeks old) C57BL6 mice. Tumor growth of young mice after i.p. treatment with 500 ng recombinant Igfbp2 or PBS (5 mice/group, treated i.p., 2 times a week, after tumors were palpable). B, IHC analysis of Igfbp2 in tumors from young mice treated with PBS or recombinant Igfbp2. C, Western blot analysis of tumor lysate from young mice treated with PBS and recombinant Igfbp2. Densitometry of phospho-AKT relative to HSP90 loading control. D, Tumor growth curve of YUMM1.7 melanoma cells subdermally injected in old (52 weeks old) mice treated i.p. with Igfbp2-neutralizing antibody (at a concentration of 1 mg/kg every day, n = 5) vs. an IgG control ( n = 5). E, IHC analysis of Igfbp2 staining in aged mice treated with either an IgG control or neutralizing Igfbp2 antibody. F, Protein expression analysis was performed on tumor lysate from aged mice treated with IgG or neutralizing Igfbp2 antibody. Quantification analysis of phospho-Akt and Fasn immunoblotting relative to Hsp90 loading control. G, Analysis of mCherry-positive cells in lungs of tumor-bearing aged mice treated with a neutralizing Igfbp2 antibody or IgG control. *, P < 0.05 student t test was used. GraphPad Prism 8 was used for plotting graphs and statistical analysis.

Article Snippet: Mice were i.p. injected with neutralizing Igfbp2 antibody (AF797 from R&D systems) or an IgG control (AB-105-C from R&D systems) at a concentration of 1 mg/kg every day until tumors reached 1,500 mm 3 .

Techniques: In Vivo, Recombinant, Western Blot, Control, Injection, Concentration Assay, Staining, Expressing

Journal: Journal of Experimental & Clinical Cancer Research : CR

Article Title: Icaritin-curcumol activates CD8 + T cells through regulation of gut microbiota and the DNMT1/IGFBP2 axis to suppress the development of prostate cancer

doi: 10.1186/s13046-024-03063-2

Figure Lengend Snippet: ICA-CUR inhibits the DNMT1/IGFBP2 pathway and activates the cytotoxic effect of CD8 + T cell. A The levels of IGFBP2 in serum were tested via ELISA. B The levels of DNMT1 and IGFBP2 in tumor tissues were tested via IHC (Magnification: ×100, scale bar = 100 μm; Magnification: ×400, scale bar = 25 μm). C The levels of DNMT1 and IGFBP2 in tumor tissues were detected by WB. D The protein changes of PD-L1, EGFR, STAT3, p-EGFR, and p-STAT3 in tumor tissues were detected by WB. E FCM was used to test the infiltration of CD8 + T cells (positivity of CD3 + CD8 + IFN-γ and CD3 + CD8 + Ki67 cells). F The levels of perforin, granzyme A, and B in sorted CD8 + T cells from tumor tissues were detected by RT-PCR. G The levels of IFN-γ and IFN-α in serum were tested via ELISA. * P < 0.05 vs. PCa

Article Snippet: According to the kit’s instructions, ELISA was utilized to evaluate levels of IGFBP2 (CSB-E04589m, CUSABIO), interferon-γ (IFN-γ) (KE10001, Proteintech), interferon-α (IFN-α) (MFNAS0, R&D Systems), Perforin (Cbic-E13429m, CUSABIO), Granzyme A (CSB-E08717m, CUSABIO, China) and Granzyme B (CSB-E08720m, CUSABIO, China).

Techniques: Enzyme-linked Immunosorbent Assay, Reverse Transcription Polymerase Chain Reaction

Journal: Journal of Experimental & Clinical Cancer Research : CR

Article Title: Icaritin-curcumol activates CD8 + T cells through regulation of gut microbiota and the DNMT1/IGFBP2 axis to suppress the development of prostate cancer

doi: 10.1186/s13046-024-03063-2

Figure Lengend Snippet: FMT from donors in the ICA-CUR treatment inhibits the development of PCa and activates the cytotoxic effects of CD8 + T cells. A Tumor imaging, volume, and weight measurements. B IF staining was utilized to examine changes in Ki67 expression in tumors (Magnification: ×400, scale bar = 25 μm). C The levels of IGFBP2 in serum were tested via ELISA. D The levels of DNMT1 and IGFBP2 in tumor tissues were detected by WB. E WB was used to detected the protein changes of PD-L1, EGFR, STAT3, p-EGFR, and p-STAT3 in tumor tissues. F The infiltration of CD8 + T cells in mouse tumor tissues (positivity of CD3 + CD8 + IFN-γ and CD3 + CD8 + Ki67 cells) was detected by FCM. G The expression of perforin, granzyme A, and B in sorted CD8 + T cells from tumor tissues was detected by RT-PCR. H The levels of IFN-γ and IFN-α in serum were tested via ELISA. * P < 0.05 vs. FMT-PCa

Article Snippet: According to the kit’s instructions, ELISA was utilized to evaluate levels of IGFBP2 (CSB-E04589m, CUSABIO), interferon-γ (IFN-γ) (KE10001, Proteintech), interferon-α (IFN-α) (MFNAS0, R&D Systems), Perforin (Cbic-E13429m, CUSABIO), Granzyme A (CSB-E08717m, CUSABIO, China) and Granzyme B (CSB-E08720m, CUSABIO, China).

Techniques: Imaging, Staining, Expressing, Enzyme-linked Immunosorbent Assay, Reverse Transcription Polymerase Chain Reaction

Journal: Journal of Experimental & Clinical Cancer Research : CR

Article Title: Icaritin-curcumol activates CD8 + T cells through regulation of gut microbiota and the DNMT1/IGFBP2 axis to suppress the development of prostate cancer

doi: 10.1186/s13046-024-03063-2

Figure Lengend Snippet: ICA-CUR inhibits tumor development and activates cytotoxic effects of CD8 + T cells by suppressing the SCFAs-IGFBP2 axis. A The levels of DNMT1 and IGFBP2 in tumor tissues were detected by WB. B The levels of IGFBP2 in serum were tested via ELISA. C The levels of DNMT1 and IGFBP2 in tumor tissues were detected by WB. * P < 0.05 vs. ICA + CUR. D Tumor imaging, volume, and weight measurements. E IF staining to examine changes in Ki67 expression in tumors (Magnification: ×400, scale bar = 25 μm). F ELISA was used to detect the levels of IGFBP2 in serum. G The protein changes of PD-L1, EGFR, STAT3, p-EGFR, and p-STAT3 in tumor tissues were detected by WB. H The infiltration of CD8 + T cells in mouse tumor tissues (positivity of CD3 + CD8 + IFN-γ and CD3 + CD8 + Ki67 cells) was detected by FCM. I The levels of perforin, granzyme A, and B in sorted CD8 + T cells from tumor tissues were detected by RT-PCR. J ELISA was utilized to detect the levels of IFN-γ and IFN-α in serum. * P < 0.05 vs. ICA + CUR + IgG, # P < 0.05 vs. ICA + CUR + anti-IGFBP2

Article Snippet: According to the kit’s instructions, ELISA was utilized to evaluate levels of IGFBP2 (CSB-E04589m, CUSABIO), interferon-γ (IFN-γ) (KE10001, Proteintech), interferon-α (IFN-α) (MFNAS0, R&D Systems), Perforin (Cbic-E13429m, CUSABIO), Granzyme A (CSB-E08717m, CUSABIO, China) and Granzyme B (CSB-E08720m, CUSABIO, China).

Techniques: Enzyme-linked Immunosorbent Assay, Imaging, Staining, Expressing, Reverse Transcription Polymerase Chain Reaction

Journal: Journal of Experimental & Clinical Cancer Research : CR

Article Title: Icaritin-curcumol activates CD8 + T cells through regulation of gut microbiota and the DNMT1/IGFBP2 axis to suppress the development of prostate cancer

doi: 10.1186/s13046-024-03063-2

Figure Lengend Snippet: ICA-CUR inhibits the development of PCa, the DNMT1/IGFBP2 pathway, and activates cytotoxic effects of CD8 + T cells in vitro. A CCK-8 was utilized to assess the proliferation ability of cells. B Transwell was applied to measure the migration and invasion ability of cells. C The level of IGFBP2 in cells was tested via ELISA. D The levels of DNMT1 and IGFBP2 in cells were detected by WB. E WB was utilized to monitor protein changes of PD-L1, EGFR, STAT3, p-EGFR, and p-STAT3. * P < 0.05 vs. Control. F FCM analysis of CD8 + IFN-γ cells. G The expression of perforin, granzyme A, and B in sorted CD8 + T cells was detected by WB. H IL-2, IFN-γ, and IFN-α levels in supernatant were measured through ELISA. I Perforin and granzyme B levels in the supernatant were tested via ELISA. & P < 0.05 vs. RM-1 + T cells

Article Snippet: According to the kit’s instructions, ELISA was utilized to evaluate levels of IGFBP2 (CSB-E04589m, CUSABIO), interferon-γ (IFN-γ) (KE10001, Proteintech), interferon-α (IFN-α) (MFNAS0, R&D Systems), Perforin (Cbic-E13429m, CUSABIO), Granzyme A (CSB-E08717m, CUSABIO, China) and Granzyme B (CSB-E08720m, CUSABIO, China).

Techniques: In Vitro, CCK-8 Assay, Migration, Enzyme-linked Immunosorbent Assay, Control, Expressing

Journal: Journal of Experimental & Clinical Cancer Research : CR

Article Title: Icaritin-curcumol activates CD8 + T cells through regulation of gut microbiota and the DNMT1/IGFBP2 axis to suppress the development of prostate cancer

doi: 10.1186/s13046-024-03063-2

Figure Lengend Snippet: ICA-CUR inhibits the development of PCa and activates the cytotoxic effects of CD8 + T cells through the inhibition of the DNMT1/IGFBP2 pathway. A Transfection efficiency detection by WB. B CCK-8 was utilized to assess the proliferation ability of cells. C Cell migration and invasion ability detection by Transwell assay. D The levels of DNMT1 and IGFBP2 in cells were detected by WB. E WB was utilized to monitor protein changes of PD-L1, EGFR, STAT3, p-EGFR, and p-STAT3. F FCM analysis of CD3 + CD8 + IFN-γ cells. G The levels of perforin, granzyme A, and B in sorted CD8 + T cells were detected by RT-qPCR. H The levels of IL-2, IFN-γ, and IFN-α in serum. I perforin, granzyme A and B levels in the supernatant were tested via ELISA. * P < 0.05 vs. Control, # P < 0.05 vs. ICR + CUR + oe-NC, & P < 0.05 vs. ICR + CUR + oe-DNMT1 + si-NC

Article Snippet: According to the kit’s instructions, ELISA was utilized to evaluate levels of IGFBP2 (CSB-E04589m, CUSABIO), interferon-γ (IFN-γ) (KE10001, Proteintech), interferon-α (IFN-α) (MFNAS0, R&D Systems), Perforin (Cbic-E13429m, CUSABIO), Granzyme A (CSB-E08717m, CUSABIO, China) and Granzyme B (CSB-E08720m, CUSABIO, China).

Techniques: Inhibition, Transfection, CCK-8 Assay, Migration, Transwell Assay, Quantitative RT-PCR, Enzyme-linked Immunosorbent Assay, Control

Journal: Journal of Experimental & Clinical Cancer Research : CR

Article Title: Icaritin-curcumol activates CD8 + T cells through regulation of gut microbiota and the DNMT1/IGFBP2 axis to suppress the development of prostate cancer

doi: 10.1186/s13046-024-03063-2

Figure Lengend Snippet: Inhibiting DNMT1 could suppress PCa development and activate the cytotoxic effects of CD8 + T cells by inhibiting the IGFBP2/EGFR/STAT3/PD-L1 pathway. A Transfection efficiency detection by WB. B CCK-8 was utilized to assess the proliferation ability of cells. C Cell migration and invasion ability detection by Transwell assay. D The levels of DNMT1 and IGFBP2 were detected by WB. E , F WB was utilized to monitor protein changes of PD-L1, EGFR, STAT3, p-EGFR, p-STAT3, DNMT1, and IGFBP2. G FCM analysis of CD3 + CD8 + IFN-γ cells. H The levels of perforin, granzyme A, and B were detected by RT-PCR. I The levels of IL-2, IFN-γ, and IFN-α in serum. J Perforin, granzyme A, and B levels in the supernatant were tested via ELISA. * P < 0.05 vs. si-NC, # P < 0.05 vs. si-DNMT1 + oe-NC

Article Snippet: According to the kit’s instructions, ELISA was utilized to evaluate levels of IGFBP2 (CSB-E04589m, CUSABIO), interferon-γ (IFN-γ) (KE10001, Proteintech), interferon-α (IFN-α) (MFNAS0, R&D Systems), Perforin (Cbic-E13429m, CUSABIO), Granzyme A (CSB-E08717m, CUSABIO, China) and Granzyme B (CSB-E08720m, CUSABIO, China).

Techniques: Transfection, CCK-8 Assay, Migration, Transwell Assay, Reverse Transcription Polymerase Chain Reaction, Enzyme-linked Immunosorbent Assay

Journal: Advanced science (Weinheim, Baden-Wurttemberg, Germany)

Article Title: Reprogramming Macrophage Polarization, Depleting ROS by Astaxanthin and Thioketal-Containing Polymers Delivering Rapamycin for Osteoarthritis Treatment.

doi: 10.1002/advs.202305363

Figure Lengend Snippet: Fig. 3 Altruistic cancer cells regenerate via epigenetic mechanism. A Mathematical model explains the persistence of altruistic subclone in breast cancer cell population (Upper), and a spatial model simulates changes in the percentage of altruistic cells over time for hypothesized genetic- and epigenetics-mediated altruism (Lower). Dotted lines link events in upper panel to corresponding points in lower panel. See Supplementary Note 2 & 3. B Indicated cancer cell lines were sorted according to Mi/EGFP levels and the fluorescence monitored over indicated time. C Mi/EGFP fluorescence of non-sorted MDA-MB-231miR-125bprom-EGFP cells, treated as indicated, as determined by FACS. D Mi/EGFPLow cells were transfected with indicated siRNAs and the Mi/EGFP fluorescence determined after four days. E Schema showing putative consensus sites for KLF2 binding along the hsa-miR-125b-1 promoter and gRNA targeting sequence of CRISPRi. F Mi/EGFPLow cells were transduced with lentivirus for expression of CRISPRi targeting KBS-1 or non-specific sequence, and Mi/EGFP fluorescence determined after four days. G,H Changes in Mi/ EGFP fluorescence as treated in (F) were monitored in indicated cell lines (G). Percentage effect of KBS-1 CRISPRi expression on cell viability relative to control CRISPRi was also measured. Cancer cells were exposed to indicated concentration of docetaxel (H). I MDA-MB-231miR-125bprom-EGFP cells, as treated in (F), were grown as xenografts in NSG mice. Upper: excised tumors at end point of monitoring period. Lower: Percentage change in tumor size with and without docetaxel treatment. J Immunoblotting to detect for IGFBP2 and CCL28 in conditioned media from MDA-MB-231miR-125bprom-EGFP cells as treated in (F) and used to establish xenograft model for (I). Ponceau S was used to visualize protein load. Quantification of band intensities (relative to Control CRISPRi) is shown. Experiments repeated two times (B, C, D, F, G, H, J). Representative data are shown for (J). Mean percentage ± s.d. cells for technical triplicates of representative set are shown in same colours as corresponding histograms (C, D, F, G) or in black only (B). Data are mean ± s.d. from two independent biological sets of triplicates (H). n = 4 independent animals per group (I). Statistical analysis was performed using two-tailed one sample t-test against 0 (H) and two-tailed unpaired t-test (I lower). NT: no treatment; DTX: docetaxel treatment. Exact P values are shown

Article Snippet: 4’,6-Diamidino-2-Phenylindole (DAPI) from Thermo Fisher Scientific; IDEAL miRNA assays from MiRXES; Superscript VILO enzyme from Thermo Fisher Scientific; RPMI and DMEM from Thermo Fisher Scientific; McCoy’s 5A modified medium, Leibovitz L-15 medium and EMEM from Lonza; Trichostatin A (T8552), 5-azacytidine (A2385), curcumin (C1386), anacardic acid (05506), MB-3 (M2449) from Sigma-Aldrich; nitro-blue tetrazolium and 5-bromo-4-chloro-3-indolyl-phosphate (NBT/ BCIP) tablets from Roche; Lipofectamine 3000 from Thermo Fisher Scientific; mirVana miR-125b oligonucleotide mimic (4464066) and negative control oligonucleotide mimic (4464058) tagged with fluorescein from Thermo Fisher Scientific; siRNA against PCAF (sc-36198), HDAC3 (sc-35538), PCAF (sc-36198), p53 (sc-29435), Ah receptor (sc-29654), Sp1 (sc-29487), TIP60 (sc-37966), FOXO3 (sc-37887), KLF2 (sc-35818), IKKβ (sc-35644), β-catenin (CTNNB1; sc-29209), SMO (sc-40161), GSK-3β (sc35527), NFκB p65 (sc-29410), NFκB p50 (sc-29408) and control (sc-37007) from Santa Cruz; azidohomoalanine (AS-63669) from AnaSpe; docetaxel from Sanofi-Aventis; VivoGlo Luciferin (P1043) from Promega; Recombinant IGFBP2 (350-06B-20) and Recombinant CCL28 (300-57- 20) from Peprotech; PI3K inhibitor LY294002 (HY-10108) from MedChemExpress,; Akt Inhibitor IV (sc-203809) from Santa Cruz Biotechnology, ; control (339121) and miR-125b miRNA LNA inhibitor (339126) from Qiagen.

Techniques: Fluorescence, Transfection, Binding Assay, Sequencing, Transduction, Expressing, Control, Concentration Assay, Western Blot, Two Tailed Test

Journal: Nature neuroscience

Article Title: Aberrant astrocyte protein secretion contributes to altered neuronal development in multiple models of neurodevelopmental disorders

doi: 10.1038/s41593-022-01150-1

Figure Lengend Snippet: a,c,e. Example images from Figure 5a,​,cc,​,ee prior to processing and analysis. Neurons cultured for 48 hours in RTT (a), FXS (c) or DS (e) ACM and immunostained with MAP2 (dendrites, green) and tau (axon, red). b,d,f. Relative frequency distribution plot of total neurite outgrowth, pooled data from 3 (b), 4 (d), 5 (f) experiments per graph, same data as Figure 5b,​,dd,​,f.f. Number of neurons: RTT (b): alone=439, WT ACM=549, RTT ACM=633, RTT ACM + Igfbp2-Ab=621; FXS (d): alone=492, WT ACM=608, FXS ACM=621, FXS ACM + Igfbp2-Ab=647; DS (f): alone=716, WT ACM=992, DS ACM=765, DS ACM + Igfbp2-Ab=858. g. Relative frequency distribution plot of neuronal cell body size, pooled data from 3 experiments, same data as Figure 5g. h-l Cell body size of upper layer cortical neurons is not different between WT and RTT mice, and unaffected by the Igfbp2 neutralizing antibody. h. Schematic of the experiment: P2 mice were injected in the visual cortex with AAV synapsin-GFP +/− antibody, and tissue collected at P7 and GFP-expressing neurons imaged, with the region to be imaged identified by the presence of the fluorescently labeled antibody. i,j. Cell body area in upper layer neurons is unaltered in RTT compared to WT mice. i. Analysis by mice, graph average ± s.e.m., individual data points represent mice, N = 4 WT and 4 RTT mice, statistics by 2-sided T-test. j. Analysis by cells, graph individual data points represent cells, dashed line represents the mean, n = 162 WT and 177 RTT cells, statistics by 2-sided T-test. k,l. Cell body area in upper layer neurons in RTT mice is unaffected by an Igfbp2 neutralizing antibody. k. Analysis by mice, graph average ± s.e.m., individual data points represent mice, N = 5 control-Ab and 5 Igfbp2-Ab mice, statistics by 2-sided T-test. l. Analysis by cells, graph individual data points represent cells, dashed line represents the mean, n = 183 control-Ab and 195 Igfbp2-Ab cells, statistics by 2-sided T-test.

Article Snippet: For Igfbp2 blocking antibody experiments injections were performed on littermate pairs of male Mecp2-/y (RTT) mice, receiving either the Igfbp2 blocking antibody (R&D MAB797) or the isotype control antibody (R&D 6–001-F) at 1mg/ml in PBS, labeled with Alexafluor 594 to visualize the injection site (Molecular Probes A20185), with the AAV-Synapsin-GFP virus diluted in the antibody solution.

Techniques: Cell Culture, Injection, Expressing, Labeling

Journal: Nature neuroscience

Article Title: Aberrant astrocyte protein secretion contributes to altered neuronal development in multiple models of neurodevelopmental disorders

doi: 10.1038/s41593-022-01150-1

Figure Lengend Snippet: a-c. BMP6-treated WT astrocytes show upregulated protein secretion (a) and gene expression (b) that overlaps with ND astrocytes. c. Heatmap of proteins increased in all ND and BMP6-treated astrocytes vs. WT, ranked by protein abundance in BMP6-treated ACM. N=6 cultures each WT, FXS, RTT, DS, plus 6 cultures WT +/− BMP6. d,e. ACM from WT astrocytes treated with BMP6 inhibits WT neurite outgrowth. d. Example images of WT neurons cultured for 48 hours, conditions as marked (merged images of MAP2 + Tau). e. Quantification of total neurite outgrowth. Data from 3 experiments, number of neurons: alone=467, WT ACM=733, BMP6 WT ACM=610. f. Blocking Igfbp2 overcomes the inhibitory effect of BMP6 WT ACM on neurite outgrowth. Data from 3 experiments, number of neurons: alone=378, WT ACM=380, BMP6 WT ACM=506, BMP6 WT ACM + Igfbp2 blocking Ab=335. g-j. Blocking BMP signaling in ND astrocytes rescues deficits in WT neurite outgrowth. g. Experimental schematic for noggin treatment of FXS or RTT astrocytes. h. Blocking BMP signaling in FXS astrocytes rescues deficits in WT neurite outgrowth. Example images of WT neurons cultured for 48 hours, conditions as marked (merged images of MAP2 + Tau). i. Quantification of total neurite outgrowth. Data from 3 experiments, number of neurons: alone=923, WT ACM=1164, FXS ACM=1132, Noggin FXS ACM=1099. j. Blocking BMP signaling in RTT astrocytes rescues deficits in WT neurite outgrowth. Quantification of total neurite outgrowth. Data from 3 experiments, number of neurons: alone=238, WT ACM=279, RTT ACM=387, Noggin RTT ACM=365. Violin plot: dashed line median, dotted lines 25th and 75th percentile. Statistics by Kruskal-Wallis one-way ANOVA on ranks with Dunn’s test for multiple comparisons. See also Extended Data Figure 7; Tables S5, S7.

Article Snippet: For Igfbp2 blocking antibody experiments injections were performed on littermate pairs of male Mecp2-/y (RTT) mice, receiving either the Igfbp2 blocking antibody (R&D MAB797) or the isotype control antibody (R&D 6–001-F) at 1mg/ml in PBS, labeled with Alexafluor 594 to visualize the injection site (Molecular Probes A20185), with the AAV-Synapsin-GFP virus diluted in the antibody solution.

Techniques: Expressing, Cell Culture, Blocking Assay

Journal: Nature neuroscience

Article Title: Aberrant astrocyte protein secretion contributes to altered neuronal development in multiple models of neurodevelopmental disorders

doi: 10.1038/s41593-022-01150-1

Figure Lengend Snippet: a. Schematic of IGF signaling via the PI3K/Akt pathway. b,c. Protein secretion (b) and gene expression (c) profiles of IGF family members in WT and ND astrocytes. Proteomics, N=6 cultures/genotype, *p<0.05, abundance >0.01%, fold change between WT and ND ≥1.5 calculated with T-fold test in Patternlab. RNASeq, N=6 cultures WT, RTT, FXS; 4 cultures DS, *adjusted p<0.05, FPKM>1, fold change between ND and WT ≥1.5 calculated with DESeq2. d,e. Excess Igfbp2 in ACM inhibits WT neurite outgrowth. d. Example images WT neurons grown 48 hours, conditions as marked (image merge of MAP2 + Tau). e. Quantification total neurite outgrowth normalized to control alone condition. Violin plot: dashed line median, dotted lines 25th and 75th percentile. Data from 3 separate experiments, number of neurons: control alone=447, control ACM=626, Igfbp2 alone=438, Igfbp2 ACM=596, Igfbp2 + Ab alone=376, Igfbp2 + Ab + ACM=562. Statistics by Kruskal-Wallis one-way ANOVA on ranks with Dunn’s test for multiple comparisons. f, g. Astrocytes express Igfbp2 mRNA in the P7 mouse visual cortex. f. smFISH against Igfbp2 mRNA in Aldh1l1-GFP mice to mark astrocytes, combined with probe for neurons (Tubb3). Left panels overview of cortex across all layers, right panel high power image from L2/3. g. Analysis of images in f, expression of Igfbp2 mRNA within astrocytes, neurons and OPCs. See Figure S4d for OPC image. N=3 WT mice. Bar graph mean±s.e.m., individual data points mice; statistics by one-way ANOVA with Tukey’s test for multiple comparisons. h-j. Immunostaining for Igfbp2 in each ND and littermate WT visual cortex at P7 (RTT - Mecp2 KO; FXS – Fmr1 KO; DS - Ts65Dn TG) reveals an increase in extracellular Igfbp2 in RTT and intracellular Igfbp2 in DS. h. Example images of L2/3 astrocytes (cyan, Aldh1l1-GFP) immunostained for Igfbp2 (magenta). i. Quantification of extracellular Igfbp2. j. Quantification of intracellular Igfbp2. N=6 littermate pairs RTT and FXS; 7 littermate pairs DS. Bar graph mean±s.e.m., individual points mice, same mouse in i and j for each genotype denoted with same shape; statistics two-sided T-test. See also Extended Data Figure 4.

Article Snippet: For Igfbp2 blocking antibody experiments injections were performed on littermate pairs of male Mecp2-/y (RTT) mice, receiving either the Igfbp2 blocking antibody (R&D MAB797) or the isotype control antibody (R&D 6–001-F) at 1mg/ml in PBS, labeled with Alexafluor 594 to visualize the injection site (Molecular Probes A20185), with the AAV-Synapsin-GFP virus diluted in the antibody solution.

Techniques: Expressing, Immunostaining

Journal: Nature neuroscience

Article Title: Aberrant astrocyte protein secretion contributes to altered neuronal development in multiple models of neurodevelopmental disorders

doi: 10.1038/s41593-022-01150-1

Figure Lengend Snippet: a-f. Application of an Igfbp2-neutralizing antibody reduces WT neurite outgrowth inhibition induced by RTT ACM. a,c,e. Example images neurons cultured for 48 hours in RTT (a), FXS (c) or DS (e) ACM (merged images of MAP2 + Tau). b,d,f. Quantification total neurite outgrowth, normalized to alone condition. Violin plot: dashed line median, dotted lines 25th and 75th percentile. Data from 3 (b), 4 (d), 5 (f) separate experiments. Number of neurons: RTT (b): alone=439, WT ACM=549, RTT ACM=633, RTT ACM + Igfbp2-Ab=621; FXS (d): alone=492, WT ACM=608, FXS ACM=621, FXS ACM + Igfbp2-Ab=647; DS (f): alone=716, WT ACM=992, DS ACM=765, DS ACM + Igfbp2-Ab=858. Statistics by Kruskal-Wallis one-way ANOVA on ranks with Dunn’s test for multiple comparisons. g. Application of an Igfbp2 neutralizing antibody reduces WT neuronal cell body size deficits induced by RTT ACM. Graph mean ± s.e.m., individual data points represent average per experiment, data from 3 experiments. Statistics by Kruskal-Wallis one-way ANOVA on ranks with Dunn’s test for multiple comparisons. h-l. Delivery of an Igfbp2-neutralizing antibody increases cell body size of deep layer neurons in P7 visual cortex of RTT mice. h. Schematic: P2 mice were injected in the visual cortex with AAV synapsin-GFP +/− antibody, tissue collected at P7 and GFP-expressing neurons imaged. i,j. Cell body area in deep layer neurons is decreased in RTT compared to WT mice. i. Analysis by mice, graph average ± s.e.m., individual data points mice, N=4 WT, 4 RTT mice, statistics by 2-sided T-test. j. Analysis by cells, graph individual data points represent cells, dashed line at mean, n=133 WT, 189 RTT cells, statistics by 2-sided T-test. k,l. Cell body area in deep layer neurons in RTT mice is increased by an Igfbp2-Ab. k. Analysis by mice, graph average ± s.e.m., individual data points mice, N=5 control-Ab, 5 Igfbp2-Ab mice, statistics by 2-sided T-test. l. Analysis by cells, graph individual data points represent cells, dashed line at mean, n=219 control-Ab, 274 Igfbp2-Ab cells, statistics by 2-sided T-test. See also Extended Data Figure 5.

Article Snippet: For Igfbp2 blocking antibody experiments injections were performed on littermate pairs of male Mecp2-/y (RTT) mice, receiving either the Igfbp2 blocking antibody (R&D MAB797) or the isotype control antibody (R&D 6–001-F) at 1mg/ml in PBS, labeled with Alexafluor 594 to visualize the injection site (Molecular Probes A20185), with the AAV-Synapsin-GFP virus diluted in the antibody solution.

Techniques: Inhibition, Cell Culture, Injection, Expressing

Journal: Nature neuroscience

Article Title: Aberrant astrocyte protein secretion contributes to altered neuronal development in multiple models of neurodevelopmental disorders

doi: 10.1038/s41593-022-01150-1

Figure Lengend Snippet: a,b. BMP6-treated WT astrocytes show protein secretion (a) and gene expression (b) downregulations that overlap with ND astrocytes. N=6 cultures WT, FXS, RTT, DS, plus 6 cultures WT +/− BMP6 proteomics; N=6 cultures WT, FXS, RTT; 4 DS, plus 6 cultures WT +/− BMP6 RNA sequencing. c. Example images from Figure 7d prior to processing and analysis. Neurons immunostained with MAP2 (dendrites, green) and tau (axon, red). d. Relative frequency distribution plot of total neurite outgrowth length, same data as Figure 7e. Data from 3 experiments, number of neurons: alone=467, WT ACM=733, BMP6 WT ACM=610.e. Relative frequency distribution plot of total neurite outgrowth length, same data as Figure 7f. Data from 3 experiments, number of neurons: alone=378, WT ACM=380, BMP6 WT ACM=506, BMP6 WT ACM + Igfbp2 blocking Ab=335. f. Example images from Figure 7h prior to processing and analysis. Neurons immunostained with MAP2 (dendrites, green) and tau (axon, red). g. Relative frequency distribution plot of total neurite outgrowth, same data as Figure 7i. Data from 3 experiments, number of neurons: alone=923, WT ACM=1164, FXS ACM=1132, Noggin FXS ACM=1099. h. Relative frequency distribution plot of total neurite outgrowth, same data as Figure 7j. Data from 3 experiments, number of neurons: alone=238, WT ACM=279, RTT ACM=387, Noggin RTT ACM=365. i. Example images of cortical neurons treated with noggin at the time of plating, ± WT ACM or ± FXS ACM (image merge of MAP2 + Tau). j. Quantification of total neurite outgrowth, data from 3 experiments. Number of neurons: alone=2197, alone + noggin=1981, WT ACM=2167, WT ACM + noggin=2421, FXS ACM=2060, FXS ACM + noggin=2523. Violin plots dashed line marks median, dotted lines 25th and 75th percentile. Statistics by Kruskal-Wallis one-way ANOVA on ranks with Dunn’s test for multiple comparisons.

Article Snippet: For Igfbp2 blocking antibody experiments injections were performed on littermate pairs of male Mecp2-/y (RTT) mice, receiving either the Igfbp2 blocking antibody (R&D MAB797) or the isotype control antibody (R&D 6–001-F) at 1mg/ml in PBS, labeled with Alexafluor 594 to visualize the injection site (Molecular Probes A20185), with the AAV-Synapsin-GFP virus diluted in the antibody solution.

Techniques: Expressing, RNA Sequencing Assay, Blocking Assay

Journal: Nature neuroscience

Article Title: Aberrant astrocyte protein secretion contributes to altered neuronal development in multiple models of neurodevelopmental disorders

doi: 10.1038/s41593-022-01150-1

Figure Lengend Snippet: a. Expression of IGF family members in cortical cell types (data from Zhang et al., 2014). b,c. Addition of Igfbp2 protein to WT ACM inhibits WT neurite outgrowth, which is reduced by adding IGF1. Addition of CPE protein to WT ACM does not inhibit WT neurite outgrowth. b. Example images of WT neurons cultured for 48 hours, conditions as marked (image merge of MAP2 + Tau). c. Quantification of total neurite outgrowth. Example experiment shown, repeated 2 times with same result, number of neurons: control alone=49, control ACM=50, CPE alone=36, CPE ACM=46, Igfbp2 alone=44, Igfbp2 ACM=48, Igfbp2 ACM + Igf1=39. d. smFISH against Igfbp2 mRNA in the P7 visual cortex in Aldh1l1-GFP mice to mark astrocytes, combined with probe for OPCs (Cspg4). See Figure 4g for quantification. N=3 WT mice. e. Example images from Figure 4d prior to processing and analysis. Neurons immunostained with MAP2 (dendrites, green) and tau (axon, red). f. Relative frequency distribution plot of total neurite outgrowth length, pooled data from 3 experiments, same data as Figure 4e. g. Adding the IgG control antibody to WT ACM does not alter neurite outgrowth. Example experiment shown, repeated twice with same result. Number of neurons: control alone=216, control ACM=333, Igfbp2-Ab alone=257, Igfbp2-Ab ACM=267, IgG con-Ab alone=277, IgG con-Ab ACM=266. Violin plots (c,g), dashed line marks median, dotted lines 25th and 75th percentile. Statistics by Kruskal-Wallis one-way ANOVA on ranks with Dunn’s test for multiple comparisons, p values compared to control alone condition (c).

Article Snippet: For Igfbp2 blocking antibody experiments injections were performed on littermate pairs of male Mecp2-/y (RTT) mice, receiving either the Igfbp2 blocking antibody (R&D MAB797) or the isotype control antibody (R&D 6–001-F) at 1mg/ml in PBS, labeled with Alexafluor 594 to visualize the injection site (Molecular Probes A20185), with the AAV-Synapsin-GFP virus diluted in the antibody solution.

Techniques: Expressing, Cell Culture

Journal: Nature neuroscience

Article Title: Aberrant astrocyte protein secretion contributes to altered neuronal development in multiple models of neurodevelopmental disorders

doi: 10.1038/s41593-022-01150-1

Figure Lengend Snippet: a,c,e. Example images from Figure 5a,​,cc,​,ee prior to processing and analysis. Neurons cultured for 48 hours in RTT (a), FXS (c) or DS (e) ACM and immunostained with MAP2 (dendrites, green) and tau (axon, red). b,d,f. Relative frequency distribution plot of total neurite outgrowth, pooled data from 3 (b), 4 (d), 5 (f) experiments per graph, same data as Figure 5b,​,dd,​,f.f. Number of neurons: RTT (b): alone=439, WT ACM=549, RTT ACM=633, RTT ACM + Igfbp2-Ab=621; FXS (d): alone=492, WT ACM=608, FXS ACM=621, FXS ACM + Igfbp2-Ab=647; DS (f): alone=716, WT ACM=992, DS ACM=765, DS ACM + Igfbp2-Ab=858. g. Relative frequency distribution plot of neuronal cell body size, pooled data from 3 experiments, same data as Figure 5g. h-l Cell body size of upper layer cortical neurons is not different between WT and RTT mice, and unaffected by the Igfbp2 neutralizing antibody. h. Schematic of the experiment: P2 mice were injected in the visual cortex with AAV synapsin-GFP +/− antibody, and tissue collected at P7 and GFP-expressing neurons imaged, with the region to be imaged identified by the presence of the fluorescently labeled antibody. i,j. Cell body area in upper layer neurons is unaltered in RTT compared to WT mice. i. Analysis by mice, graph average ± s.e.m., individual data points represent mice, N = 4 WT and 4 RTT mice, statistics by 2-sided T-test. j. Analysis by cells, graph individual data points represent cells, dashed line represents the mean, n = 162 WT and 177 RTT cells, statistics by 2-sided T-test. k,l. Cell body area in upper layer neurons in RTT mice is unaffected by an Igfbp2 neutralizing antibody. k. Analysis by mice, graph average ± s.e.m., individual data points represent mice, N = 5 control-Ab and 5 Igfbp2-Ab mice, statistics by 2-sided T-test. l. Analysis by cells, graph individual data points represent cells, dashed line represents the mean, n = 183 control-Ab and 195 Igfbp2-Ab cells, statistics by 2-sided T-test.

Article Snippet: Primary antibodies, goat anti-Igfbp2 (1:500, R&D AF797) or rabbit anti-pSMAD (1:800, Cell Signaling 9516), were incubated in antibody buffer + 0.3% Triton X-100 overnight at 4°C.

Techniques: Cell Culture, Injection, Expressing, Labeling

Journal: Nature neuroscience

Article Title: Aberrant astrocyte protein secretion contributes to altered neuronal development in multiple models of neurodevelopmental disorders

doi: 10.1038/s41593-022-01150-1

Figure Lengend Snippet: a-c. BMP6-treated WT astrocytes show upregulated protein secretion (a) and gene expression (b) that overlaps with ND astrocytes. c. Heatmap of proteins increased in all ND and BMP6-treated astrocytes vs. WT, ranked by protein abundance in BMP6-treated ACM. N=6 cultures each WT, FXS, RTT, DS, plus 6 cultures WT +/− BMP6. d,e. ACM from WT astrocytes treated with BMP6 inhibits WT neurite outgrowth. d. Example images of WT neurons cultured for 48 hours, conditions as marked (merged images of MAP2 + Tau). e. Quantification of total neurite outgrowth. Data from 3 experiments, number of neurons: alone=467, WT ACM=733, BMP6 WT ACM=610. f. Blocking Igfbp2 overcomes the inhibitory effect of BMP6 WT ACM on neurite outgrowth. Data from 3 experiments, number of neurons: alone=378, WT ACM=380, BMP6 WT ACM=506, BMP6 WT ACM + Igfbp2 blocking Ab=335. g-j. Blocking BMP signaling in ND astrocytes rescues deficits in WT neurite outgrowth. g. Experimental schematic for noggin treatment of FXS or RTT astrocytes. h. Blocking BMP signaling in FXS astrocytes rescues deficits in WT neurite outgrowth. Example images of WT neurons cultured for 48 hours, conditions as marked (merged images of MAP2 + Tau). i. Quantification of total neurite outgrowth. Data from 3 experiments, number of neurons: alone=923, WT ACM=1164, FXS ACM=1132, Noggin FXS ACM=1099. j. Blocking BMP signaling in RTT astrocytes rescues deficits in WT neurite outgrowth. Quantification of total neurite outgrowth. Data from 3 experiments, number of neurons: alone=238, WT ACM=279, RTT ACM=387, Noggin RTT ACM=365. Violin plot: dashed line median, dotted lines 25th and 75th percentile. Statistics by Kruskal-Wallis one-way ANOVA on ranks with Dunn’s test for multiple comparisons. See also Extended Data Figure 7; Tables S5, S7.

Article Snippet: Primary antibodies, goat anti-Igfbp2 (1:500, R&D AF797) or rabbit anti-pSMAD (1:800, Cell Signaling 9516), were incubated in antibody buffer + 0.3% Triton X-100 overnight at 4°C.

Techniques: Expressing, Cell Culture, Blocking Assay

Journal: Nature neuroscience

Article Title: Aberrant astrocyte protein secretion contributes to altered neuronal development in multiple models of neurodevelopmental disorders

doi: 10.1038/s41593-022-01150-1

Figure Lengend Snippet: a. Schematic of IGF signaling via the PI3K/Akt pathway. b,c. Protein secretion (b) and gene expression (c) profiles of IGF family members in WT and ND astrocytes. Proteomics, N=6 cultures/genotype, *p<0.05, abundance >0.01%, fold change between WT and ND ≥1.5 calculated with T-fold test in Patternlab. RNASeq, N=6 cultures WT, RTT, FXS; 4 cultures DS, *adjusted p<0.05, FPKM>1, fold change between ND and WT ≥1.5 calculated with DESeq2. d,e. Excess Igfbp2 in ACM inhibits WT neurite outgrowth. d. Example images WT neurons grown 48 hours, conditions as marked (image merge of MAP2 + Tau). e. Quantification total neurite outgrowth normalized to control alone condition. Violin plot: dashed line median, dotted lines 25th and 75th percentile. Data from 3 separate experiments, number of neurons: control alone=447, control ACM=626, Igfbp2 alone=438, Igfbp2 ACM=596, Igfbp2 + Ab alone=376, Igfbp2 + Ab + ACM=562. Statistics by Kruskal-Wallis one-way ANOVA on ranks with Dunn’s test for multiple comparisons. f, g. Astrocytes express Igfbp2 mRNA in the P7 mouse visual cortex. f. smFISH against Igfbp2 mRNA in Aldh1l1-GFP mice to mark astrocytes, combined with probe for neurons (Tubb3). Left panels overview of cortex across all layers, right panel high power image from L2/3. g. Analysis of images in f, expression of Igfbp2 mRNA within astrocytes, neurons and OPCs. See Figure S4d for OPC image. N=3 WT mice. Bar graph mean±s.e.m., individual data points mice; statistics by one-way ANOVA with Tukey’s test for multiple comparisons. h-j. Immunostaining for Igfbp2 in each ND and littermate WT visual cortex at P7 (RTT - Mecp2 KO; FXS – Fmr1 KO; DS - Ts65Dn TG) reveals an increase in extracellular Igfbp2 in RTT and intracellular Igfbp2 in DS. h. Example images of L2/3 astrocytes (cyan, Aldh1l1-GFP) immunostained for Igfbp2 (magenta). i. Quantification of extracellular Igfbp2. j. Quantification of intracellular Igfbp2. N=6 littermate pairs RTT and FXS; 7 littermate pairs DS. Bar graph mean±s.e.m., individual points mice, same mouse in i and j for each genotype denoted with same shape; statistics two-sided T-test. See also Extended Data Figure 4.

Article Snippet: Primary antibodies, goat anti-Igfbp2 (1:500, R&D AF797) or rabbit anti-pSMAD (1:800, Cell Signaling 9516), were incubated in antibody buffer + 0.3% Triton X-100 overnight at 4°C.

Techniques: Expressing, Immunostaining

Journal: Nature neuroscience

Article Title: Aberrant astrocyte protein secretion contributes to altered neuronal development in multiple models of neurodevelopmental disorders

doi: 10.1038/s41593-022-01150-1

Figure Lengend Snippet: a-f. Application of an Igfbp2-neutralizing antibody reduces WT neurite outgrowth inhibition induced by RTT ACM. a,c,e. Example images neurons cultured for 48 hours in RTT (a), FXS (c) or DS (e) ACM (merged images of MAP2 + Tau). b,d,f. Quantification total neurite outgrowth, normalized to alone condition. Violin plot: dashed line median, dotted lines 25th and 75th percentile. Data from 3 (b), 4 (d), 5 (f) separate experiments. Number of neurons: RTT (b): alone=439, WT ACM=549, RTT ACM=633, RTT ACM + Igfbp2-Ab=621; FXS (d): alone=492, WT ACM=608, FXS ACM=621, FXS ACM + Igfbp2-Ab=647; DS (f): alone=716, WT ACM=992, DS ACM=765, DS ACM + Igfbp2-Ab=858. Statistics by Kruskal-Wallis one-way ANOVA on ranks with Dunn’s test for multiple comparisons. g. Application of an Igfbp2 neutralizing antibody reduces WT neuronal cell body size deficits induced by RTT ACM. Graph mean ± s.e.m., individual data points represent average per experiment, data from 3 experiments. Statistics by Kruskal-Wallis one-way ANOVA on ranks with Dunn’s test for multiple comparisons. h-l. Delivery of an Igfbp2-neutralizing antibody increases cell body size of deep layer neurons in P7 visual cortex of RTT mice. h. Schematic: P2 mice were injected in the visual cortex with AAV synapsin-GFP +/− antibody, tissue collected at P7 and GFP-expressing neurons imaged. i,j. Cell body area in deep layer neurons is decreased in RTT compared to WT mice. i. Analysis by mice, graph average ± s.e.m., individual data points mice, N=4 WT, 4 RTT mice, statistics by 2-sided T-test. j. Analysis by cells, graph individual data points represent cells, dashed line at mean, n=133 WT, 189 RTT cells, statistics by 2-sided T-test. k,l. Cell body area in deep layer neurons in RTT mice is increased by an Igfbp2-Ab. k. Analysis by mice, graph average ± s.e.m., individual data points mice, N=5 control-Ab, 5 Igfbp2-Ab mice, statistics by 2-sided T-test. l. Analysis by cells, graph individual data points represent cells, dashed line at mean, n=219 control-Ab, 274 Igfbp2-Ab cells, statistics by 2-sided T-test. See also Extended Data Figure 5.

Article Snippet: Primary antibodies, goat anti-Igfbp2 (1:500, R&D AF797) or rabbit anti-pSMAD (1:800, Cell Signaling 9516), were incubated in antibody buffer + 0.3% Triton X-100 overnight at 4°C.

Techniques: Inhibition, Cell Culture, Injection, Expressing

Journal: Nature neuroscience

Article Title: Aberrant astrocyte protein secretion contributes to altered neuronal development in multiple models of neurodevelopmental disorders

doi: 10.1038/s41593-022-01150-1

Figure Lengend Snippet: a,b. BMP6-treated WT astrocytes show protein secretion (a) and gene expression (b) downregulations that overlap with ND astrocytes. N=6 cultures WT, FXS, RTT, DS, plus 6 cultures WT +/− BMP6 proteomics; N=6 cultures WT, FXS, RTT; 4 DS, plus 6 cultures WT +/− BMP6 RNA sequencing. c. Example images from Figure 7d prior to processing and analysis. Neurons immunostained with MAP2 (dendrites, green) and tau (axon, red). d. Relative frequency distribution plot of total neurite outgrowth length, same data as Figure 7e. Data from 3 experiments, number of neurons: alone=467, WT ACM=733, BMP6 WT ACM=610.e. Relative frequency distribution plot of total neurite outgrowth length, same data as Figure 7f. Data from 3 experiments, number of neurons: alone=378, WT ACM=380, BMP6 WT ACM=506, BMP6 WT ACM + Igfbp2 blocking Ab=335. f. Example images from Figure 7h prior to processing and analysis. Neurons immunostained with MAP2 (dendrites, green) and tau (axon, red). g. Relative frequency distribution plot of total neurite outgrowth, same data as Figure 7i. Data from 3 experiments, number of neurons: alone=923, WT ACM=1164, FXS ACM=1132, Noggin FXS ACM=1099. h. Relative frequency distribution plot of total neurite outgrowth, same data as Figure 7j. Data from 3 experiments, number of neurons: alone=238, WT ACM=279, RTT ACM=387, Noggin RTT ACM=365. i. Example images of cortical neurons treated with noggin at the time of plating, ± WT ACM or ± FXS ACM (image merge of MAP2 + Tau). j. Quantification of total neurite outgrowth, data from 3 experiments. Number of neurons: alone=2197, alone + noggin=1981, WT ACM=2167, WT ACM + noggin=2421, FXS ACM=2060, FXS ACM + noggin=2523. Violin plots dashed line marks median, dotted lines 25th and 75th percentile. Statistics by Kruskal-Wallis one-way ANOVA on ranks with Dunn’s test for multiple comparisons.

Article Snippet: Primary antibodies, goat anti-Igfbp2 (1:500, R&D AF797) or rabbit anti-pSMAD (1:800, Cell Signaling 9516), were incubated in antibody buffer + 0.3% Triton X-100 overnight at 4°C.

Techniques: Expressing, RNA Sequencing Assay, Blocking Assay

Journal: Nature neuroscience

Article Title: Aberrant astrocyte protein secretion contributes to altered neuronal development in multiple models of neurodevelopmental disorders

doi: 10.1038/s41593-022-01150-1

Figure Lengend Snippet: a. Expression of IGF family members in cortical cell types (data from Zhang et al., 2014). b,c. Addition of Igfbp2 protein to WT ACM inhibits WT neurite outgrowth, which is reduced by adding IGF1. Addition of CPE protein to WT ACM does not inhibit WT neurite outgrowth. b. Example images of WT neurons cultured for 48 hours, conditions as marked (image merge of MAP2 + Tau). c. Quantification of total neurite outgrowth. Example experiment shown, repeated 2 times with same result, number of neurons: control alone=49, control ACM=50, CPE alone=36, CPE ACM=46, Igfbp2 alone=44, Igfbp2 ACM=48, Igfbp2 ACM + Igf1=39. d. smFISH against Igfbp2 mRNA in the P7 visual cortex in Aldh1l1-GFP mice to mark astrocytes, combined with probe for OPCs (Cspg4). See Figure 4g for quantification. N=3 WT mice. e. Example images from Figure 4d prior to processing and analysis. Neurons immunostained with MAP2 (dendrites, green) and tau (axon, red). f. Relative frequency distribution plot of total neurite outgrowth length, pooled data from 3 experiments, same data as Figure 4e. g. Adding the IgG control antibody to WT ACM does not alter neurite outgrowth. Example experiment shown, repeated twice with same result. Number of neurons: control alone=216, control ACM=333, Igfbp2-Ab alone=257, Igfbp2-Ab ACM=267, IgG con-Ab alone=277, IgG con-Ab ACM=266. Violin plots (c,g), dashed line marks median, dotted lines 25th and 75th percentile. Statistics by Kruskal-Wallis one-way ANOVA on ranks with Dunn’s test for multiple comparisons, p values compared to control alone condition (c).

Article Snippet: Primary antibodies, goat anti-Igfbp2 (1:500, R&D AF797) or rabbit anti-pSMAD (1:800, Cell Signaling 9516), were incubated in antibody buffer + 0.3% Triton X-100 overnight at 4°C.

Techniques: Expressing, Cell Culture