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: Wound repair and regeneration : official publication of the Wound Healing Society [and] the European Tissue Repair Society

Article Title: The lysosomal trafficking regulator is necessary for normal wound healing

doi: 10.1111/wrr.12984

Figure Lengend Snippet: Assessment of protein levels in conditioned media from primary fibroblast culture. (A) Representative blots of relevant cytokines, chemokines, and growth factors from RayBiotech Mouse Cytokine Antibody Arrays demonstrating WT and Bg fibroblasts’ relative protein secretion in conditioned media. Fibroblasts lines were derived from 2 to 3 mice from each genotype and experiments were done with 3–5 technical replicates. (B) Quantification of the integrated densities (n = 3 per group). Integrated densities are relative to manufacturer controls. Relative integrated densities of MCP-1, IGF-1, and IGFBP-2 were significantly decreased in Bg samples (p = 0.0083, p = 0.0159, and p = 0.0111, respectively). (C) Confirmatory ELISAs of MCP-1 and IGFBP-2 (n = 5 per group) show decreased protein secretion from Bg fibroblasts (p = 0.0068 and p = 0.0498, respectively). *p ≤ 0.05, **p ≤ 0.01

Article Snippet: Taqman Fast Advanced Master Mix (Cat# 4444557, Thermo Fisher Scientific) with the following hybridization probes were utilised for qPCR of cDNA samples: LYST (Cat# Mm00465000_m1), CCL2 (Cat# Mm00441242_m1), IGF1 (Cat# Mm00439560_m1), IGFBP2 (Cat# Mm00492632_m1), Col1a1 (Cat# Mm00801666_g1), Col1a2 (Cat# Mm00483888_m1), and Col3a1 (Mm00802331_m1).

Techniques: Derivative Assay

Journal: Wound repair and regeneration : official publication of the Wound Healing Society [and] the European Tissue Repair Society

Article Title: The lysosomal trafficking regulator is necessary for normal wound healing

doi: 10.1111/wrr.12984

Figure Lengend Snippet: Protein localization and gene expression in fibroblasts. (A) LAMP-1 immunofluorescent (IF) images of WT and Bg fibroblasts demonstrate enlarged lysosomes in Bg fibroblasts. (B) RT-qPCR analysis shows no change in relative gene expression of lysosomal trafficking regulator (LYST) between WT and Bg fibroblasts. (C) MCP-1 staining shows primarily cytoplasmic staining of similar degree between genotypes. (D) Gene expression of MCP-1 is comparable between genotypes at all time points. (E) Representative images of IGF-1 also show high nuclear localization with presence in nucleoli in both genotypes. (F) IGF-1 gene expression between cell types is similar at 24 and 48 h. At 72 h, Bg cells show a higher degree of gene expression (p = 0.0156). (G) Representative IF images of IGFBP-2 show a high degree of nuclear localization in both genotypes. (H) Relative gene expression of IGFBP-2 is decreased in Bg fibroblasts at 24 h (p = 0.0083) but is comparable to WT cells starting at 48 h. Fibroblasts lines were derived from 2 to 3 mice from each genotype and experiments were done with 5–6 technical replicates. DAPI was used to visualise nuclei (blue). Actin is represented with green, and proteins of interest are red. Scale bar = 20 μm. *p ≤ 0.05, **p ≤ 0.01 (n = 5–6 per group)

Article Snippet: Taqman Fast Advanced Master Mix (Cat# 4444557, Thermo Fisher Scientific) with the following hybridization probes were utilised for qPCR of cDNA samples: LYST (Cat# Mm00465000_m1), CCL2 (Cat# Mm00441242_m1), IGF1 (Cat# Mm00439560_m1), IGFBP2 (Cat# Mm00492632_m1), Col1a1 (Cat# Mm00801666_g1), Col1a2 (Cat# Mm00483888_m1), and Col3a1 (Mm00802331_m1).

Techniques: Gene Expression, Quantitative RT-PCR, Staining, Derivative Assay

Journal: Wound repair and regeneration : official publication of the Wound Healing Society [and] the European Tissue Repair Society

Article Title: The lysosomal trafficking regulator is necessary for normal wound healing

doi: 10.1111/wrr.12984

Figure Lengend Snippet: Localization of IGF associated proteins in fibroblasts. (A) Co-stain of IGF-1 (red) and IGFBP-2 (green) shows nuclear colocalization (yellow) of the proteins based on the microscopic resolution (0.24 μm). Colocalization is not seen in the cytoplasm or in the nucleoli of the nucleus. (B) IGF-1 (red) and IGF-1 receptor (IGF-1R) (green) do not appear to colocalize. IGF-1R appears mostly cytoplasmic. (C) Lysosomes stained with LAMP-1 (red) and IGF-1R (green) show colocalization associated with the membrane of lysosomes. DAPI (blue) is used to visualise nuclei and void regions in nuclei are nucleoli. Scale bar = 20 μm

Article Snippet: Taqman Fast Advanced Master Mix (Cat# 4444557, Thermo Fisher Scientific) with the following hybridization probes were utilised for qPCR of cDNA samples: LYST (Cat# Mm00465000_m1), CCL2 (Cat# Mm00441242_m1), IGF1 (Cat# Mm00439560_m1), IGFBP2 (Cat# Mm00492632_m1), Col1a1 (Cat# Mm00801666_g1), Col1a2 (Cat# Mm00483888_m1), and Col3a1 (Mm00802331_m1).

Techniques: Staining, Membrane

Journal: PLoS ONE

Article Title: IGFBP2 promotes immunosuppression associated with its mesenchymal induction and FcγRIIB phosphorylation in glioblastoma

doi: 10.1371/journal.pone.0222999

Figure Lengend Snippet: The interfering RNA sequences used for IGFBP2 knockdown.

Article Snippet: For anti-IGFBP2 antibody treatment, 2.0 μg/ml of a rabbit anti-IGFBP2 monoclonal antibody (anti-IGFBP2mAb) (bs-1108R, Bioss) or isotype IgG control (bs-0295P, Bioss) was added to GL261 culture for 3 days and collected for western blotting.

Techniques: Sequencing

Journal: PLoS ONE

Article Title: IGFBP2 promotes immunosuppression associated with its mesenchymal induction and FcγRIIB phosphorylation in glioblastoma

doi: 10.1371/journal.pone.0222999

Figure Lengend Snippet: (A) An association of the enrichment score of the MES signature with the expression of IGFBP2 using the TCGA-gliomas database. R = Pearson’s correlation coefficient. (B-C) Effect of IGFBP2 knockdown with shRNA (IGFBP2KD) (B) or an anti-IGFBP2 antibody (anti-IGFBP2) (C) on mesenchymal marker protein levels in mouse GBM GL261 cells. (D) Effect of overexpressed IGFBP2 wildtype (IGFBP2OE) or mutant (IGFBP2mt) with the change of RGD to RGE on mesenchymal marker protein levels. The experiments were repeated at least three times. A statistical significance was calculated using an unpaired Welch’s t test. * P<0.05, ** P<0.01, *** P<0.001.

Article Snippet: For anti-IGFBP2 antibody treatment, 2.0 μg/ml of a rabbit anti-IGFBP2 monoclonal antibody (anti-IGFBP2mAb) (bs-1108R, Bioss) or isotype IgG control (bs-0295P, Bioss) was added to GL261 culture for 3 days and collected for western blotting.

Techniques: Expressing, shRNA, Marker, Mutagenesis

Journal: PLoS ONE

Article Title: IGFBP2 promotes immunosuppression associated with its mesenchymal induction and FcγRIIB phosphorylation in glioblastoma

doi: 10.1371/journal.pone.0222999

Figure Lengend Snippet: (A-C) The mouse splenocytes (SPCs) were co-cultured with GL261 cells for 6 days and then collected to analyze the number of CD8 + and CD4 + T cells by fluorescence activated cell sorting (FACS) under the setting of IGFBP2KD (A), anti-IGFBP2 (B) or overexpressed IGFBP2 and its mutant (C). The experiments were repeated at least three times. Representative IHC images of CD8 + T cells (D-E), CD163 + M2 macrophages (G-H), and pY513-CD19 + cells (J-K) in tumor-bearing brains of mice treated with anti-IGFBP2 or IgG. 100X (left), 400X (right). N = 3. (F, I) The percentage of tumor infiltrating CD8 + T cells (F) and CD163 + M2 macrophages in F4/80 + macrophages (I) analyzed by FACS after anti-IGFBP2 or IgG treatment. N = 5. The data are mean ± SD. A statistical significance was calculated by an unpaired Welch’s t test. * P<0.05, ** p < 0.01, *** p < 0.001.

Article Snippet: For anti-IGFBP2 antibody treatment, 2.0 μg/ml of a rabbit anti-IGFBP2 monoclonal antibody (anti-IGFBP2mAb) (bs-1108R, Bioss) or isotype IgG control (bs-0295P, Bioss) was added to GL261 culture for 3 days and collected for western blotting.

Techniques: Cell Culture, Fluorescence, FACS, Mutagenesis

Journal: PLoS ONE

Article Title: IGFBP2 promotes immunosuppression associated with its mesenchymal induction and FcγRIIB phosphorylation in glioblastoma

doi: 10.1371/journal.pone.0222999

Figure Lengend Snippet: ( A) An association of the enrichment score of the MES signature with FcγRIIB expression analyzed by using the TCGA-human glioma database. R = Pearson’s correlation coefficient. (B) Representative IHC images of IGFBP2 protein and phosphorylated FcγRIIB (p-FcγRIIB) levels in human GBMs (hGBMs) or normal brains stained in tissue microarrays. (C) An association of IGFBP2 protein and p-FcγRIIB levels in human GBMs. R, the Pearson correlation coefficient. (D-I), The percentage of p-FcγRIIB + cells in the SPCs after co-cultured with GL261 cells treated with sh1RNA-IGFBP2 (D-E), anti-IGFBP2 (F-G) or overexpressing IGFBP2 or IGFBP2mt (H-I) analyzed by FACS. (J-K), Representative IHC images of p-FcγRIIB staining in tumor-bearing brains treated with anti-IGFBP2 or IgG. N = 3. (L) The percentage of p-FcγRIIB + cells in infiltrating immune cells in GL261 tumors analyzed by FACS. N = 5. The data are mean ± SD. A statistical significance was computed by an unpaired Welch’s t test. * P < 0.05, ** p < 0.01.

Article Snippet: For anti-IGFBP2 antibody treatment, 2.0 μg/ml of a rabbit anti-IGFBP2 monoclonal antibody (anti-IGFBP2mAb) (bs-1108R, Bioss) or isotype IgG control (bs-0295P, Bioss) was added to GL261 culture for 3 days and collected for western blotting.

Techniques: Expressing, Staining, Cell Culture

Journal: PLoS ONE

Article Title: IGFBP2 promotes immunosuppression associated with its mesenchymal induction and FcγRIIB phosphorylation in glioblastoma

doi: 10.1371/journal.pone.0222999

Figure Lengend Snippet: (A-B) The time course induction of FcγRIIB phosphorylation on CD19 + B cells in SPCs co-cultured with IGFBP2OE GL261 or control cells. The percentage of CD19 L p-FcγRIIB H , CD19 L p-FcγRIIB - , and CD19 H p-FcγRIIB L B cell subsets was analyzed by FACS. L is low, H is high. (C-H) The percentage of p-FcγRIIB + cells in CD19 + B cells after co-cultured with GL261 cells treated with shRNA (C-D), anti-IGFBP2 (E-F) or overexpressing IGFBP2 or its mutant (G-H). (I-J) The percentage of CD19 + p-FcγRIIB + B cells in infiltrating immune cells (I) and p-FcγRIIB + cells in CD19 + B cells in anti-IGFBP2 or IgG tumors analyzed by FACS(J). (K) The ratio of CD19 + B cells versus CD19 + p-FcγRIIB + B cells. (L-M) The percentage of F4/80 + p-FcγRIIB + macrophages in infiltrating immune cells (L) and p-FcγRIIB + cells in F4/80 + macrophages (M) in anti-IGFBP2 or IgG tumors analyzed by FACS. N = 5. The data are mean ± SD. A statistical significance was calculated by an unpaired Welch's t test. * P < 0.05, ** P < 0.01.

Article Snippet: For anti-IGFBP2 antibody treatment, 2.0 μg/ml of a rabbit anti-IGFBP2 monoclonal antibody (anti-IGFBP2mAb) (bs-1108R, Bioss) or isotype IgG control (bs-0295P, Bioss) was added to GL261 culture for 3 days and collected for western blotting.

Techniques: Cell Culture, shRNA, Mutagenesis

Journal: PLoS ONE

Article Title: IGFBP2 promotes immunosuppression associated with its mesenchymal induction and FcγRIIB phosphorylation in glioblastoma

doi: 10.1371/journal.pone.0222999

Figure Lengend Snippet: P values in Fig 4C–4H .

Article Snippet: For anti-IGFBP2 antibody treatment, 2.0 μg/ml of a rabbit anti-IGFBP2 monoclonal antibody (anti-IGFBP2mAb) (bs-1108R, Bioss) or isotype IgG control (bs-0295P, Bioss) was added to GL261 culture for 3 days and collected for western blotting.

Techniques:

Journal: PLoS ONE

Article Title: IGFBP2 promotes immunosuppression associated with its mesenchymal induction and FcγRIIB phosphorylation in glioblastoma

doi: 10.1371/journal.pone.0222999

Figure Lengend Snippet: ( A) Representative T2-weighted MRI of GL261-bearing brains of mice treated with anti-IGFBP2 or IgG after 3 and 5 weeks of GL261cell injection. (B) The tumor growth curve in anti-IGFBP2 and IgG groups. (C) The Kaplan-Meier survival plot of GL261-bearing mice treated with anti-IGFBP2 or IgG. Log-rank test was used to compare the difference of the median survival time between the two groups. N = 5.

Article Snippet: For anti-IGFBP2 antibody treatment, 2.0 μg/ml of a rabbit anti-IGFBP2 monoclonal antibody (anti-IGFBP2mAb) (bs-1108R, Bioss) or isotype IgG control (bs-0295P, Bioss) was added to GL261 culture for 3 days and collected for western blotting.

Techniques: Injection

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: Frontiers in Immunology

Article Title: Comprehensive analysis of m6A RNA methylation modification patterns and the immune microenvironment in osteoarthritis

doi: 10.3389/fimmu.2023.1128459

Figure Lengend Snippet: Relationship analysis of m6A regulators with immune characteristics in OA. (A) Dot plot showing the relationship of infiltrating immune cells with hub m6A regulators in the OA immune microenvironment. The most positively related immunocyte-m6A regulator pair is YTHDF2-Treg cell (r = 0.429), and the most negative is IGFBP2-DC cell (r = -0.325), with expression and fraction status presented in the upper panel. (B) Dot plot showing the relationship of immune response pathways with hub m6A regulators in the OA immune microenvironment. The most positively associated pair is the YTHDF2-HLA pathway (r = 0.369), the most negatively correlated is HNRNPA2B1-Type I IFN response (r = -0.355), with the violin plot in the bottom panel showing expression or activity.

Article Snippet: IGFBP2 polyclonal antibody (11065-3-AP, Proteintech Group, Inc.) was utilized to detect IGFBP2.

Techniques: Expressing, Activity Assay

Journal: Frontiers in Immunology

Article Title: Comprehensive analysis of m6A RNA methylation modification patterns and the immune microenvironment in osteoarthritis

doi: 10.3389/fimmu.2023.1128459

Figure Lengend Snippet: Correlations between the expression of IGFBP2 and DCs marker CD1a, YTHDF2 and Treg marker FOXP3 in OA and normal tissues. (A) Representative images for the IHC staining of IGFBP2 and CD1a in OA and normal tissues (n = 10). (B) Representative images for the IHC staining of YTHDF2 and FOXP3 in OA and normal tissues (n = 10). (C) Relation between protein levels of IGFBP2 and CD1a in OA and normal tissues via Spearman correlation. (D) Relation between protein levels of YTHDF2 and FOXP3 in OA and normal tissues via Spearman correlation.

Article Snippet: IGFBP2 polyclonal antibody (11065-3-AP, Proteintech Group, Inc.) was utilized to detect IGFBP2.

Techniques: Expressing, Marker, Immunohistochemistry

Journal: Frontiers in Immunology

Article Title: Comprehensive analysis of m6A RNA methylation modification patterns and the immune microenvironment in osteoarthritis

doi: 10.3389/fimmu.2023.1128459

Figure Lengend Snippet: qRT-PCR validation of key m6A regulators. (A) X ray images and intraoperative macroscopic views (arthroplasty and arthroscopy image) of knee joint from OA patients and healthy donors. (B, C) Validation of IGFBP2 and YTHDF2 mRNA expression by qRT-PCR between OA (n = 10) and normal group (n = 10). **p < 0.01, and ***p < 0.001.

Article Snippet: IGFBP2 polyclonal antibody (11065-3-AP, Proteintech Group, Inc.) was utilized to detect IGFBP2.

Techniques: Quantitative RT-PCR, Biomarker Discovery, Expressing