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: Molecular and Cellular Biology

Article Title: Disruption of the Fbxw8 Gene Results in Pre- and Postnatal Growth Retardation in Mice

doi: 10.1128/mcb.01665-07

Figure Lengend Snippet: FIG. 5. IGFBP2 up-regulation in Cul7/ and Fbxw8/ MEFs. (A) Passage 2 MEFs (105) derived from littermates from Fbxw8/ in- tercrosses were replica plated into 60-mm dishes and counted in duplicate every 24 h. F, Fbxw8/; , Fbxw8/; Œ, Fbxw8/. (B) MEFs from a confluent primary culture were plated into 100-mm dishes at a density of 106/dish, grown for 3 days, and counted, and the N3/N0 ratios were determined. The cells were transferred to a new dish at the original cell density, and the N3/N0 ratios were repeatedly calculated. F, Fbxw8/; , Fbxw8/. (C) Cul7/ and wild-type MEFs were cultured in Opti-MEM for 24 h, and proteins in the tissue culture supernatant were precipitated with trichloroacetic acid, resolved by SDS-PAGE, and detected by Coo- massie blue staining. Mass spectrometry analysis of the band indicated by the arrow revealed IGFBP2. (D) Expression of secreted IGFBP2 in MEFs. Aliquots from the samples in panel C were analyzed by immuno- blotting with anti-IGFBP2 antibody. (E) Cell lysates prepared from wild- type (/), Cul7/, and Fbxw8/ MEFs were blotted with IGFBP2 and vinculin antibodies. (F) Cell lysates were prepared from MEFs that were serum starved [IGF1()] and subsequently treated with IGF1 [IFG1()] (C7, Cul7/; F8, Fbxw8/). Lysates were immunoprecipitated (IP) with IGF1R antibody and immunoblotted with phosphotyrosine (P-Tyr) and IGF1R antibodies.

Article Snippet: Additional antibodies used were vinculin (hVIN1; Sigma), SKP1 (Neomarkers), IGFBP2 (R&D Systems), IGF1 receptor (IGF1R [C-20; Santa Cruz]), and phosphotyrosine (PY99; Santa Cruz). siRNA transfection.

Techniques: Derivative Assay, Cell Culture, SDS Page, Staining, Mass Spectrometry, Expressing, Immunoprecipitation

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 Molecular Medicine (Berlin, Germany)

Article Title: IGFBP2 function as a novel biomarker for active lupus nephritis

doi: 10.1007/s00109-022-02241-z

Figure Lengend Snippet: TMT detection of differential protein expression between the active LN and HC groups. A Statistical histograms of GO ontology biological processes, cellular components, and molecular functions. B Bar graph displaying the top 10 enriched GO biological process functions. C Network analysis diagram. D Scatter plot of DE proteins and corresponding transcripts analyzed by the proteome and transcriptome. The x -axis is the transcript log 2 (fold-change), and the y -axis is the protein log 2 (fold-change). R = 0.0244 was the Pearson correlation coefficient of the two sets of data. The transcript was differentially expressed according to whether it was expressed in different colors. E Heatmap of proteins that were differentially expressed at the proteome and transcriptome levels and were consistently up- or downregulated. F Real-time PCR validation tests of IGFBP2 gene expression were performed in peripheral blood mononuclear cells (PBMCs) of the control ( n = 10) and LN ( n = 24) groups. ELISA validation tests were performed to verify IGFBP2 expression in the plasma of the controls ( n = 18), inactive LN ( n = 18), and active LN patients ( n = 23). G Validation of IGFBP2 expression in the PBMCs of HC and LN patients by WB ( n = 3). * P < 0.05; ** P < 0.01; *** P < 0.001; **** P < 0.0001

Article Snippet: Briefly, protein samples (20 μg) were resolved by 12% SDS–PAGE, electroblotted on nitrocellulose membranes (Bio-Rad), incubated overnight at 4 °C with the following antibodies: IGFBP2 (11065–3-AP, Proteintech), antibodies used for Western blotting, were bought from AiFang biological and listed as follows: p-RPS6KB1(AF14512), RPS6KB1(AF11049), p-4E-BP1(AF01102), 4E-BP1(AF03855), p-AKT(AF00453), AKT(AF01499), p-mTOR (AF00658), mTOR (AF02824).

Techniques: Expressing, Real-time Polymerase Chain Reaction, Biomarker Discovery, Gene Expression, Control, Enzyme-linked Immunosorbent Assay, Clinical Proteomics

Journal: Journal of Molecular Medicine (Berlin, Germany)

Article Title: IGFBP2 function as a novel biomarker for active lupus nephritis

doi: 10.1007/s00109-022-02241-z

Figure Lengend Snippet: The level of plasma IGFBP2 expression in LNs and correlation analysis with clinical indicators. A Level of IGFBP2 expression in the plasma of RA ( n = 16), SS ( n = 16), active LN ( n = 17), and control ( n = 18) patients. B The level of urine IGFBP2 expression in LN patients ( n = 12) was higheer than that in HC ( n = 12) patients ( P < 0.01). C There was no significant difference between urinary IGFBP2 in patients with inactive ( n = 19) and active SLE ( n = 23). D There was no correlation between plasma and urinary IGFBP2 levels in LN patients ( n = 26). E Correlation analysis between plasma IGFBP2 levels and SLE activity-related indicators. F Correlation analysis between the plasma IGFBP2 and SLEDAI score at 3 months after treatment and change in SLEDAI before and after treatment. G The ROC curve revealed that IGFBP2 showed an AUC value of 0.992, with a sensitivity of 0.909 and specificity of 1.0 (95% CI 0.974–1.0, P < 0.001) at the cutoff of 512 ng/mL between inactive and active LN. * P < 0.05; ** P < 0.01; *** P < 0.001; **** P < 0.0001. ns , not statistically significant

Article Snippet: Briefly, protein samples (20 μg) were resolved by 12% SDS–PAGE, electroblotted on nitrocellulose membranes (Bio-Rad), incubated overnight at 4 °C with the following antibodies: IGFBP2 (11065–3-AP, Proteintech), antibodies used for Western blotting, were bought from AiFang biological and listed as follows: p-RPS6KB1(AF14512), RPS6KB1(AF11049), p-4E-BP1(AF01102), 4E-BP1(AF03855), p-AKT(AF00453), AKT(AF01499), p-mTOR (AF00658), mTOR (AF02824).

Techniques: Clinical Proteomics, Expressing, Control, Activity Assay

Journal: Journal of Molecular Medicine (Berlin, Germany)

Article Title: IGFBP2 function as a novel biomarker for active lupus nephritis

doi: 10.1007/s00109-022-02241-z

Figure Lengend Snippet: The level of IGFBP2 expression in the kidneys of LN. A IGFBP2 expression in normal kidney issues and LNs by IF staining ( n = 3). B IHC staining of IGFBP2 in HC ( n = 3), MN ( n = 3), and II–V type LN kidneys ( n = 3). Original magnification, × 400. The positive area is quantitatively displayed as a histogram on the right. * P < 0.05; ** P < 0.01

Article Snippet: Briefly, protein samples (20 μg) were resolved by 12% SDS–PAGE, electroblotted on nitrocellulose membranes (Bio-Rad), incubated overnight at 4 °C with the following antibodies: IGFBP2 (11065–3-AP, Proteintech), antibodies used for Western blotting, were bought from AiFang biological and listed as follows: p-RPS6KB1(AF14512), RPS6KB1(AF11049), p-4E-BP1(AF01102), 4E-BP1(AF03855), p-AKT(AF00453), AKT(AF01499), p-mTOR (AF00658), mTOR (AF02824).

Techniques: Expressing, Staining, Immunohistochemistry

Journal: Journal of Molecular Medicine (Berlin, Germany)

Article Title: IGFBP2 function as a novel biomarker for active lupus nephritis

doi: 10.1007/s00109-022-02241-z

Figure Lengend Snippet: The distribution of IGFBP2 in the organs of mice. A The level of IGFBP2 expression in the blood of MRL mice (16 weeks) ( n = 4) was higher than that of C57BL/6 mice ( n = 3). B ELISA detection of the IGFBP2 content in the various organs of C57BL/6 and MRL/lpr mice revealed that in most organs of MRL/lpr mice, the protein content of IGFBP2 was higher than that of the C57BL/6 mice. C WB showed the expression of IGFBP2 in the lymph nodes of MRL/lpr and C57BL/6 mice ( n = 3). D IHC showed that the level of IGFBP2 in the lymph nodes of MRL/lpr was significantly higher than that in the lymph nodes of C57BL/6 mice (intracellular tan deposition) ( n = 3). * P < 0.05

Article Snippet: Briefly, protein samples (20 μg) were resolved by 12% SDS–PAGE, electroblotted on nitrocellulose membranes (Bio-Rad), incubated overnight at 4 °C with the following antibodies: IGFBP2 (11065–3-AP, Proteintech), antibodies used for Western blotting, were bought from AiFang biological and listed as follows: p-RPS6KB1(AF14512), RPS6KB1(AF11049), p-4E-BP1(AF01102), 4E-BP1(AF03855), p-AKT(AF00453), AKT(AF01499), p-mTOR (AF00658), mTOR (AF02824).

Techniques: Expressing, Enzyme-linked Immunosorbent Assay

Journal: Journal of Molecular Medicine (Berlin, Germany)

Article Title: IGFBP2 function as a novel biomarker for active lupus nephritis

doi: 10.1007/s00109-022-02241-z

Figure Lengend Snippet: The IGFBP2 blocking effect in mice following treatment. A MRL/lpr mice were divided into the PBS group ( n = 4), ISO group ( n = 4), and anti-IGFBP2 group (n = 4). The ELISA results showed that compared with PBS and ISO groups, the level of plasma IGFBP2 in the anti-IGFBP2 group was decreased. B PCR and C WB showed the level of renal IGFBP2 protein expression in each of the three groups. * P < 0.05; ** P < 0.01; *** P < 0.001. ns , not statistically significant

Article Snippet: Briefly, protein samples (20 μg) were resolved by 12% SDS–PAGE, electroblotted on nitrocellulose membranes (Bio-Rad), incubated overnight at 4 °C with the following antibodies: IGFBP2 (11065–3-AP, Proteintech), antibodies used for Western blotting, were bought from AiFang biological and listed as follows: p-RPS6KB1(AF14512), RPS6KB1(AF11049), p-4E-BP1(AF01102), 4E-BP1(AF03855), p-AKT(AF00453), AKT(AF01499), p-mTOR (AF00658), mTOR (AF02824).

Techniques: Blocking Assay, Enzyme-linked Immunosorbent Assay, Clinical Proteomics, Expressing

Journal: Journal of Molecular Medicine (Berlin, Germany)

Article Title: IGFBP2 function as a novel biomarker for active lupus nephritis

doi: 10.1007/s00109-022-02241-z

Figure Lengend Snippet: Improvement of LN after a IGFBP2 blockade. A MRL/lpr mice were divided into the PBS group ( n = 4), ISO group ( n = 4), and anti-IGFBP2 group ( n = 4). The skin lesions in the anti-IGFBP2 group were significantly improved. B Compared with the PBS and ISO groups, the lymph/body weight ratio was decreased in the anti-IGFBP2 group. C Trend in the reduction of urine protein following treatment. D HE, PAS, and Masson staining (original magnification, × 400), quantified histogram of glomerulus, tubulointerstitium and perivascular pathology. * P < 0.05; ** P < 0.01

Article Snippet: Briefly, protein samples (20 μg) were resolved by 12% SDS–PAGE, electroblotted on nitrocellulose membranes (Bio-Rad), incubated overnight at 4 °C with the following antibodies: IGFBP2 (11065–3-AP, Proteintech), antibodies used for Western blotting, were bought from AiFang biological and listed as follows: p-RPS6KB1(AF14512), RPS6KB1(AF11049), p-4E-BP1(AF01102), 4E-BP1(AF03855), p-AKT(AF00453), AKT(AF01499), p-mTOR (AF00658), mTOR (AF02824).

Techniques: Staining

Journal: Journal of Molecular Medicine (Berlin, Germany)

Article Title: IGFBP2 function as a novel biomarker for active lupus nephritis

doi: 10.1007/s00109-022-02241-z

Figure Lengend Snippet: IGFBP2 blockade affects T cells and AKT/mTOR pathway. A Effect on peripheral blood T cell immunity by flow cytometry in PBS group ( n = 4), ISO group ( n = 4), and anti-IGFBP2 group ( n = 4). B Protein expression and phosphorylation levels of AKT, mTOR, RPS6KB1, and 4E-BPI in anti-IGFBP2 group ( n = 4) and ISO group ( n = 3) were detected by Western blotting. The quantitative analysis of proteins was performed using ImageJ software. * P < 0.05; ** P < 0.01; ns , not statistically significant

Article Snippet: Briefly, protein samples (20 μg) were resolved by 12% SDS–PAGE, electroblotted on nitrocellulose membranes (Bio-Rad), incubated overnight at 4 °C with the following antibodies: IGFBP2 (11065–3-AP, Proteintech), antibodies used for Western blotting, were bought from AiFang biological and listed as follows: p-RPS6KB1(AF14512), RPS6KB1(AF11049), p-4E-BP1(AF01102), 4E-BP1(AF03855), p-AKT(AF00453), AKT(AF01499), p-mTOR (AF00658), mTOR (AF02824).

Techniques: Flow Cytometry, Expressing, Phospho-proteomics, Western Blot, Software