Journal: Molecular Medicine Reports

Article Title: IGFBP-rP1-silencing promotes hypoxia-induced angiogenic potential of choroidal endothelial cells via the RAF/MEK/ERK signaling pathway

doi: 10.3892/mmr.2020.11578

Figure Lengend Snippet: IGFBP-rP1-knockdown in RF/6A cells by siIGFBP-rP1. (A) Reverse transcription-quantitative PCR analysis of IGFBP-rP1 transcript expression in RF/6A cells. GAPDH served as an internal reference for control. Both siIGFBP-rP1 duplex 1 and 2 significantly inhibited the expression IGFBP-rP1 compared with controls ( # P<0.05). Furthermore, the inhibitory effect of siIGFBP-rP1 duplex 2 was significantly increased compared with siIGFBP-rP1 duplex 1 (*P<0.05). (B) IGFBP-rP1 expression was measured by western blotting and normalized to that of β-actin. The inhibitory effect of siIGFBP-rP1 on the IGFBP-rP1 protein expression was consistent with that of the RNA expression. The membranes were stripped off and probed for the proteins. Data are presented as the mean ± standard deviation of three independent experiments with similar results and calculated as the integrated optical density of IGFBP-rP1 relative to the internal reference. # P<0.01 vs. the blank control group. *P<0.05 vs. the siIGFBP-rP1 duplex 1 group. IGFBP-rP1, insulin-like growth factor binding protein-related protein 1; siIGFBP-rP1, IGFBP-rP1 specific siRNA. Lane M, marker; lane 1, blank control; lane 2, transfection reagent; lane 3, scrambled control siRNA; lane 4, siRNA duplex 1; lane 5, siRNA duplex 2.

Article Snippet: Recombinant human IGFBP-rP1 and goat anti-human IGFBP-rP1 polyclonal antibody (cat. no. AF1334) were obtained from R&D Systems, Inc. Rabbit anti-human GAPDH polyclonal (cat. no. 10494-1-AP) and mouse anti-human β-actin monoclonal (cat. no. 66009-1-Ig) antibodies were obtained from ProteinTech Group, Inc. Rabbit anti-human VEGF polyclonal (cat. no. ab150766) antibodies were purchased from Abcam.

Techniques: Knockdown, Reverse Transcription, Real-time Polymerase Chain Reaction, Expressing, Control, Western Blot, RNA Expression, Standard Deviation, Binding Assay, Marker, Transfection

Journal: Molecular Medicine Reports

Article Title: IGFBP-rP1-silencing promotes hypoxia-induced angiogenic potential of choroidal endothelial cells via the RAF/MEK/ERK signaling pathway

doi: 10.3892/mmr.2020.11578

Figure Lengend Snippet: Cell growth curves of siIGFBP-rP1-transfected cells and untransfected cells cultured in normoxic conditions for 6, 12, 24, 48 and 72 h. The OD values of transfected cells at 12, 24 and 48 h were significantly higher compared with untransfected cells (*P<0.01). Furthermore, the OD value of siIGFBP-rP1 duplex 2-transfected cells was significantly higher compared with siIGFBP-rP1 duplex 1-transfected cells at 24 h ( # P<0.01). No significant differences were observed among the groups at 6 and 72 h. OD values are presented as the mean ± standard deviation of 4 wells/group and experiments were performed in triplicate. IGFBP-rP1, insulin-like growth factor binding protein-related protein 1; siIGFBP-rP1, IGFBP-rP1 specific siRNA; OD, optical density.

Article Snippet: Recombinant human IGFBP-rP1 and goat anti-human IGFBP-rP1 polyclonal antibody (cat. no. AF1334) were obtained from R&D Systems, Inc. Rabbit anti-human GAPDH polyclonal (cat. no. 10494-1-AP) and mouse anti-human β-actin monoclonal (cat. no. 66009-1-Ig) antibodies were obtained from ProteinTech Group, Inc. Rabbit anti-human VEGF polyclonal (cat. no. ab150766) antibodies were purchased from Abcam.

Techniques: Transfection, Cell Culture, Standard Deviation, Binding Assay

Journal: Molecular Medicine Reports

Article Title: IGFBP-rP1-silencing promotes hypoxia-induced angiogenic potential of choroidal endothelial cells via the RAF/MEK/ERK signaling pathway

doi: 10.3892/mmr.2020.11578

Figure Lengend Snippet: Cell growth curves of siIGFBP-rP1-transfected or untransfected cells cultured under normoxic or hypoxic conditions for 6, 12, 24, 48 and 72 h, as detected by MTS colorimetric assays. The OD values of hypoxic groups (CoCl 2 and 1% O 2 ) decreased significantly at 12, 24, 48 and 72 h compared with the control group ( # P<0.01). There was no significant difference between the hypoxic groups (P>0.05). The OD value of the siRNA group were significantly increased at 12, 24 and 48 h compared with controls (**P<0.01). Furthermore, the OD values of the transfected cells cultured in hypoxic conditions (CoCl 2 + siRNA group and 1% O 2 + siRNA group) for 12, 24, 48 and 72 h were significantly lower compared with the siRNA group; additionally, the values were significantly higher compared with the hypoxic groups (*P<0.01). No significant differences were identified between the CoCl 2 + siRNA, 1% O 2 + siRNA and control groups (P>0.05). OD values are presented as the mean ± standard deviation of 4 wells/group and experiments were performed in triplicate. IGFBP-rP1, insulin-like growth factor binding protein-related protein 1; siIGFBP-rP1, IGFBP-rP1 specific siRNA; CoCl 2 , cobalt chloride; OD, optical density.

Article Snippet: Recombinant human IGFBP-rP1 and goat anti-human IGFBP-rP1 polyclonal antibody (cat. no. AF1334) were obtained from R&D Systems, Inc. Rabbit anti-human GAPDH polyclonal (cat. no. 10494-1-AP) and mouse anti-human β-actin monoclonal (cat. no. 66009-1-Ig) antibodies were obtained from ProteinTech Group, Inc. Rabbit anti-human VEGF polyclonal (cat. no. ab150766) antibodies were purchased from Abcam.

Techniques: Transfection, Cell Culture, Control, Standard Deviation, Binding Assay

Journal: Molecular Medicine Reports

Article Title: IGFBP-rP1-silencing promotes hypoxia-induced angiogenic potential of choroidal endothelial cells via the RAF/MEK/ERK signaling pathway

doi: 10.3892/mmr.2020.11578

Figure Lengend Snippet: Cell motility of siIGFBP-rP1-transfected or untransfected cells cultured under normoxic or hypoxic conditions for 24 h is detected by wound and Transwell assays. (A) Representative images showing that RF/6A cells migrated across the wound boundary to the blank area (light microscopy; magnification, ×400) and (B) passed across the filter toward the lower surface (light microscopy; magnification, ×200). Hypoxia significantly promoted cell mobility compared with the controls ( # P<0.01). siIGFBP-rP1 transfection (siIGFBP-rP1 group) further enhanced cell migration, as compared with untransfected cells cultured in hypoxic conditions (hypoxia group; *P<0.01). The transfected cells cultured in hypoxic conditions (hypoxia + siRNA group) exhibited a significantly increased migration ability compared with the transfected cells cultured in normoxic conditions (siIGFBP-rP1 group; **P<0.01). Values are presented as the mean ± standard deviation of 4 samples/group and experiments were performed in triplicate and are quantified as the ratio relative to the control group. IGFBP-rP1, insulin-like growth factor binding protein-related protein 1; siIGFBP-rP1, IGFBP-rP1 specific siRNA.

Article Snippet: Recombinant human IGFBP-rP1 and goat anti-human IGFBP-rP1 polyclonal antibody (cat. no. AF1334) were obtained from R&D Systems, Inc. Rabbit anti-human GAPDH polyclonal (cat. no. 10494-1-AP) and mouse anti-human β-actin monoclonal (cat. no. 66009-1-Ig) antibodies were obtained from ProteinTech Group, Inc. Rabbit anti-human VEGF polyclonal (cat. no. ab150766) antibodies were purchased from Abcam.

Techniques: Transfection, Cell Culture, Light Microscopy, Migration, Standard Deviation, Control, Binding Assay

Journal: Molecular Medicine Reports

Article Title: IGFBP-rP1-silencing promotes hypoxia-induced angiogenic potential of choroidal endothelial cells via the RAF/MEK/ERK signaling pathway

doi: 10.3892/mmr.2020.11578

Figure Lengend Snippet: IGFBP-rP1-silencing stimulates hypoxia-induced tube formation of RF/6A cells. Representative images (inverted phase contrast microscopy; magnification, ×50) demonstrating that RF/6A cells formed capillary-like tube structures within the Matrigel layer in different mediums. RF/6A cells in hypoxic conditions or siIGFBP-rP1-transfected cells significantly formed completely enclosed capillary-like tubes compared with the controls ( # P<0.01 the hypoxia group vs. the control group; *P<0.01 the siIGFBP-rP1 group vs. the control group). Moreover, siIGFBP-rP1 transfection further promoted tube formation in RF/6A cells in the hypoxia + siIGFBP-rP1 group compared with the siIGFBP-rP1 group (**P<0.01). The values are presented as the mean ± standard deviation of 4 samples/group and experiments were performed in triplicate and are quantified as the ratio relative to the control group. IGFBP-rP1, insulin-like growth factor binding protein-related protein 1; siIGFBP-rP1, IGFBP-rP1 specific small interfering RNA.

Article Snippet: Recombinant human IGFBP-rP1 and goat anti-human IGFBP-rP1 polyclonal antibody (cat. no. AF1334) were obtained from R&D Systems, Inc. Rabbit anti-human GAPDH polyclonal (cat. no. 10494-1-AP) and mouse anti-human β-actin monoclonal (cat. no. 66009-1-Ig) antibodies were obtained from ProteinTech Group, Inc. Rabbit anti-human VEGF polyclonal (cat. no. ab150766) antibodies were purchased from Abcam.

Techniques: Microscopy, Transfection, Control, Standard Deviation, Binding Assay, Small Interfering RNA

Journal: Molecular Medicine Reports

Article Title: IGFBP-rP1-silencing promotes hypoxia-induced angiogenic potential of choroidal endothelial cells via the RAF/MEK/ERK signaling pathway

doi: 10.3892/mmr.2020.11578

Figure Lengend Snippet: IGFBP-rP1-silencing upregulates the hypoxia-induced RAF/MEK/ERK signaling pathway activation and VEGF expression. Representative images and quantified data demonstrated that hypoxic stress upregulated B-RAF, p-MEK, p-ERK and VEGF expression in RF/6A cells compared with controls ( # P<0.05). siIGFBP-rP1 transfection significantly promoted hypoxia-induced B-RAF, p-MEK, p-ERK and VEGF expression in RF/6A compared with the hypoxia group ( # P<0.05). IGFBP-rP1 restoration significantly downregulated the expression of B-RAF, p-MEK, p-ERK and VEGF in siIGFBP-rP1-transfected cells, under both normoxic and hypoxic conditions, compared with the siIGFBP-rP1 and hypoxia + siIGFBP-rP1 groups, respectively (*P<0.01). The membranes were stripped off and probed for the proteins. Values are presented as the mean ± SD of 3 independent experiments with similar results and are presented as the integrated optical density of studied proteins relative to GAPDH. IGFBP-rP1, insulin-like growth factor binding protein-related protein 1; siIGFBP-rP1, IGFBP-rP1 specific siRNA. Lane 1, control; lane 2, siIGFBP-rP1; lane 3, siIGFBP-rP1 + IGFBP-rP1; lane 4, hypoxia; lane 5, hypoxia + siIGFBP-rP1; lane 6, hypoxia + siIGFBP-rP1 + IGFBP-rP1.

Article Snippet: Recombinant human IGFBP-rP1 and goat anti-human IGFBP-rP1 polyclonal antibody (cat. no. AF1334) were obtained from R&D Systems, Inc. Rabbit anti-human GAPDH polyclonal (cat. no. 10494-1-AP) and mouse anti-human β-actin monoclonal (cat. no. 66009-1-Ig) antibodies were obtained from ProteinTech Group, Inc. Rabbit anti-human VEGF polyclonal (cat. no. ab150766) antibodies were purchased from Abcam.

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

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

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

Journal: Cell reports. Medicine

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

doi: 10.1016/j.xcrm.2023.100945

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

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

Techniques: Expressing, In Vitro, Western Blot, Control

Journal: Cell reports. Medicine

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

doi: 10.1016/j.xcrm.2023.100945

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

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

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

Journal: Cell reports. Medicine

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

doi: 10.1016/j.xcrm.2023.100945

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

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

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

Journal: Cell reports. Medicine

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

doi: 10.1016/j.xcrm.2023.100945

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

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

Techniques: Recombinant, Saline

Journal: Cell reports. Medicine

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

doi: 10.1016/j.xcrm.2023.100945

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

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

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

Journal: Cell reports. Medicine

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

doi: 10.1016/j.xcrm.2023.100945

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

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

Techniques: Expressing, Isolation, Staining, Two Tailed Test

Journal: Molecular and Cellular Biology

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

doi: 10.1128/mcb.01665-07

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

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

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