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: 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: 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 Antibodies Rat-anti-IGFBP2 R&D systems Cat# MAB7971; RRID:AB_2264598 Rabbit anti-IGFBP2 for IHC Abcam Cat# ab188200 Rabbit anti-P21 Abcam Cat# ab188224; RRID:AB_2734729 Rabbit anti-Collagen 1 EMD Millipore Cat# AB765P; RRID:AB_92259 Rabbit anti-PPARa LSBio Cat# LS-C312574 Rabbit anti-Fibronectin Abcam Cat# ab2413; RRID:AB_2262874 Rabbit anti-Vimentin CST Cat# 5741S; RRID:AB_10695459 HRP conjugated-b actin Santa Cruz Cat# sc-47778HRP; RRID:AB_2714189 Mouse anti-a tubulin CST Cat# 3873; RRID:AB_1904178 Rabbit anti-H3 Histone CST Cat# 4499; RRID:AB_10544537 Rabbit anti-phospho-Histone 2AX CST Cat# 2577; RRID:AB_2118010 Rabbit anti-Prosurfactant Protein C Abcam Cat# ab211326; RRID:AB_2927746 Bacterial and virus strains Ad-Null Vector Biolabs Cat#1240 Ad-m-TGFb1 Vector Biolabs Cat#ADV-274099 mouse Igfbp2 lentiviral particle Origene Cat#: MR204287L3V Lenticontrol particles Origene Cat# PS100092V Chemicals, peptides, and recombinant proteins human IGFBP2 recombinant protein R&D Systems Cat# 797-B2-025 Bleomycin EMD Millipore Cat# 203401 DMSO Sigma- Aldrich Cat# D8418 4% PFA Santa Cruz Cat# sc-281692 DMEM/F12 Gibco Cat# 11-320-033 Plasmocin invivogen Cat# ant-mpp Collagenase type 1 Worthington Biochemical Cat# LS004197 Dispase Corning Cat# 354235 Green chromogen Leica Cat# DC9913 Bond polymer refine system Leica Cat# DS9800 BondTM Primary antibody diluent Leica Cat# AR9352 BOND Epitope Retrieval Solution 1 Leica Cat# AR9961 BOND Epitope Retrieval Solution 2 Leica Cat# AR9640 Wash solution 10X concentration Leica Cat# AR9590 BOND Universal Covertile Leica Cat# S21.4611 Tween-20 Sigma- Aldrich Cat# P9416 PVDF membrane EMD Millipore Cat# IPVH00010 SuperSignalTM Pico or Femto substrate Thermo Fisher Scientific Cat# 34095 RIPA buffer Thermo Fisher scientific Cat# 89901 Igfbp2 Life science technology Hs01040719_m1 18S rRNA Life science technology Hs99999901_s1 Ppara Life science technology Mm00440939_m1 and Hs00231882_m1 Igfbp2 siRNA Horizon Discovery L-062198-01-0005 Ppara siRNA Santa Cruz sc-36308 Critical commercial assays High-Capacity RNA-to-cDNATM Kit Thermo Fisher scientific Cat# 4387406 (Continued on next page) Cell Reports Medicine 4, 100945, March 21, 2023 e1

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 Antibodies Rat-anti-IGFBP2 R&D systems Cat# MAB7971; RRID:AB_2264598 Rabbit anti-IGFBP2 for IHC Abcam Cat# ab188200 Rabbit anti-P21 Abcam Cat# ab188224; RRID:AB_2734729 Rabbit anti-Collagen 1 EMD Millipore Cat# AB765P; RRID:AB_92259 Rabbit anti-PPARa LSBio Cat# LS-C312574 Rabbit anti-Fibronectin Abcam Cat# ab2413; RRID:AB_2262874 Rabbit anti-Vimentin CST Cat# 5741S; RRID:AB_10695459 HRP conjugated-b actin Santa Cruz Cat# sc-47778HRP; RRID:AB_2714189 Mouse anti-a tubulin CST Cat# 3873; RRID:AB_1904178 Rabbit anti-H3 Histone CST Cat# 4499; RRID:AB_10544537 Rabbit anti-phospho-Histone 2AX CST Cat# 2577; RRID:AB_2118010 Rabbit anti-Prosurfactant Protein C Abcam Cat# ab211326; RRID:AB_2927746 Bacterial and virus strains Ad-Null Vector Biolabs Cat#1240 Ad-m-TGFb1 Vector Biolabs Cat#ADV-274099 mouse Igfbp2 lentiviral particle Origene Cat#: MR204287L3V Lenticontrol particles Origene Cat# PS100092V Chemicals, peptides, and recombinant proteins human IGFBP2 recombinant protein R&D Systems Cat# 797-B2-025 Bleomycin EMD Millipore Cat# 203401 DMSO Sigma- Aldrich Cat# D8418 4% PFA Santa Cruz Cat# sc-281692 DMEM/F12 Gibco Cat# 11-320-033 Plasmocin invivogen Cat# ant-mpp Collagenase type 1 Worthington Biochemical Cat# LS004197 Dispase Corning Cat# 354235 Green chromogen Leica Cat# DC9913 Bond polymer refine system Leica Cat# DS9800 BondTM Primary antibody diluent Leica Cat# AR9352 BOND Epitope Retrieval Solution 1 Leica Cat# AR9961 BOND Epitope Retrieval Solution 2 Leica Cat# AR9640 Wash solution 10X concentration Leica Cat# AR9590 BOND Universal Covertile Leica Cat# S21.4611 Tween-20 Sigma- Aldrich Cat# P9416 PVDF membrane EMD Millipore Cat# IPVH00010 SuperSignalTM Pico or Femto substrate Thermo Fisher Scientific Cat# 34095 RIPA buffer Thermo Fisher scientific Cat# 89901 Igfbp2 Life science technology Hs01040719_m1 18S rRNA Life science technology Hs99999901_s1 Ppara Life science technology Mm00440939_m1 and Hs00231882_m1 Igfbp2 siRNA Horizon Discovery L-062198-01-0005 Ppara siRNA Santa Cruz sc-36308 Critical commercial assays High-Capacity RNA-to-cDNATM Kit Thermo Fisher scientific Cat# 4387406 (Continued on next page) Cell Reports Medicine 4, 100945, March 21, 2023 e1

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 Antibodies Rat-anti-IGFBP2 R&D systems Cat# MAB7971; RRID:AB_2264598 Rabbit anti-IGFBP2 for IHC Abcam Cat# ab188200 Rabbit anti-P21 Abcam Cat# ab188224; RRID:AB_2734729 Rabbit anti-Collagen 1 EMD Millipore Cat# AB765P; RRID:AB_92259 Rabbit anti-PPARa LSBio Cat# LS-C312574 Rabbit anti-Fibronectin Abcam Cat# ab2413; RRID:AB_2262874 Rabbit anti-Vimentin CST Cat# 5741S; RRID:AB_10695459 HRP conjugated-b actin Santa Cruz Cat# sc-47778HRP; RRID:AB_2714189 Mouse anti-a tubulin CST Cat# 3873; RRID:AB_1904178 Rabbit anti-H3 Histone CST Cat# 4499; RRID:AB_10544537 Rabbit anti-phospho-Histone 2AX CST Cat# 2577; RRID:AB_2118010 Rabbit anti-Prosurfactant Protein C Abcam Cat# ab211326; RRID:AB_2927746 Bacterial and virus strains Ad-Null Vector Biolabs Cat#1240 Ad-m-TGFb1 Vector Biolabs Cat#ADV-274099 mouse Igfbp2 lentiviral particle Origene Cat#: MR204287L3V Lenticontrol particles Origene Cat# PS100092V Chemicals, peptides, and recombinant proteins human IGFBP2 recombinant protein R&D Systems Cat# 797-B2-025 Bleomycin EMD Millipore Cat# 203401 DMSO Sigma- Aldrich Cat# D8418 4% PFA Santa Cruz Cat# sc-281692 DMEM/F12 Gibco Cat# 11-320-033 Plasmocin invivogen Cat# ant-mpp Collagenase type 1 Worthington Biochemical Cat# LS004197 Dispase Corning Cat# 354235 Green chromogen Leica Cat# DC9913 Bond polymer refine system Leica Cat# DS9800 BondTM Primary antibody diluent Leica Cat# AR9352 BOND Epitope Retrieval Solution 1 Leica Cat# AR9961 BOND Epitope Retrieval Solution 2 Leica Cat# AR9640 Wash solution 10X concentration Leica Cat# AR9590 BOND Universal Covertile Leica Cat# S21.4611 Tween-20 Sigma- Aldrich Cat# P9416 PVDF membrane EMD Millipore Cat# IPVH00010 SuperSignalTM Pico or Femto substrate Thermo Fisher Scientific Cat# 34095 RIPA buffer Thermo Fisher scientific Cat# 89901 Igfbp2 Life science technology Hs01040719_m1 18S rRNA Life science technology Hs99999901_s1 Ppara Life science technology Mm00440939_m1 and Hs00231882_m1 Igfbp2 siRNA Horizon Discovery L-062198-01-0005 Ppara siRNA Santa Cruz sc-36308 Critical commercial assays High-Capacity RNA-to-cDNATM Kit Thermo Fisher scientific Cat# 4387406 (Continued on next page) Cell Reports Medicine 4, 100945, March 21, 2023 e1

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 Antibodies Rat-anti-IGFBP2 R&D systems Cat# MAB7971; RRID:AB_2264598 Rabbit anti-IGFBP2 for IHC Abcam Cat# ab188200 Rabbit anti-P21 Abcam Cat# ab188224; RRID:AB_2734729 Rabbit anti-Collagen 1 EMD Millipore Cat# AB765P; RRID:AB_92259 Rabbit anti-PPARa LSBio Cat# LS-C312574 Rabbit anti-Fibronectin Abcam Cat# ab2413; RRID:AB_2262874 Rabbit anti-Vimentin CST Cat# 5741S; RRID:AB_10695459 HRP conjugated-b actin Santa Cruz Cat# sc-47778HRP; RRID:AB_2714189 Mouse anti-a tubulin CST Cat# 3873; RRID:AB_1904178 Rabbit anti-H3 Histone CST Cat# 4499; RRID:AB_10544537 Rabbit anti-phospho-Histone 2AX CST Cat# 2577; RRID:AB_2118010 Rabbit anti-Prosurfactant Protein C Abcam Cat# ab211326; RRID:AB_2927746 Bacterial and virus strains Ad-Null Vector Biolabs Cat#1240 Ad-m-TGFb1 Vector Biolabs Cat#ADV-274099 mouse Igfbp2 lentiviral particle Origene Cat#: MR204287L3V Lenticontrol particles Origene Cat# PS100092V Chemicals, peptides, and recombinant proteins human IGFBP2 recombinant protein R&D Systems Cat# 797-B2-025 Bleomycin EMD Millipore Cat# 203401 DMSO Sigma- Aldrich Cat# D8418 4% PFA Santa Cruz Cat# sc-281692 DMEM/F12 Gibco Cat# 11-320-033 Plasmocin invivogen Cat# ant-mpp Collagenase type 1 Worthington Biochemical Cat# LS004197 Dispase Corning Cat# 354235 Green chromogen Leica Cat# DC9913 Bond polymer refine system Leica Cat# DS9800 BondTM Primary antibody diluent Leica Cat# AR9352 BOND Epitope Retrieval Solution 1 Leica Cat# AR9961 BOND Epitope Retrieval Solution 2 Leica Cat# AR9640 Wash solution 10X concentration Leica Cat# AR9590 BOND Universal Covertile Leica Cat# S21.4611 Tween-20 Sigma- Aldrich Cat# P9416 PVDF membrane EMD Millipore Cat# IPVH00010 SuperSignalTM Pico or Femto substrate Thermo Fisher Scientific Cat# 34095 RIPA buffer Thermo Fisher scientific Cat# 89901 Igfbp2 Life science technology Hs01040719_m1 18S rRNA Life science technology Hs99999901_s1 Ppara Life science technology Mm00440939_m1 and Hs00231882_m1 Igfbp2 siRNA Horizon Discovery L-062198-01-0005 Ppara siRNA Santa Cruz sc-36308 Critical commercial assays High-Capacity RNA-to-cDNATM Kit Thermo Fisher scientific Cat# 4387406 (Continued on next page) Cell Reports Medicine 4, 100945, March 21, 2023 e1

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 Antibodies Rat-anti-IGFBP2 R&D systems Cat# MAB7971; RRID:AB_2264598 Rabbit anti-IGFBP2 for IHC Abcam Cat# ab188200 Rabbit anti-P21 Abcam Cat# ab188224; RRID:AB_2734729 Rabbit anti-Collagen 1 EMD Millipore Cat# AB765P; RRID:AB_92259 Rabbit anti-PPARa LSBio Cat# LS-C312574 Rabbit anti-Fibronectin Abcam Cat# ab2413; RRID:AB_2262874 Rabbit anti-Vimentin CST Cat# 5741S; RRID:AB_10695459 HRP conjugated-b actin Santa Cruz Cat# sc-47778HRP; RRID:AB_2714189 Mouse anti-a tubulin CST Cat# 3873; RRID:AB_1904178 Rabbit anti-H3 Histone CST Cat# 4499; RRID:AB_10544537 Rabbit anti-phospho-Histone 2AX CST Cat# 2577; RRID:AB_2118010 Rabbit anti-Prosurfactant Protein C Abcam Cat# ab211326; RRID:AB_2927746 Bacterial and virus strains Ad-Null Vector Biolabs Cat#1240 Ad-m-TGFb1 Vector Biolabs Cat#ADV-274099 mouse Igfbp2 lentiviral particle Origene Cat#: MR204287L3V Lenticontrol particles Origene Cat# PS100092V Chemicals, peptides, and recombinant proteins human IGFBP2 recombinant protein R&D Systems Cat# 797-B2-025 Bleomycin EMD Millipore Cat# 203401 DMSO Sigma- Aldrich Cat# D8418 4% PFA Santa Cruz Cat# sc-281692 DMEM/F12 Gibco Cat# 11-320-033 Plasmocin invivogen Cat# ant-mpp Collagenase type 1 Worthington Biochemical Cat# LS004197 Dispase Corning Cat# 354235 Green chromogen Leica Cat# DC9913 Bond polymer refine system Leica Cat# DS9800 BondTM Primary antibody diluent Leica Cat# AR9352 BOND Epitope Retrieval Solution 1 Leica Cat# AR9961 BOND Epitope Retrieval Solution 2 Leica Cat# AR9640 Wash solution 10X concentration Leica Cat# AR9590 BOND Universal Covertile Leica Cat# S21.4611 Tween-20 Sigma- Aldrich Cat# P9416 PVDF membrane EMD Millipore Cat# IPVH00010 SuperSignalTM Pico or Femto substrate Thermo Fisher Scientific Cat# 34095 RIPA buffer Thermo Fisher scientific Cat# 89901 Igfbp2 Life science technology Hs01040719_m1 18S rRNA Life science technology Hs99999901_s1 Ppara Life science technology Mm00440939_m1 and Hs00231882_m1 Igfbp2 siRNA Horizon Discovery L-062198-01-0005 Ppara siRNA Santa Cruz sc-36308 Critical commercial assays High-Capacity RNA-to-cDNATM Kit Thermo Fisher scientific Cat# 4387406 (Continued on next page) Cell Reports Medicine 4, 100945, March 21, 2023 e1

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 Antibodies Rat-anti-IGFBP2 R&D systems Cat# MAB7971; RRID:AB_2264598 Rabbit anti-IGFBP2 for IHC Abcam Cat# ab188200 Rabbit anti-P21 Abcam Cat# ab188224; RRID:AB_2734729 Rabbit anti-Collagen 1 EMD Millipore Cat# AB765P; RRID:AB_92259 Rabbit anti-PPARa LSBio Cat# LS-C312574 Rabbit anti-Fibronectin Abcam Cat# ab2413; RRID:AB_2262874 Rabbit anti-Vimentin CST Cat# 5741S; RRID:AB_10695459 HRP conjugated-b actin Santa Cruz Cat# sc-47778HRP; RRID:AB_2714189 Mouse anti-a tubulin CST Cat# 3873; RRID:AB_1904178 Rabbit anti-H3 Histone CST Cat# 4499; RRID:AB_10544537 Rabbit anti-phospho-Histone 2AX CST Cat# 2577; RRID:AB_2118010 Rabbit anti-Prosurfactant Protein C Abcam Cat# ab211326; RRID:AB_2927746 Bacterial and virus strains Ad-Null Vector Biolabs Cat#1240 Ad-m-TGFb1 Vector Biolabs Cat#ADV-274099 mouse Igfbp2 lentiviral particle Origene Cat#: MR204287L3V Lenticontrol particles Origene Cat# PS100092V Chemicals, peptides, and recombinant proteins human IGFBP2 recombinant protein R&D Systems Cat# 797-B2-025 Bleomycin EMD Millipore Cat# 203401 DMSO Sigma- Aldrich Cat# D8418 4% PFA Santa Cruz Cat# sc-281692 DMEM/F12 Gibco Cat# 11-320-033 Plasmocin invivogen Cat# ant-mpp Collagenase type 1 Worthington Biochemical Cat# LS004197 Dispase Corning Cat# 354235 Green chromogen Leica Cat# DC9913 Bond polymer refine system Leica Cat# DS9800 BondTM Primary antibody diluent Leica Cat# AR9352 BOND Epitope Retrieval Solution 1 Leica Cat# AR9961 BOND Epitope Retrieval Solution 2 Leica Cat# AR9640 Wash solution 10X concentration Leica Cat# AR9590 BOND Universal Covertile Leica Cat# S21.4611 Tween-20 Sigma- Aldrich Cat# P9416 PVDF membrane EMD Millipore Cat# IPVH00010 SuperSignalTM Pico or Femto substrate Thermo Fisher Scientific Cat# 34095 RIPA buffer Thermo Fisher scientific Cat# 89901 Igfbp2 Life science technology Hs01040719_m1 18S rRNA Life science technology Hs99999901_s1 Ppara Life science technology Mm00440939_m1 and Hs00231882_m1 Igfbp2 siRNA Horizon Discovery L-062198-01-0005 Ppara siRNA Santa Cruz sc-36308 Critical commercial assays High-Capacity RNA-to-cDNATM Kit Thermo Fisher scientific Cat# 4387406 (Continued on next page) Cell Reports Medicine 4, 100945, March 21, 2023 e1

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 Antibodies Rat-anti-IGFBP2 R&D systems Cat# MAB7971; RRID:AB_2264598 Rabbit anti-IGFBP2 for IHC Abcam Cat# ab188200 Rabbit anti-P21 Abcam Cat# ab188224; RRID:AB_2734729 Rabbit anti-Collagen 1 EMD Millipore Cat# AB765P; RRID:AB_92259 Rabbit anti-PPARa LSBio Cat# LS-C312574 Rabbit anti-Fibronectin Abcam Cat# ab2413; RRID:AB_2262874 Rabbit anti-Vimentin CST Cat# 5741S; RRID:AB_10695459 HRP conjugated-b actin Santa Cruz Cat# sc-47778HRP; RRID:AB_2714189 Mouse anti-a tubulin CST Cat# 3873; RRID:AB_1904178 Rabbit anti-H3 Histone CST Cat# 4499; RRID:AB_10544537 Rabbit anti-phospho-Histone 2AX CST Cat# 2577; RRID:AB_2118010 Rabbit anti-Prosurfactant Protein C Abcam Cat# ab211326; RRID:AB_2927746 Bacterial and virus strains Ad-Null Vector Biolabs Cat#1240 Ad-m-TGFb1 Vector Biolabs Cat#ADV-274099 mouse Igfbp2 lentiviral particle Origene Cat#: MR204287L3V Lenticontrol particles Origene Cat# PS100092V Chemicals, peptides, and recombinant proteins human IGFBP2 recombinant protein R&D Systems Cat# 797-B2-025 Bleomycin EMD Millipore Cat# 203401 DMSO Sigma- Aldrich Cat# D8418 4% PFA Santa Cruz Cat# sc-281692 DMEM/F12 Gibco Cat# 11-320-033 Plasmocin invivogen Cat# ant-mpp Collagenase type 1 Worthington Biochemical Cat# LS004197 Dispase Corning Cat# 354235 Green chromogen Leica Cat# DC9913 Bond polymer refine system Leica Cat# DS9800 BondTM Primary antibody diluent Leica Cat# AR9352 BOND Epitope Retrieval Solution 1 Leica Cat# AR9961 BOND Epitope Retrieval Solution 2 Leica Cat# AR9640 Wash solution 10X concentration Leica Cat# AR9590 BOND Universal Covertile Leica Cat# S21.4611 Tween-20 Sigma- Aldrich Cat# P9416 PVDF membrane EMD Millipore Cat# IPVH00010 SuperSignalTM Pico or Femto substrate Thermo Fisher Scientific Cat# 34095 RIPA buffer Thermo Fisher scientific Cat# 89901 Igfbp2 Life science technology Hs01040719_m1 18S rRNA Life science technology Hs99999901_s1 Ppara Life science technology Mm00440939_m1 and Hs00231882_m1 Igfbp2 siRNA Horizon Discovery L-062198-01-0005 Ppara siRNA Santa Cruz sc-36308 Critical commercial assays High-Capacity RNA-to-cDNATM Kit Thermo Fisher scientific Cat# 4387406 (Continued on next page) Cell Reports Medicine 4, 100945, March 21, 2023 e1

Techniques: Expressing, Isolation, Staining, Two Tailed Test

Journal: Respiratory research

Article Title: Alveolar epithelial type 2 cell specific loss of IGFBP2 activates inflammation in COVID-19.

doi: 10.1186/s12931-025-03187-9

Figure Lengend Snippet: Fig. 3 Reduced IGFBP2 protein expression in AEC2 cells of both COVID-infected lungs and lungs without COVID. Multicolor immunohistochemistry images showing fibrotic lung sections stained with SPC (green) and (A) IGFBP2; C IGF1; and E IGF2 (brown) from donor (healthy) controls and patients with COVID-ARDS, idiopathic pulmonary fibrosis (IPF), or IPF with a history of COVID. 25 – 30 images were analyzed per human subject. Scale bars, 10 μm. Quantification of double positive cells for (B) IGFBP2; D IGF1; and (F) IGF2 in SPC + cells in the fibrotic lung regions of patients with COVID-ARDS, IPF alone, or IPF with COVID history and healthy (donor) controls. Data are presented as mean ± SEM. NS, not significant; *p < 0.05, **p < 0.01, and ****p < 0.0001, One-way ANOVA followed by Tukey post-hoc test. N = 3 for each group (Donor controls, COVID-ARDS, IPF alone, and IPF with COVID history)

Article Snippet: Igfbp2 lentivirus was commercially obtained (OriGene Technologies; catalog# MR204287L3V) and was used as previously reported [18].

Techniques: Expressing, Infection, Immunohistochemistry, Staining

Journal: Respiratory research

Article Title: Alveolar epithelial type 2 cell specific loss of IGFBP2 activates inflammation in COVID-19.

doi: 10.1186/s12931-025-03187-9

Figure Lengend Snippet: Fig. 4 Lentiviral Igfbp2 expression inhibits COVID S2 spike glycoprotein-induced inflammatory phenotype in MLE-12 cells. Quantitative polymerase chain reaction (QPCR analyses of mRNA expression of a panel of cytokines and chemokines in lentiviral mediated transduction of MLE-12 cells in the absence or presence of SARS-COV-2 spike protein S2 treatment. Relative mRNA expression of cytokines and chemokines in MLE-12 cells expressing (A) IGFBP2 versus mock (B) IGF1 versus mock (C) IGF2 versus mock after S2 spike protein (500 ng/ml) injury at 24 h. Data are representative of minimum of 3 independent experiments. Data are presented as mean ± SEM. *p < 0.05, **p < 0.01, and ***p < 0.001, Student’s unpaired two-tailed t test

Article Snippet: Igfbp2 lentivirus was commercially obtained (OriGene Technologies; catalog# MR204287L3V) and was used as previously reported [18].

Techniques: Expressing, Real-time Polymerase Chain Reaction, Transduction, Two Tailed Test

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:

Article Title: Serum complexes of insulin-like growth factor-1 modulate skeletal integrity and carbohydrate metabolism

doi: 10.1096/fj.08-118976

Figure Lengend Snippet: IGF-binding proteins and IGF-1 clearance. A) Half-life of [125I]IGF-1 in sera obtained from control, LID, ALSKO, and BP3KO mice. Data are means ± se; n >12 mice/group. *P < 0.05 vs. control. B) Ligand blot assay shows IGF-1 binding capacity of serum from control, LID, ALSKO, BP3KO, and LAB mice. Reactive bands corresponding to IGFBP-3 were not detectable in BP3KO and LAB mice and significantly reduced in LID and ALSKO mice. C) Serum IGFBP-2 levels, determined by RIA in sera from 8- to 10-wk-old control, LID, ALSKO, BP3KO, and LAB mice. Data are means ± se (number per group in parentheses). D) Serum levels of IGFBP-5 detected by Western immunoblot and (E) quantified by densitometry, showing twofold increase in BP3KO mice. Data are mean ± se; n = 5/group.

Article Snippet: Serum levels of mouse IGFBP-2, IGFBP-3, and ALS were determined using ELISA assays developed at UCLA, with recombinant mouse proteins from R&D Systems (Minneapolis, MN, USA) and monoclonal antibodies as described previously (29) .

Techniques: Binding Assay, Western Blot

Journal: Cell Reports Medicine

Article Title: IGFBP-2 partly mediates the early metabolic improvements caused by bariatric surgery

doi: 10.1016/j.xcrm.2021.100248

Figure Lengend Snippet:

Article Snippet: Mouse and rat plasma IGFBP-2 levels ELISA , ALPCO, Canada , #22-BP2MS-E01.

Techniques: Clinical Proteomics, Enzyme-linked Immunosorbent Assay, Bioassay, Mutagenesis, Software