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: Cell reports. Medicine

Article Title: Loss of IGFBP2 mediates alveolar type 2 cell senescence and promotes lung fibrosis.

doi: 10.1016/j.xcrm.2023.100945

Figure Lengend Snippet: Figure 2. IGFBP2 downregulation in adenoviral TGF-b1-induced pulmonary fibrosis in aged mice (A) Sirius red (top)- or Mason’s trichrome (bottom)-stained lung sections of aged (78–82 weeks old) WT mice 28 days after intratracheal administration of Ad-null or Ad-TGF-b1 virus (5 3 108 PFU). Scale bars, 50 mm (n = 6 Ad-null; n = 6 Ad-TGF-b1). (B) Hydroxyproline content (mg per mg of lung) in the lungs of 18-month-old mice 28 days after intratracheal administration of Ad-null or Ad-TGF-b1 virus (n = 6 Ad-null; n = 6 Ad-TGF-b1). (C) Representative double-color immunohistochemistry lung images of aged WT mice challenged with Ad-Null or Ad-TGF-b1 virus. Green color indicates SPC expression; brown color indicates IGFBP2 or P21 or phospho-H2AX expression. Black arrowheads indicate the double-positive AEC2 cells. Scale bars, 10 mm (n = 6 Ad-null; n = 6 Ad-TGF-b1). (D) Quantification of percentages of double-positive cells for IGFBP2, P21, and phospho-H2AX expression in SPC + cells, respectively. Data are mean ± SEM **p < 0.01, and ***p < 0.001, Student’s unpaired two-tailed t test.

Article Snippet: REAGENT or RESOURCE SOURCE IDENTIFIER TaqManTM Fast Advanced Master Mix Thermo Fisher scientific Cat# 44-445-56 Hydroxyproline assay Cell Biolab Cat# STA-675 Beta-galactosidase activity assay Biovision Cat# K821- 100 RT2 profiler PCR assay Qiagen Cat# PAMM-050ZC-6 IGFBP2 ELSA assay R&D systems Cat# DY797 BCA protein assay Thermo Fisher Scientific Cat# 23227 Experimental models: Cell lines MLE-12 ATCC CRL-2110 Experimental models: Organisms/strains Igfbp2flx/flxtransgenic mouse Leeds University Sftpc-CreERT2 The Jackson Laboratory RRID#IMSR_JAX:028054 C57BL6/J The Jackson Laboratory RRID#IMSR_JAX:000664 Oligonucleotides IL-1bCCH PrimerBank ID 6680415a1 50-GCAACTGTTCCTGAACTCAACT-30 and 50-ATCTTTTGGGGTCCGTCAACT-30 IL-4 PrimerBank ID 10946584a1 50- GGTCTCAACCCCCAGCTAGT-30 50- GCCGATGATCTCTCTCAAGTGAT-30 IL-6 PrimerBank ID 13624311a1 50-TAGTCCTTCC-30 50-TACCCCAATTTCC-30 MCP-1 PrimerBank ID 6755430a1 50- TTAAAAACCTGGATCGGAACCAA-30 50- GCATTAGCTTCAGATTTACGGGT-30 STAT-3 PrimerBank ID 13277852a1 50- CAATACCATTGACCTGCCGAT-30 50- GAGCGACTCAAACTGCCCT-30 STAT6 PrimerBank ID 6678155a1 50- CTCTGTGGGGCCTAATTTCCA-30 50- CATCTGAACCGACCAGGAACT-30 TNF-a PrimerBank ID 7305585a1 50- CCCTCACACTCAGATCATCTTCT-30 50- GCTACGACGTGGGCTACAG-30 Software and algorithms ImageJ 1.53t FIJI https://imagej.nih.gov/ij/ Graphpad Prism 9.0 Graphpad software LLC https://www.graphpad.com

Techniques: Staining, Virus, Immunohistochemistry, Expressing, Two Tailed Test

Journal: Cell reports. Medicine

Article Title: Loss of IGFBP2 mediates alveolar type 2 cell senescence and promotes lung fibrosis.

doi: 10.1016/j.xcrm.2023.100945

Figure Lengend Snippet: Figure 3. IGFBP2 deficiency increases P21 expression in response to fibrotic stimuli in vitro (A) Western blot for the expression of IGFBP2, P21, and phospho-H2AX in MLE-12 cells pretreated with atazanavir (ATZ; 20 mg/mL) for 1 h and exposed to hypoxia treatment for 72 h. (B) Western blot for the expression of IGFBP2, P21, and phospho-H2AX in MLE-12 cells exposed to absence or presence of chronic exposure to bleomycin (two- hit model; 10 mg/mL). (C) Western blot for the expression of IGFBP2 and P21 in MLE-12 cells exposed to absence or presence of hypoxia treatment at 4 h. b-Actin served as an internal control. (D) Western blot for the expression of IGFBP2 and P21 in the cytosolic and nuclear fractions of MLE-12 cells that were exposed to absence or presence of hypoxia treatment. a-Tubulin and histone-3 served as internal controls. (E) Western blot for the expression of IGFBP2 and P21 in MLE-12 cells exposed to absence or presence of cigarette smoke treatment (100 mg/mL). (F) Non-targeting or Igfbp2 siRNA-transduced MLE-12 cells were exposed to absence or presence of hypoxia treatment at 4 h. Western blot for the expression of IGFBP2 and P21. (G) Non-targeting or Igfbp2 siRNA-transduced MLE-12 cells were challenged with absence or presence of bleomycin exposure (10 mg/mL). Western blot for the expression of IGFBP2, P21, and phospho-H2AX in MLE-12 cells subjected to bleomycin exposure at 4 h. b-Actin served as an internal control. Data are representative of minimum of 3 independent experiments.

Article Snippet: REAGENT or RESOURCE SOURCE IDENTIFIER TaqManTM Fast Advanced Master Mix Thermo Fisher scientific Cat# 44-445-56 Hydroxyproline assay Cell Biolab Cat# STA-675 Beta-galactosidase activity assay Biovision Cat# K821- 100 RT2 profiler PCR assay Qiagen Cat# PAMM-050ZC-6 IGFBP2 ELSA assay R&D systems Cat# DY797 BCA protein assay Thermo Fisher Scientific Cat# 23227 Experimental models: Cell lines MLE-12 ATCC CRL-2110 Experimental models: Organisms/strains Igfbp2flx/flxtransgenic mouse Leeds University Sftpc-CreERT2 The Jackson Laboratory RRID#IMSR_JAX:028054 C57BL6/J The Jackson Laboratory RRID#IMSR_JAX:000664 Oligonucleotides IL-1bCCH PrimerBank ID 6680415a1 50-GCAACTGTTCCTGAACTCAACT-30 and 50-ATCTTTTGGGGTCCGTCAACT-30 IL-4 PrimerBank ID 10946584a1 50- GGTCTCAACCCCCAGCTAGT-30 50- GCCGATGATCTCTCTCAAGTGAT-30 IL-6 PrimerBank ID 13624311a1 50-TAGTCCTTCC-30 50-TACCCCAATTTCC-30 MCP-1 PrimerBank ID 6755430a1 50- TTAAAAACCTGGATCGGAACCAA-30 50- GCATTAGCTTCAGATTTACGGGT-30 STAT-3 PrimerBank ID 13277852a1 50- CAATACCATTGACCTGCCGAT-30 50- GAGCGACTCAAACTGCCCT-30 STAT6 PrimerBank ID 6678155a1 50- CTCTGTGGGGCCTAATTTCCA-30 50- CATCTGAACCGACCAGGAACT-30 TNF-a PrimerBank ID 7305585a1 50- CCCTCACACTCAGATCATCTTCT-30 50- GCTACGACGTGGGCTACAG-30 Software and algorithms ImageJ 1.53t FIJI https://imagej.nih.gov/ij/ Graphpad Prism 9.0 Graphpad software LLC https://www.graphpad.com

Techniques: Expressing, In Vitro, Western Blot, Control

Journal: Cell reports. Medicine

Article Title: Loss of IGFBP2 mediates alveolar type 2 cell senescence and promotes lung fibrosis.

doi: 10.1016/j.xcrm.2023.100945

Figure Lengend Snippet: Figure 4. Stable transduction with Igfbp2 lentivirus vector decreased P21 expression and b-galactosidase activity in vitro (A) Mock-virus- and Igfbp2 lentivirus-transduced MLE-12 cells in the absence or presence of hypoxia treatment at 4 h. Western blot for the expression of IGFBP2 and P21. b-Actin served as internal control. (B) Western blot for the expression of IGFBP2 and P21 in the cytosolic and nuclear fractions of Igfbp2 lentivirus-transduced MLE-12 cells in the absence or presence of hypoxia treatment at 4 h. a-Tubulin and histone-3 served as internal controls. (C) Western blot for the expression of IGFBP2, P21, and phosph-H2AX in Igfbp2 lentivirus-transduced MLE-12 cells in the absence or presence of cigarette smoke treatment (100 mg/mL). (D) Western blot for the expression of IGFBP2, P21, and phospho-H2AX in Igfbp2 lentivirus-transduced MLE-12 cells in the absence or presence of bleomycin (10 mg/mL). b-Actin served as internal control. (E) Bar graph showing the b-galactosidase activity of MLE-12 cells pretreated with ATZ for 1 h and subjected to hypoxia for 96 h. (F) Bar graph showing the b-galactosidase activity of MLE-12 cells treated with bleomycin for 48 h. Data are representative of minimum of 3 independent ex- periments. Data are mean ± SEM. *p < 0.05, **p < 0.01, ****p < 0.0001, one-way ANOVA with Tukey’s post-hoc test.

Article Snippet: REAGENT or RESOURCE SOURCE IDENTIFIER TaqManTM Fast Advanced Master Mix Thermo Fisher scientific Cat# 44-445-56 Hydroxyproline assay Cell Biolab Cat# STA-675 Beta-galactosidase activity assay Biovision Cat# K821- 100 RT2 profiler PCR assay Qiagen Cat# PAMM-050ZC-6 IGFBP2 ELSA assay R&D systems Cat# DY797 BCA protein assay Thermo Fisher Scientific Cat# 23227 Experimental models: Cell lines MLE-12 ATCC CRL-2110 Experimental models: Organisms/strains Igfbp2flx/flxtransgenic mouse Leeds University Sftpc-CreERT2 The Jackson Laboratory RRID#IMSR_JAX:028054 C57BL6/J The Jackson Laboratory RRID#IMSR_JAX:000664 Oligonucleotides IL-1bCCH PrimerBank ID 6680415a1 50-GCAACTGTTCCTGAACTCAACT-30 and 50-ATCTTTTGGGGTCCGTCAACT-30 IL-4 PrimerBank ID 10946584a1 50- GGTCTCAACCCCCAGCTAGT-30 50- GCCGATGATCTCTCTCAAGTGAT-30 IL-6 PrimerBank ID 13624311a1 50-TAGTCCTTCC-30 50-TACCCCAATTTCC-30 MCP-1 PrimerBank ID 6755430a1 50- TTAAAAACCTGGATCGGAACCAA-30 50- GCATTAGCTTCAGATTTACGGGT-30 STAT-3 PrimerBank ID 13277852a1 50- CAATACCATTGACCTGCCGAT-30 50- GAGCGACTCAAACTGCCCT-30 STAT6 PrimerBank ID 6678155a1 50- CTCTGTGGGGCCTAATTTCCA-30 50- CATCTGAACCGACCAGGAACT-30 TNF-a PrimerBank ID 7305585a1 50- CCCTCACACTCAGATCATCTTCT-30 50- GCTACGACGTGGGCTACAG-30 Software and algorithms ImageJ 1.53t FIJI https://imagej.nih.gov/ij/ Graphpad Prism 9.0 Graphpad software LLC https://www.graphpad.com

Techniques: Transduction, Plasmid Preparation, Expressing, Activity Assay, In Vitro, Virus, Western Blot, Control

Journal: Cell reports. Medicine

Article Title: Loss of IGFBP2 mediates alveolar type 2 cell senescence and promotes lung fibrosis.

doi: 10.1016/j.xcrm.2023.100945

Figure Lengend Snippet: Figure 5. Reduced PPARA expression in the AEC2 cells from fibrotic lung regions of patients with IPF (A) Western blot for the expression of PPARa and b-actin in MLE-12 cells exposed to absence or presence of bleomycin at 4 h. (B) Non-targeting or Ppara siRNA-transduced MLE-12 cells were exposed to absence or presence of bleomycin treatment at 4 h. Western blot for the expression of PPARa and IGFBP2. b-Actin served as internal control. Data are representative of minimum of 3 independent experiments. (C) Ppara mRNA expression in the primary AEC2 cells isolated from aged mice subjected to low-dose bleomycin challenge after 14 days. Eukaryotic 18S rRNA was used as an endogenous control (n = 5 WT saline; n = 5 WT bleomycin). (D) Representative multicolor color immunohistochemistry of lung sections from aged WT mice 28 days after bleomycin injury. Green color indicates SPC expression; brown color indicates PPARa expression. Scale bars, 10 mm (n = 5 WT saline; n = 5 WT bleomycin). (E) Quantification of percentages of double-positive cells for SPC and PPARa in the lungs of aged WT mice subjected to low-dose bleomycin after 28 days. (F) Ppara mRNA expression was determined by qPCR in the primary AEC2 cells of patients with IPF (n = 21) compared with HP (n = 5) or COPD (n = 9). (G) Representative multicolor immunohistological staining of PPARA and SPC. Arrows indicate examples of SPC-positive and PPARA-positive cells. Staining was performed with lung sections from 2 healthy controls and 2 patients with IPF. (H) Quantification of percentages of double-positive cells for SPC and PPARA in the fibrotic lung regions of patients with IPF and donor (healthy) controls. Data are mean ± SEM. NS, not significant; **p < 0.01, ***p < 0.001, ****p < 0.0001, one-way ANOVA with Tukey’s post-hoc test (for multiple group comparisons) or Student’s unpaired two-tailed t test (for two group comparisons).

Article Snippet: REAGENT or RESOURCE SOURCE IDENTIFIER TaqManTM Fast Advanced Master Mix Thermo Fisher scientific Cat# 44-445-56 Hydroxyproline assay Cell Biolab Cat# STA-675 Beta-galactosidase activity assay Biovision Cat# K821- 100 RT2 profiler PCR assay Qiagen Cat# PAMM-050ZC-6 IGFBP2 ELSA assay R&D systems Cat# DY797 BCA protein assay Thermo Fisher Scientific Cat# 23227 Experimental models: Cell lines MLE-12 ATCC CRL-2110 Experimental models: Organisms/strains Igfbp2flx/flxtransgenic mouse Leeds University Sftpc-CreERT2 The Jackson Laboratory RRID#IMSR_JAX:028054 C57BL6/J The Jackson Laboratory RRID#IMSR_JAX:000664 Oligonucleotides IL-1bCCH PrimerBank ID 6680415a1 50-GCAACTGTTCCTGAACTCAACT-30 and 50-ATCTTTTGGGGTCCGTCAACT-30 IL-4 PrimerBank ID 10946584a1 50- GGTCTCAACCCCCAGCTAGT-30 50- GCCGATGATCTCTCTCAAGTGAT-30 IL-6 PrimerBank ID 13624311a1 50-TAGTCCTTCC-30 50-TACCCCAATTTCC-30 MCP-1 PrimerBank ID 6755430a1 50- TTAAAAACCTGGATCGGAACCAA-30 50- GCATTAGCTTCAGATTTACGGGT-30 STAT-3 PrimerBank ID 13277852a1 50- CAATACCATTGACCTGCCGAT-30 50- GAGCGACTCAAACTGCCCT-30 STAT6 PrimerBank ID 6678155a1 50- CTCTGTGGGGCCTAATTTCCA-30 50- CATCTGAACCGACCAGGAACT-30 TNF-a PrimerBank ID 7305585a1 50- CCCTCACACTCAGATCATCTTCT-30 50- GCTACGACGTGGGCTACAG-30 Software and algorithms ImageJ 1.53t FIJI https://imagej.nih.gov/ij/ Graphpad Prism 9.0 Graphpad software LLC https://www.graphpad.com

Techniques: Expressing, Western Blot, Control, Isolation, Saline, Immunohistochemistry, Staining, Two Tailed Test

Journal: Cell reports. Medicine

Article Title: Loss of IGFBP2 mediates alveolar type 2 cell senescence and promotes lung fibrosis.

doi: 10.1016/j.xcrm.2023.100945

Figure Lengend Snippet: Figure 6. Intranasal treatment of recombinant IGFBP2 alleviates bleomycin-induced pulmonary fibrosis in aged mice (A) Schematic representation of the experimental approach. Aged WT mice were exposed to saline or bleomycin treated with or without recombinant IGFBP2 protein (25 mg/kgwt), containing Curosurf (50 mg/kgwt), by intranasal instillation and euthanized 14 and 28 days later. (B) Body weights of IGFBP2-treated and vehicle-treated mice were measured and represented as bar graph (n = 8 per group). ***p < 0.001 and *p < 0.05, two-way ANOVA.

Article Snippet: REAGENT or RESOURCE SOURCE IDENTIFIER TaqManTM Fast Advanced Master Mix Thermo Fisher scientific Cat# 44-445-56 Hydroxyproline assay Cell Biolab Cat# STA-675 Beta-galactosidase activity assay Biovision Cat# K821- 100 RT2 profiler PCR assay Qiagen Cat# PAMM-050ZC-6 IGFBP2 ELSA assay R&D systems Cat# DY797 BCA protein assay Thermo Fisher Scientific Cat# 23227 Experimental models: Cell lines MLE-12 ATCC CRL-2110 Experimental models: Organisms/strains Igfbp2flx/flxtransgenic mouse Leeds University Sftpc-CreERT2 The Jackson Laboratory RRID#IMSR_JAX:028054 C57BL6/J The Jackson Laboratory RRID#IMSR_JAX:000664 Oligonucleotides IL-1bCCH PrimerBank ID 6680415a1 50-GCAACTGTTCCTGAACTCAACT-30 and 50-ATCTTTTGGGGTCCGTCAACT-30 IL-4 PrimerBank ID 10946584a1 50- GGTCTCAACCCCCAGCTAGT-30 50- GCCGATGATCTCTCTCAAGTGAT-30 IL-6 PrimerBank ID 13624311a1 50-TAGTCCTTCC-30 50-TACCCCAATTTCC-30 MCP-1 PrimerBank ID 6755430a1 50- TTAAAAACCTGGATCGGAACCAA-30 50- GCATTAGCTTCAGATTTACGGGT-30 STAT-3 PrimerBank ID 13277852a1 50- CAATACCATTGACCTGCCGAT-30 50- GAGCGACTCAAACTGCCCT-30 STAT6 PrimerBank ID 6678155a1 50- CTCTGTGGGGCCTAATTTCCA-30 50- CATCTGAACCGACCAGGAACT-30 TNF-a PrimerBank ID 7305585a1 50- CCCTCACACTCAGATCATCTTCT-30 50- GCTACGACGTGGGCTACAG-30 Software and algorithms ImageJ 1.53t FIJI https://imagej.nih.gov/ij/ Graphpad Prism 9.0 Graphpad software LLC https://www.graphpad.com

Techniques: Recombinant, Saline

Journal: Cell reports. Medicine

Article Title: Loss of IGFBP2 mediates alveolar type 2 cell senescence and promotes lung fibrosis.

doi: 10.1016/j.xcrm.2023.100945

Figure Lengend Snippet: Figure 7. Effects of aged human Igfbp2 transgenic mice challenged with bleomycin treatment (A) Line plot showing the change in body weights of aged (36 weeks) WT and human Igfbp2 transgenic (Tg) mice subjected to intratracheal administration of bleomycin treatment (0.75 U/kg bodyweight) (n = 7 Igfbp2 fx/fx; n = 7 Igfbp2 Tg). ***p < 0.001 and **p < 0.01, two-way ANOVA. (B) Sirius red (top)- or Mason’s trichrome (middle)-stained lung sections and whole-lung images (trichrome; below) of aged Igfbp2 fx/fx and human Igfbp2 Tg mice 28 days after intratracheal administration of bleomycin treatment. Scale bars, 50 mm (top and middle) and 1 mm (below) (n = 8 Igfbp2 fx/fx; n = 8 Igfbp2 Tg). (C) Total lung collagen content measured by hydroxyproline assay in aged Igfbp2 fx/fx and human Igfbp2 Tg mice 28 days after intratracheal administration of bleomycin treatment (n = 4 Igfbp2 fx/fx; n = 8 Igfbp2 Tg). **p < 0.01 and *p < 0.05, one way ANOVA with Tukey’s post-hoc test. (D) Western blot for the expression of IGFBP2, P21, collagen-I, fibronectin, and vimentin (n = 6 Igfbp2 fx/fx; n = 8 Igfbp2 Tg). (E) qPCR analysis for mRNA expression of tumor necrosis factor a (TNF-a), IL-1b, MCP-1, IL-6, STAT3, STAT6, and IL-4 in aged WT and human Igfbp2 Tg mice 14 days after intratracheal administration of bleomycin. Each sample is obtained from 4 mice lungs (n = 6 Igfbp2 fx/fx; n = 6 Igfbp2 Tg). ****p < 0.0001, ***p < 0.001, **p < 0.01, and *p < 0.05. Student’s unpaired two-tailed t test. (F) Representative double-color immunohistochemistry-stained lung images of SPC (green) and phospho-H2AX (brown) expression from aged Igfbp2 fx/fx and Igfbp2 Tg mice 28 days after bleomycin injury. Black arrowheads indicate the double-positive AEC2 cells. Scale bars, 10 mm. (G) Bar graph showing the percentages of double-positive p-H2AX and SPC AEC2 cells that were quantified. Data are mean ± SEM. NS, not significant; ****p < 0.001, one way ANOVA with Tukey’s post-hoc test. (H) Schema represents molecular regulation of IGFBP2 signaling involving senescence in the AEC2 cells of the aged lung.

Article Snippet: REAGENT or RESOURCE SOURCE IDENTIFIER TaqManTM Fast Advanced Master Mix Thermo Fisher scientific Cat# 44-445-56 Hydroxyproline assay Cell Biolab Cat# STA-675 Beta-galactosidase activity assay Biovision Cat# K821- 100 RT2 profiler PCR assay Qiagen Cat# PAMM-050ZC-6 IGFBP2 ELSA assay R&D systems Cat# DY797 BCA protein assay Thermo Fisher Scientific Cat# 23227 Experimental models: Cell lines MLE-12 ATCC CRL-2110 Experimental models: Organisms/strains Igfbp2flx/flxtransgenic mouse Leeds University Sftpc-CreERT2 The Jackson Laboratory RRID#IMSR_JAX:028054 C57BL6/J The Jackson Laboratory RRID#IMSR_JAX:000664 Oligonucleotides IL-1bCCH PrimerBank ID 6680415a1 50-GCAACTGTTCCTGAACTCAACT-30 and 50-ATCTTTTGGGGTCCGTCAACT-30 IL-4 PrimerBank ID 10946584a1 50- GGTCTCAACCCCCAGCTAGT-30 50- GCCGATGATCTCTCTCAAGTGAT-30 IL-6 PrimerBank ID 13624311a1 50-TAGTCCTTCC-30 50-TACCCCAATTTCC-30 MCP-1 PrimerBank ID 6755430a1 50- TTAAAAACCTGGATCGGAACCAA-30 50- GCATTAGCTTCAGATTTACGGGT-30 STAT-3 PrimerBank ID 13277852a1 50- CAATACCATTGACCTGCCGAT-30 50- GAGCGACTCAAACTGCCCT-30 STAT6 PrimerBank ID 6678155a1 50- CTCTGTGGGGCCTAATTTCCA-30 50- CATCTGAACCGACCAGGAACT-30 TNF-a PrimerBank ID 7305585a1 50- CCCTCACACTCAGATCATCTTCT-30 50- GCTACGACGTGGGCTACAG-30 Software and algorithms ImageJ 1.53t FIJI https://imagej.nih.gov/ij/ Graphpad Prism 9.0 Graphpad software LLC https://www.graphpad.com

Techniques: Transgenic Assay, Staining, Hydroxyproline Assay, Western Blot, Expressing, Two Tailed Test, Immunohistochemistry

Journal: Cell reports. Medicine

Article Title: Loss of IGFBP2 mediates alveolar type 2 cell senescence and promotes lung fibrosis.

doi: 10.1016/j.xcrm.2023.100945

Figure Lengend Snippet: Figure 8. IGFBP2 expression was suppressed in the primary AEC2 cells of fibrotic lungs obtained from patients with IPF (A) IGFBP2 mRNA expression was determined by qPCR in the primary AEC2 cells isolated from fibrotic lung regions of patients with IPF (n = 27) compared with patients with COPD (n = 9) or HP (n = 5). *p < 0.05 and **p < 0.01, one-way ANOVA with Tukey’s post-hoc test. (B) IGFBP2 mRNA expression in primary AEC2 cells obtained from patients with IPF with smoking history (n = 19) compared with patients with IPF with non- smoking history (n = 6). (C) IGFBP2 mRNA expression in primary AEC2 cells obtained from patients with IPF with type 2 diabetes (n = 4) compared with patients with IPF with no type 2 diabetes (n = 7). (D) IGFBP2 mRNA expression determined by qPCR in the primary AEC2 cells obtained from patients with IPF with pulmonary hypertension (MPAP R 25 mmHg) (n = 13) compared with patients with IPF with no pulmonary hypertension (n = 14). MPAP, mean pulmonary artery pressure. (E) Representative multicolor immunohistological staining of SPC and IGFBP2. Arrows indicate examples of SPC-positive and IGFBP2-positive cells. Staining was performed with lung sections from 2 healthy controls and 2 patients with IPF. (F) Quantification of percentages of double-positive cells for SPC and IGFBP2 in the fibrotic lung regions of patients with IPF and donor (healthy) controls. Data are expressed as mean ± SEM. NS, not significant; *p < 0.05, **p < 0.01, and ****p < 0.0001, Student’s unpaired two-tailed t test.

Article Snippet: REAGENT or RESOURCE SOURCE IDENTIFIER TaqManTM Fast Advanced Master Mix Thermo Fisher scientific Cat# 44-445-56 Hydroxyproline assay Cell Biolab Cat# STA-675 Beta-galactosidase activity assay Biovision Cat# K821- 100 RT2 profiler PCR assay Qiagen Cat# PAMM-050ZC-6 IGFBP2 ELSA assay R&D systems Cat# DY797 BCA protein assay Thermo Fisher Scientific Cat# 23227 Experimental models: Cell lines MLE-12 ATCC CRL-2110 Experimental models: Organisms/strains Igfbp2flx/flxtransgenic mouse Leeds University Sftpc-CreERT2 The Jackson Laboratory RRID#IMSR_JAX:028054 C57BL6/J The Jackson Laboratory RRID#IMSR_JAX:000664 Oligonucleotides IL-1bCCH PrimerBank ID 6680415a1 50-GCAACTGTTCCTGAACTCAACT-30 and 50-ATCTTTTGGGGTCCGTCAACT-30 IL-4 PrimerBank ID 10946584a1 50- GGTCTCAACCCCCAGCTAGT-30 50- GCCGATGATCTCTCTCAAGTGAT-30 IL-6 PrimerBank ID 13624311a1 50-TAGTCCTTCC-30 50-TACCCCAATTTCC-30 MCP-1 PrimerBank ID 6755430a1 50- TTAAAAACCTGGATCGGAACCAA-30 50- GCATTAGCTTCAGATTTACGGGT-30 STAT-3 PrimerBank ID 13277852a1 50- CAATACCATTGACCTGCCGAT-30 50- GAGCGACTCAAACTGCCCT-30 STAT6 PrimerBank ID 6678155a1 50- CTCTGTGGGGCCTAATTTCCA-30 50- CATCTGAACCGACCAGGAACT-30 TNF-a PrimerBank ID 7305585a1 50- CCCTCACACTCAGATCATCTTCT-30 50- GCTACGACGTGGGCTACAG-30 Software and algorithms ImageJ 1.53t FIJI https://imagej.nih.gov/ij/ Graphpad Prism 9.0 Graphpad software LLC https://www.graphpad.com

Techniques: Expressing, Isolation, Staining, Two Tailed Test

Journal: Cell Reports Medicine

Article Title: Loss of IGFBP2 mediates alveolar type 2 cell senescence and promotes lung fibrosis

doi: 10.1016/j.xcrm.2023.100945

Figure Lengend Snippet:

Article Snippet: mouse Igfbp2 lentiviral particle , Origene , Cat#: MR204287L3V.

Techniques: Virus, Plasmid Preparation, Recombinant, Polymer, Concentration Assay, Membrane, Hydroxyproline Assay, Activity Assay, Bicinchoninic Acid Protein Assay, Transgenic Assay, Software

Journal: BMC Neuroscience

Article Title: Spatial distribution of insulin-like growth factor binding protein-2 following hypoxic-ischemic injury

doi: 10.1186/1471-2202-14-158

Figure Lengend Snippet: IGFBP-2 in ischemic cerebrocortical neurons and astrocytes. Primary neuron or astrocyte cultures were subjected to 1 h of oxygen-glucose deprivation (OGD) followed by 24 h of re-oxygenation. (A) Neurons are co-labeled with anti-MAP2 and anti-IGFBP-2. (B) Astrocytes are co-labeled with anti-GFAP and anti-IGFBP-2. Bar represents 10 μm.

Article Snippet: Sections were then permeabilized (0.1% Triton-X/PBS, 5 m, RT), blocked (5% Bovine Serum Albumin/PBS, 1.5 h, RT) and incubated in primary (IGFBP-2, R&D Systems, AF797; Neuronal Nuclei (NeuN), Millipore, MAB377; GFAP, Sigma, G6171; Iba-1, Wako, 019–19741) and secondary antibodies (Alexa Fluors, 1:500, Invitrogen) in blocking buffer.

Techniques: Labeling

Journal: BMC Neuroscience

Article Title: Spatial distribution of insulin-like growth factor binding protein-2 following hypoxic-ischemic injury

doi: 10.1186/1471-2202-14-158

Figure Lengend Snippet: IGFBP-2 protein in adult mouse brain. IGFBP-2 is detectable in the olfactory bulb, cortex and cerebellum of adult mice. The olfactory bulb has the highest levels of IGFBP-2 compared to the rest of the tissue. (A) Western blot of IGFBP-2 in brain lysates. (Rcb - recombinant protein, OB - olfactory blulb, CTX - cortex, CB - cerebellum). (B) IGFBP-2 ELISA using brain lysates. Results are mean ± S.D. n = 5; * p < 0.001 by ANOVA, olfactory bulb versus cortex or cerebellum.

Article Snippet: Sections were then permeabilized (0.1% Triton-X/PBS, 5 m, RT), blocked (5% Bovine Serum Albumin/PBS, 1.5 h, RT) and incubated in primary (IGFBP-2, R&D Systems, AF797; Neuronal Nuclei (NeuN), Millipore, MAB377; GFAP, Sigma, G6171; Iba-1, Wako, 019–19741) and secondary antibodies (Alexa Fluors, 1:500, Invitrogen) in blocking buffer.

Techniques: Western Blot, Recombinant, Enzyme-linked Immunosorbent Assay

Journal: BMC Neuroscience

Article Title: Spatial distribution of insulin-like growth factor binding protein-2 following hypoxic-ischemic injury

doi: 10.1186/1471-2202-14-158

Figure Lengend Snippet: IGFBP-2 Expression Following Hypoxic-Ischemic Injury. Brain sections of mice from either the Sham group or the MCAO group were used to visualize IGFBP-2 in neurons, astrocytes and microglia. All images were taken of cells in the cortex that formed the penumbra. (A, B) Neurons are co-labeled with anti-NeuN and anti-IGFBP-2. (C) Astrocytes are co-labeled with anti-GFAP and anti-IGFBP-2. (D) Microglia are co-labeled with anti-Iba-1 and anti-IGFBP-2. Bars represent 20 μm.

Article Snippet: Sections were then permeabilized (0.1% Triton-X/PBS, 5 m, RT), blocked (5% Bovine Serum Albumin/PBS, 1.5 h, RT) and incubated in primary (IGFBP-2, R&D Systems, AF797; Neuronal Nuclei (NeuN), Millipore, MAB377; GFAP, Sigma, G6171; Iba-1, Wako, 019–19741) and secondary antibodies (Alexa Fluors, 1:500, Invitrogen) in blocking buffer.

Techniques: Expressing, Labeling

Journal: BMC Neuroscience

Article Title: Spatial distribution of insulin-like growth factor binding protein-2 following hypoxic-ischemic injury

doi: 10.1186/1471-2202-14-158

Figure Lengend Snippet: IGFBP-2 protein levels following hypoxic-lschemic injury. IGFBP-2 levels of sham animals are not significantly different between the left and the right hemispheres for the brain regions analyzed (A, C) . For the brain regions analyzed, IGFBP-2 levels are not significantly different between the contralateral and stroke hemispheres 24 h post-stroke (B) . IGFBP-2 levels on the stroke hemisphere increase significantly (*) in the penumbra (5-fold) and core (3-fold) when compared to the contralateral hemisphere 72 h post-stroke (D) . For statistical analysis, for each brain region, IGFBP-2 levels of the two hemispheres were compared. (*) on the graphs denotes the brain regions where the statistical analysis revealed a significant difference between the stroke and the contralateral hemisphere. Results are mean ± S.D. n = 5; * p < 0.05 by ANOVA contralateral hemisphere vs. stroke hemisphere.

Article Snippet: Sections were then permeabilized (0.1% Triton-X/PBS, 5 m, RT), blocked (5% Bovine Serum Albumin/PBS, 1.5 h, RT) and incubated in primary (IGFBP-2, R&D Systems, AF797; Neuronal Nuclei (NeuN), Millipore, MAB377; GFAP, Sigma, G6171; Iba-1, Wako, 019–19741) and secondary antibodies (Alexa Fluors, 1:500, Invitrogen) in blocking buffer.

Techniques:

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: Nature Communications

Article Title: Redox-dependent Igfbp2 signaling controls Brca1 DNA damage response to govern neural stem cell fate

doi: 10.1038/s41467-023-36174-z

Figure Lengend Snippet: a – c Non-adherent cultures of primary postnatal forebrain NSCs were established from WT and Ncf1 –/– mice and 2 nd passage NSCs were grown for 11 days. a Photomicrographs of NSCs after 11 days in culture. b Quantification of neurospheres ≥60 µm in diameter ( n = 5 donors). c Quantification of total cells in each NSC culture ( n = 10 donors). d – f Monolayer cultures of 2 nd passage neonatal NSCs were grown for 3 days with L-VNIO (100 µM) or vehicle. NSCs were pulsed with EdU (5 µM) for 4 h before fixation. d Photomicrographs of EdU (Red) and TUNEL (Green) labeling. e Quantification of EdU + NSCs. f Quantification of cell death ( n = 6 donors). g Photomicrographs of NSC cultures 5 days after plating 2 nd passage of Ncf1 –/– Tomato + NSCs, in either direct coculture or transwell insert coculture with Ncf1 +/+ Tomato – NSCs. h Quantification of neurospheres ≥60 µm in diameter ( n = 8, 12, 10, and 6 donors in WT, coculture, transwell coculture, and Ncf1 –/– groups, respectively). i Western blot analysis and quantification comparing Igfbp2 levels in the conditioned medium 5 days after plating ( n = 5 donors). The samples were derived from the same experiment and blots were processed in parallel. kD = kilodalton. Two-tailed Mann–Whitney U test ( b , c , i ) and Kruskal–Wallis (see Source data for full details) followed by Benjamini–Hochberg FDR multiple comparison posttest ( e , f , h ). Error bars indicate s.e.m. Scale: 200 µm. Source data are provided as a Source data file.

Article Snippet: The membrane was then incubated with rabbit polyclonal anti-Igfbp2 (Millipore, 1:1000), goat polyclonal anti-Igfbp2 (R & D systems, 1:500), rabbit monoclonal anti-γ-H2ax (Abcam, 1:1000), goat polyclonal anti-Gapdh (ThermoFisher, 1:1000), Rad51 (mouse, 1:1000, Abcam), Nestin (chicken, 1:750, Novus Biologicals), or mouse monoclonal anti-Flag (Sigma, 1:500) in 0.5% BSA in filtered 1 x TBS, overnight at 4 °C.

Techniques: TUNEL Assay, Labeling, Western Blot, Derivative Assay, Two Tailed Test, MANN-WHITNEY, Comparison

Journal: Nature Communications

Article Title: Redox-dependent Igfbp2 signaling controls Brca1 DNA damage response to govern neural stem cell fate

doi: 10.1038/s41467-023-36174-z

Figure Lengend Snippet: a , b Second-passage WT and Ncf1 –/– NSCs were treated with mouse recombinant insulin-like growth factor-binding protein 2 (Igfbp2) or H 2 O 2 24–28 h after plating. a Photomicrographs of NSCs 5 days after treatment with Igfbp2 (60 ng ml –1 ) or H 2 O 2 (10 µM). b Quantification of neurospheres ≥60 µm in diameter ( n = 4 donors). Western blot below the bar graph shows Igfbp2 levels in the medium 5 days post-treatment. c Photomicrographs of NSCs 8 days after treatment with 100 ng ml –1 native Igfbp2, H 2 O 2 -treated (Oxi), DTT-treated (Red), or DTT then H 2 O 2 sequentially treated (Red → Oxi) Igfbp2. d Quantification of neurospheres ≥60 µm in diameter ( n = 6 donors). e , f Igfbp2 –/– NSCs were transfected with empty or Igfbp2 expressing plasmid 24 h after plating. e Photomicrographs of NSCs 11 days after transfection. f Quantification of neurospheres ≥60 µm in diameter ( n = 6 donors). g , h Second passage WT and Igfbp2 –/– NSCs were treated with H 2 O 2 24 h after plating. g Photomicrographs of NSCs 10 days after H 2 O 2 (10 µM) treatment. h Quantification of neurospheres ≥60 µm in diameter ( n = 6 donors). Two-tailed students t-test ( f ), Two-way ANOVA ( b ), and One-way ANOVA ( d , h ), followed by Bonferroni multiple comparison posttest. Error bars indicate s.e.m. Scale: 200 µm ( a , c , e ) and 300 µm ( g ). Source data are provided as a Source data file.

Article Snippet: The membrane was then incubated with rabbit polyclonal anti-Igfbp2 (Millipore, 1:1000), goat polyclonal anti-Igfbp2 (R & D systems, 1:500), rabbit monoclonal anti-γ-H2ax (Abcam, 1:1000), goat polyclonal anti-Gapdh (ThermoFisher, 1:1000), Rad51 (mouse, 1:1000, Abcam), Nestin (chicken, 1:750, Novus Biologicals), or mouse monoclonal anti-Flag (Sigma, 1:500) in 0.5% BSA in filtered 1 x TBS, overnight at 4 °C.

Techniques: Recombinant, Binding Assay, Western Blot, Transfection, Expressing, Plasmid Preparation, Two Tailed Test, Comparison

Journal: Nature Communications

Article Title: Redox-dependent Igfbp2 signaling controls Brca1 DNA damage response to govern neural stem cell fate

doi: 10.1038/s41467-023-36174-z

Figure Lengend Snippet: NSCs prepared from 4 WT (A, B, C, and D) and 4 Ncf1 –/– mice were treated with vehicle (1, 2, 3, and 4) or Igfbp2 (1I, 2I, 3I, and 4I) 48 h after plating. Vehicle- and Igfbp2-treated Ncf1 –/– groups are matched for NSC preparation by the number with “I” indicating Igfbp2-treatment. After additional 48 h, total RNA was collected from all groups and ribosome-depleted RNAseq was performed. a – c Heat maps represent clustering based on Euclidean distance of all genes that were significantly differentially expressed between any of the three groups (WT, Ncf1 –/– , and Igfbp2-treated Ncf1 –/– NSCs) ( a ), WT and Ncf1 –/– ( b ), and WT and Ncf1 –/– for which expression was restored towards WT levels after treatment with Igfbp2 ( c ). Statistical analysis of changes in gene expression used Benjamini–Hochberg FDR corrected one-way ANOVAs ( a ) and Tukey’s post hoc tests ( b , c ). d Ingenuity Pathway Analysis (IPA) performed on the gene sets in ( c ) using the absolute fold change for Ncf1 –/– vs. WT and Ncf1 –/– + Igfbp2 vs. Ncf1 –/– revealed the top pathway: Role of Brca1 in DNA Damage Response ( P = 0.0010 and P = 0.0017, respectively) (Supplementary Data ). Subset of genes in the Brca1 pathway that are expressed differentially in each of the experimental groups. TPM: transcript per million. Benjamini–Hochberg corrected One-way ANOVA followed by Tukey’s multiple comparison post hoc test, N = 4 donors. Source data are provided as a Source data file.

Article Snippet: The membrane was then incubated with rabbit polyclonal anti-Igfbp2 (Millipore, 1:1000), goat polyclonal anti-Igfbp2 (R & D systems, 1:500), rabbit monoclonal anti-γ-H2ax (Abcam, 1:1000), goat polyclonal anti-Gapdh (ThermoFisher, 1:1000), Rad51 (mouse, 1:1000, Abcam), Nestin (chicken, 1:750, Novus Biologicals), or mouse monoclonal anti-Flag (Sigma, 1:500) in 0.5% BSA in filtered 1 x TBS, overnight at 4 °C.

Techniques: Expressing, Comparison

Journal: Nature Communications

Article Title: Redox-dependent Igfbp2 signaling controls Brca1 DNA damage response to govern neural stem cell fate

doi: 10.1038/s41467-023-36174-z

Figure Lengend Snippet: a , b Photomicrographs ( a ) and quantification ( b ) of neurospheres ≥60 µm in diameter 7 days after transfection of Ncf1 –/– NSCs with siRNA targeting Fanca, Fancd2 or Rad51 or negative control ( n = 6 donors). c , d Western blot analysis ( c ) and quantification ( d ) of γ-H2ax levels (normalized to Gapdh) in NSC lysates 3 days after Igfbp2 treatment ( n = 7 donors from two different experiments). e , f Western blot ( e ) and quantification ( f ) of γ-H2ax levels (relative to Gapdh) in cell lysates 3 days after transfection ( n = 3 donors). The samples were derived from the same experiment and blots were processed in parallel. kD = kilodalton. g – i WT NSCs were transduced with Peggy Back transposase and transposon to overexpress GFP or Fanca. g Photomicrographs and h quantification of neurospheres ≥60 µm in diameter ten days after plating P4 NSCs overexpressing GFP or Fanca ( n = 6 donors). i qPCR of Fanca mRNA 3 days after plating P5 WT NSCs overexpressing GFP or Fanca ( n = 6 and 5 donors in GFP and Fanca OE NSCs respectively). ΔΔCT: the difference in threshold cycles normalized to β-Actin. Kruskal–Wallis test and FDR method of Benjamini and Hochberg multiple comparison posttest ( d ), One-way ANOVA and FDR method of Benjamini and Hochberg ( b ) or Bonferroni multiple comparison posttest for marked comparisons in ( f ) and two-tailed Mann–Whitney U test ( h , i ). Error bars indicate s.e.m. Scale: 120 and 250 µm in ( a ) and ( g ) respectively. Source data are provided as a Source data file.

Article Snippet: The membrane was then incubated with rabbit polyclonal anti-Igfbp2 (Millipore, 1:1000), goat polyclonal anti-Igfbp2 (R & D systems, 1:500), rabbit monoclonal anti-γ-H2ax (Abcam, 1:1000), goat polyclonal anti-Gapdh (ThermoFisher, 1:1000), Rad51 (mouse, 1:1000, Abcam), Nestin (chicken, 1:750, Novus Biologicals), or mouse monoclonal anti-Flag (Sigma, 1:500) in 0.5% BSA in filtered 1 x TBS, overnight at 4 °C.

Techniques: Transfection, Negative Control, Western Blot, Derivative Assay, Transduction, Comparison, Two Tailed Test, MANN-WHITNEY

Journal: Nature Communications

Article Title: Redox-dependent Igfbp2 signaling controls Brca1 DNA damage response to govern neural stem cell fate

doi: 10.1038/s41467-023-36174-z

Figure Lengend Snippet: a Experimental design. b Western blot analysis of untreated or TCEP- or H 2 O 2 -treated recombinant mouse Igfbp2 (rmIgfbp2) after labeling with biotinylated iodoacetamide (BIAM). c – h Igfbp2 –/– NSCs were transfected with Igfbp2, Igfbp2 C263A , Igfbp2 C43A , Igfbp2 C43/263A , or GFP expressing plasmid 24 h after plating. c Photomicrographs of NSCs 11 days after transfection. d Quantification of neurospheres ≥60 µm in diameter ( n = 6 donors). e – g qPCR of Fanca ( e ), Fancd2 ( f ) and Rad51 ( g ) in Igfbp2 –/– NSCs 5 days after transfection with Igfbp2, Igfbp2 C263A , Igfbp2 C43A , Igfbp2 C43&263A , or GFP expressing plasmid ( n = 12 donors). ΔΔCT: the difference in threshold cycles normalized to β-Actin and compared to untransfected Igfbp2 –/– NSCs. h Western blot analysis of Igfbp2 and flag-tag in conditioned medium 3 days after transfection. kD = kilodalton. One-way ANOVA followed by Bonferroni multiple comparison posttest for marked comparisons in ( d ) or comparing the mean of each group to the mean of untransfected Igfbp2 –/– NSCs in ( e – g ). Error bars indicate s.e.m. Scale: 100 µm. Source data are provided as a Source data file.

Article Snippet: The membrane was then incubated with rabbit polyclonal anti-Igfbp2 (Millipore, 1:1000), goat polyclonal anti-Igfbp2 (R & D systems, 1:500), rabbit monoclonal anti-γ-H2ax (Abcam, 1:1000), goat polyclonal anti-Gapdh (ThermoFisher, 1:1000), Rad51 (mouse, 1:1000, Abcam), Nestin (chicken, 1:750, Novus Biologicals), or mouse monoclonal anti-Flag (Sigma, 1:500) in 0.5% BSA in filtered 1 x TBS, overnight at 4 °C.

Techniques: Western Blot, Recombinant, Labeling, Transfection, Expressing, Plasmid Preparation, FLAG-tag, Comparison