igfbp2 recombinant protein  (MedChemExpress)

Journal: Science advances

Article Title: IGFBP2 secretion by mammary adipocytes limits breast cancer invasion.

doi: 10.1126/sciadv.adg1840

Figure Lengend Snippet: Fig. 2. Stromal-derived IGFBP2 is sufficient to inhibit MM231 breast cancer cell invasion. (A and B) Representative images (A) and quantification (B) of MM231 breast cancer cell invasion in inverted collagen/fibronectin matrices in the presence of conditioned medium from HUVECs transfected with siRNAs against IGFBP2 (siIGFBP2_1 and siIGFBP2_2) or siNTC. Scale bars, 50 μm. n = 3 biological replicates performed in triplicate, with three stacksper transwell; one-way ANOVAwith Tukey correction; **P < 0.01 and ***P < 0.001; ns, not significant. (C and D) Representative Western blot (C) and densitometry analysis (D) of TIFs stably overexpressing mTurquoise2 (mT2) or IGFBP2 (n = 4; one-sample t test; *P < 0.05). (E) Enzyme-linked immunosorbent assay (ELISA) for human IGFBP2 in concentrated conditioned media from TIFs overex- pressing mT2 or IGFBP2, measured in duplicate (n = 3 biological replicates; two-tailed Student’s t test; **P < 0.01). (F) Representative images and quantification of fibro- blast-contracted 3D collagen invasion assays of MM231 cancer cell invasion in mT2 or IGFBP2 TIF-contracted matrices after 14 days and stained for Pan-CK (brown). Scale bars, 100 μm. n = 3 biological replicates, triplicate matrices, eight regions per condition per replicate; one-way ANOVA with Tukey correction; ***P < 0.001. (G and H) Representative hematoxylin and eosin (H&E) images (G) and quantification (H) of MM231 cell invasion into the mouse dermis from subcutaneous xenografts of MM231 co-injected with TIFs overexpressing either mT2 (control) or IGFBP2. Scale bars, 500 μm; insets, 50 μm. n = 10 mice (mT2) or 12 mice (IGFBP2).

Article Snippet: The bottom chamber was then filled with 1- ml complete medium and either PBS, recombinant IGFBP2 (5 μM; R&D Systems, 674-B2), anti–IGF-II (10 μg/ml; SigmaAldrich, 05-166), or IgG1κ (10 μg/ml; Sigma-Aldrich, M7894).

Techniques: Derivative Assay, Transfection, Western Blot, Stable Transfection, Enzyme-linked Immunosorbent Assay, Two Tailed Test, Staining, Injection, Control

Journal: Science advances

Article Title: IGFBP2 secretion by mammary adipocytes limits breast cancer invasion.

doi: 10.1126/sciadv.adg1840

Figure Lengend Snippet: Fig. 4. IGFBP2 acts through depletion of IGF-II from the cancer microenvironment. (A) Sche- matic of the proteomics experimental setup. LC- MS/MS, liquid chromatography–tandem mass spectrometry. IP, immunoprecipitation. (B and C) Representative Western blot (B) and densitome- try analysis (C) after silencing of IGF2 (IGF-II gene) using siRNAs in MM231 cells (n = 4 biological replicates; one-sample t test; *P < 0.05 and ***P < 0.001). (D and E) Representative images (D) and quantification (E) of MM231 cell invasion in in- verted collagen/fibronectin matrices after IGF2 silencing. Scale bars, 50 μm. n = 3 biological replicates, performed in duplicate with three stacks per transwell; one-way ANOVA with Tukey correction; ***P < 0.001. (F and G) Representative images (F) and quantification (G) of MM231 cell invasion in inverted collagen/fibronectin matri- ces treated with PBS, IGF-II (10 ng/ml), IgG1κ (10 μg/ml), or anti–IGF-II (10 μg/ml). Scale bars, 50 μm. n = 3 biological replicates, performed in duplicate with three stacks per transwell; one- way ANOVA with Tukey correction; ***P < 0.001. (H and I) Representative IGF-II Western blot (H) and quantification (I) of TIF, MM231, MM468, and HCC1937 cells (n = 6 biological replicates; one- sample t test; ***P < 0.001). (J) ELISA for human IGF-II in conditioned media from TIF, MM231, MM468, and HCC1937 cells (n = 4 biological replicates; one-way ANOVA with Dunnett’s cor- rection; *P < 0.05 and ***P < 0.001). (K and L) Matrigel invasion assays for MM231 (K) and MM468 (L) cells treated with IgG1κ or anti–IGF-II (n = 3, eight fields of view (FOVs) per chamber, two to three invasion chambers per condition per replicate; two-tailed Student’s t test with Welch’s correction; **P < 0.01). Scale bars, 100 μm. (M) Schematic of the proposed mechanism for IGFBP2 inhibition of invasion through dis- ruption of breast cancer IGF-II autocrine signaling.

Article Snippet: The bottom chamber was then filled with 1- ml complete medium and either PBS, recombinant IGFBP2 (5 μM; R&D Systems, 674-B2), anti–IGF-II (10 μg/ml; SigmaAldrich, 05-166), or IgG1κ (10 μg/ml; Sigma-Aldrich, M7894).

Techniques: Liquid Chromatography with Mass Spectroscopy, Liquid Chromatography, Mass Spectrometry, Immunoprecipitation, Western Blot, Enzyme-linked Immunosorbent Assay, Two Tailed Test, Inhibition

Journal: Science advances

Article Title: IGFBP2 secretion by mammary adipocytes limits breast cancer invasion.

doi: 10.1126/sciadv.adg1840

Figure Lengend Snippet: Fig. 5. IGFBP2 expression during breast cancer progression. (A) Representative H&E-stained breast tissue samples from healthy patients, patients with DCIS, or pa- tients with IDC. Scale bars, 200 μm. (B) Quantification of adipocytes per section from healthy (n = 8 patients, three to eight sections per patient), DCIS (n = 6 patients, one to four sections per patient), and IDC (n = 3 patients, one to two sections per patient). Kruskal-Wallis test using Dunn’s test to correct for multiple comparisons (**P < 0.01 and ***P < 0.001). (C) Representative image from a patient sample stained for IGFBP2 (magenta) and counterstained with DAPI (cyan) and keratin-8/keratin-14 (KRT8/14; red). Scale bar, 100 μm. n = 8 normal reduction mammoplasty patient samples. Autofluorescence is given in green. (D) Representative image of human DCIS breast cancer tissue stained for IGFBP2 (magenta) and counterstained with DAPI (cyan) and KRT8/14 (red). Scale bar, 100 μm (n = 4 DCIS patient samples). Autofluorescence is given in green. (E) Representative image of human IDC breast cancer tissue stained for IGFBP2 (magenta) and counterstained with DAPI (cyan) and KRT8/14 (red; n = 3 IDC patient samples). Scale bar, 100 μm. Autofluorescence is given in green. The brightness of IGFBP2 staining was increased for display purposes only. (F) Quantification of IGFBP2 per adipocyte from healthy (n = 8 patients, 143 to 280 adipocytes per patient), DCIS (n = 6 patients, 39 to 410 adipocytes per patient), and IDC (n = 3 patients, 75 to 283 adipocytes per patient). Kruskal-Wallis test using Dunn’s test to correct for multiple comparisons (***P < 0.001).

Article Snippet: The bottom chamber was then filled with 1- ml complete medium and either PBS, recombinant IGFBP2 (5 μM; R&D Systems, 674-B2), anti–IGF-II (10 μg/ml; SigmaAldrich, 05-166), or IgG1κ (10 μg/ml; Sigma-Aldrich, M7894).

Techniques: Expressing, Staining

Journal: CNS Neuroscience & Therapeutics

Article Title: Insulin‐Like Growth Factor Binding Protein 2 Drives Neurodegeneration in Parkinson's Disease: Insights From In Vivo and In Vitro Studies

doi: 10.1111/cns.70076

Figure Lengend Snippet: Effect of IGFBP2 on PD‐like phenotypes induced by 6‐OHDA in rats. (A) Schematic representation showing the experiment process of this study. (B, C) RT‐qPCR and western blotting analysis revealed that IGFBP2 expression was downregulated in the substantia nigra pars compacta (SNpc) of 6‐OHDA‐induced PD rats. (D) Behavioral tests showing the effect of IGFBP2 on 6‐OHDA‐induced behavioral impairment (Rotation, F (2, 15) = 745.7; Latency time, F (2, 15) = 59.48; T ‐turn time, F (2, 15) = 33.01; T ‐total time, F (2, 15) = 95.94). (E) Representative images showed IHC staining of TH in the SNpc of 6‐OHDA‐lesioned rats, followed by quantification of the number of TH‐positive cells ( F (2, 15) = 188.2). (F) Western blotting analysis depicting α‐synuclein expression in the SNpc tissues ( F (2, 15) = 102.3). Data were expressed as mean ± SD. Group sizes were: N = 6 animals per group. *** p < 0.001, # p < 0.05, ## p < 0.01, ### p < 0.001. * = control versus 6‐OHDA, # = 6‐OHDA versus rIGFBP2 + 6‐OHDA.

Article Snippet: Recombinant IGFBP2 protein (rIGFBP2) was purchased from Cloud‐Clone Corp (Wuhan, China).

Techniques: Quantitative RT-PCR, Western Blot, Expressing, Immunohistochemistry, Control

Journal: CNS Neuroscience & Therapeutics

Article Title: Insulin‐Like Growth Factor Binding Protein 2 Drives Neurodegeneration in Parkinson's Disease: Insights From In Vivo and In Vitro Studies

doi: 10.1111/cns.70076

Figure Lengend Snippet: Differential expression of mRNAs in PD rats. (A) The rats were injected with 6‐OHDA to induce the PD animal model. Apomorphine‐induced rotational behavior was tested at 2 and 3 weeks after modeling. (B) IHC staining of TH in OHDA‐lesioned rats (3 weeks). Bar = 100 µm. (C) Bidimensional principal component analysis (PCA) showing distinct clustering of gene profiles in 6‐OHDA‐induced PD rats and control rats. (D, E) Volcano plot and heatmap showing differential expression of mRNAs (|log 2 fold change (FC)| > 1 and p < 0.05). (F) Venn graph depicting the common genes of downregulated differentially expressed genes (DEGs, log 2 FC < −1.5 and p < 0.001) and parkinson‐related genes from the GeneCards database. (G) The FPKM value of IGFBP2 was shown. Group sizes were: N = 3 animals per group. ** p < 0.01, *** p < 0.001. * = control vs. 6‐OHDA.

Article Snippet: Recombinant IGFBP2 protein (rIGFBP2) was purchased from Cloud‐Clone Corp (Wuhan, China).

Techniques: Expressing, Injection, Animal Model, Immunohistochemistry, Control

Journal: CNS Neuroscience & Therapeutics

Article Title: Insulin‐Like Growth Factor Binding Protein 2 Drives Neurodegeneration in Parkinson's Disease: Insights From In Vivo and In Vitro Studies

doi: 10.1111/cns.70076

Figure Lengend Snippet: Effect of IGFBP2 on 6‐OHDA‐induced oxidative stress and mitochondrial impairment in rats. (A) ROS production, MDA content, and SOD and GSH‐Px activities in the SNpc were tested using corresponding commercial assay kits (ROS, F (2, 15) = 37; MDA, F (2, 15) = 42.56; SOD, F (2, 15) = 25.42; GSH‐Px, F (2, 15) = 45.15). (B) JC‐1 staining indicating the changes in mitochondrial membrane potential (Ψm; Q2‐2, F (2, 15) = 175.4; Q2‐4, F (2, 15) = 175.5). (C) ATP level was assessed by ATP detection kit ( F (2, 15) = 26.63). (D) Western blotting analysis revealing cytochrome c expression in cytoplasm and mitochondria in the SNpc of rats (cytochrome c in cytoplasm, F (2, 15) = 38.49; cytochrome c in mitochondria, F (2, 15) = 193.7). (E) Representative images of TUNEL‐positive staining in the SNpc tissues. Data were expressed as mean ± SD. Group sizes were: N = 6 animals per group. * p < 0.05, ** p < 0.01, *** p < 0.001, # p < 0.05, ## p < 0.01, ### p < 0.001. * = control versus 6‐OHDA, # = 6‐OHDA versus rIGFBP2 + 6‐OHDA.

Article Snippet: Recombinant IGFBP2 protein (rIGFBP2) was purchased from Cloud‐Clone Corp (Wuhan, China).

Techniques: Staining, Membrane, Western Blot, Expressing, TUNEL Assay, Control

Journal: CNS Neuroscience & Therapeutics

Article Title: Insulin‐Like Growth Factor Binding Protein 2 Drives Neurodegeneration in Parkinson's Disease: Insights From In Vivo and In Vitro Studies

doi: 10.1111/cns.70076

Figure Lengend Snippet: Effect of IGFBP2 on IGF‐1R/AKT signaling pathway. (A, B) Relative expression levels of IGF‐1R, p‐IGF‐1R, AKT, and p‐AKT in the SNpc were measured by western blotting (p‐IGF‐1R, F (2, 15) = 345.4; IGF‐1R, F (2, 15) = 229.7; p‐IGF‐1R/IGF‐1R, F (2, 15) = 92.01; p‐AKT, F (2, 15) = 811.5; AKT, F (2, 15) = 0.2335; p‐AKT/AKT, F (2, 15) = 851.2). Data were expressed as mean ± SD. Group sizes were: N = 6 animals per group. *** p < 0.001, ### p < 0.001. * = control versus 6‐OHDA, # = 6‐OHDA versus rIGFBP2 + 6‐OHDA.

Article Snippet: Recombinant IGFBP2 protein (rIGFBP2) was purchased from Cloud‐Clone Corp (Wuhan, China).

Techniques: Expressing, Western Blot, Control

Journal: CNS Neuroscience & Therapeutics

Article Title: Insulin‐Like Growth Factor Binding Protein 2 Drives Neurodegeneration in Parkinson's Disease: Insights From In Vivo and In Vitro Studies

doi: 10.1111/cns.70076

Figure Lengend Snippet: Effect of IGFBP2 on apoptosis induced by 6‐OHDA in differentiated PC12 cells. The differentiated PC12 cells were co‐treated with rIGFBP2 and rIGF‐1 for 24 h, and then subjected to 6‐OHDA for 24 h. (A) CCK‐8 assay was performed to determine cell viability ( F (4, 10) = 45.19). (B) LDH content was evaluated using a LDH detection kit ( F (4, 10) = 45.50). (C) Apoptosis induced by 6‐OHDA was determined with Hoechst 33258 staining ( F (4, 10) = 160.7). (D) Bax, Bcl‐2, cleaved caspase‐9, and cleaved caspase‐3 expression were determined by western blotting in PC12 cells (Bax, F (4, 10) = 82.57; Bcl‐2, F (4, 10) = 136.6; cleaved caspase‐9, F (4, 10) = 53.36; cleaved caspase‐3, F (4, 10) = 65.57). Data were expressed as mean ± SD. Group sizes were: N = 3 wells per group. * p < 0.05, *** p < 0.001, ^ p < 0.05, ^^ p < 0.01, ^^^ p < 0.001, # p < 0.05, ## p < 0.01, ### p < 0.001, + p < 0.05, +++ p < 0.001. * = control versus 6‐OHDA, ^ = 6‐OHDA versus rIGFBP2 + 6‐OHDA, # = 6‐OHDA versus rIGF‐1 + 6‐OHDA, + = rIGFBP2 + 6‐OHDA versus rIGF‐1 + rIGFBP2 + 6‐OHDA.

Article Snippet: Recombinant IGFBP2 protein (rIGFBP2) was purchased from Cloud‐Clone Corp (Wuhan, China).

Techniques: CCK-8 Assay, Staining, Expressing, Western Blot, Control

Journal: CNS Neuroscience & Therapeutics

Article Title: Insulin‐Like Growth Factor Binding Protein 2 Drives Neurodegeneration in Parkinson's Disease: Insights From In Vivo and In Vitro Studies

doi: 10.1111/cns.70076

Figure Lengend Snippet: Effect of IGFBP2 on oxidative stress and mitochondrial function in 6‐OHDA‐lesioned PC12 cells. (A) ROS and MDA levels, SOD and GSH‐Px activities were determined by the commercial assay kits (ROS, F (4, 10) = 37.55; MDA, F (4, 10) = 41.91; SOD, F (4, 10) = 55.79; GSH‐Px, F (4, 10) = 75.03). (B) Changes in Ψm were evaluated by JC‐1 staining. (C) Relative expression of Cytochrome c in cytoplasm and mitochondria was detected by western blotting analysis (cytochrome c in cytoplasm, F (4, 10) = 47.39; cytochrome c in mitochondria, F (4, 10) = 62). Data were expressed as mean ± SD. Group sizes were: N = 3 wells per group. *** p < 0.001, ^ p < 0.05, ^^ p < 0.01, ^^^ p < 0.001, ## p < 0.01, ### p < 0.001, ++ p < 0.01, +++ p < 0.001. * = control versus 6‐OHDA, ^ = 6‐OHDA versus rIGFBP2 + 6‐OHDA, # = 6‐OHDA versus rIGF‐1 + 6‐OHDA, + = rIGFBP2 + 6‐OHDA versus rIGF‐1 + rIGFBP2 + 6‐OHDA.

Article Snippet: Recombinant IGFBP2 protein (rIGFBP2) was purchased from Cloud‐Clone Corp (Wuhan, China).

Techniques: Staining, Expressing, Western Blot, Control

Journal: Science advances

Article Title: IGFBP2 secretion by mammary adipocytes limits breast cancer invasion.

doi: 10.1126/sciadv.adg1840

Figure Lengend Snippet: Fig. 2. Stromal-derived IGFBP2 is sufficient to inhibit MM231 breast cancer cell invasion. (A and B) Representative images (A) and quantification (B) of MM231 breast cancer cell invasion in inverted collagen/fibronectin matrices in the presence of conditioned medium from HUVECs transfected with siRNAs against IGFBP2 (siIGFBP2_1 and siIGFBP2_2) or siNTC. Scale bars, 50 μm. n = 3 biological replicates performed in triplicate, with three stacksper transwell; one-way ANOVAwith Tukey correction; **P < 0.01 and ***P < 0.001; ns, not significant. (C and D) Representative Western blot (C) and densitometry analysis (D) of TIFs stably overexpressing mTurquoise2 (mT2) or IGFBP2 (n = 4; one-sample t test; *P < 0.05). (E) Enzyme-linked immunosorbent assay (ELISA) for human IGFBP2 in concentrated conditioned media from TIFs overex- pressing mT2 or IGFBP2, measured in duplicate (n = 3 biological replicates; two-tailed Student’s t test; **P < 0.01). (F) Representative images and quantification of fibro- blast-contracted 3D collagen invasion assays of MM231 cancer cell invasion in mT2 or IGFBP2 TIF-contracted matrices after 14 days and stained for Pan-CK (brown). Scale bars, 100 μm. n = 3 biological replicates, triplicate matrices, eight regions per condition per replicate; one-way ANOVA with Tukey correction; ***P < 0.001. (G and H) Representative hematoxylin and eosin (H&E) images (G) and quantification (H) of MM231 cell invasion into the mouse dermis from subcutaneous xenografts of MM231 co-injected with TIFs overexpressing either mT2 (control) or IGFBP2. Scale bars, 500 μm; insets, 50 μm. n = 10 mice (mT2) or 12 mice (IGFBP2).

Article Snippet: For the exogenous protein inverse invasion screen, cells were treated with either PBS or one of the following recombinant proteins (5 μM): SPARC (R&D Systems, 941-SP), DKK1 (R&D Systems, 5439-DK), DKK3 (R&D Systems, 1118-DK), TFPI (R&D Systems, 2974-PI), TFPI2 (R&D Systems, 2545-PI), HHIP (R&D Systems, 9280-HP), PAI1 (R&D Systems, 1786-PI), IGFBP2 (R&D Systems, 674-B2), IGFBP7 (R&D Systems, 1334-B7), and GSN (Novus, H00002934-Q01).

Techniques: Derivative Assay, Transfection, Western Blot, Stable Transfection, Enzyme-linked Immunosorbent Assay, Two Tailed Test, Staining, Injection, Control

Journal: Science advances

Article Title: IGFBP2 secretion by mammary adipocytes limits breast cancer invasion.

doi: 10.1126/sciadv.adg1840

Figure Lengend Snippet: Fig. 4. IGFBP2 acts through depletion of IGF-II from the cancer microenvironment. (A) Sche- matic of the proteomics experimental setup. LC- MS/MS, liquid chromatography–tandem mass spectrometry. IP, immunoprecipitation. (B and C) Representative Western blot (B) and densitome- try analysis (C) after silencing of IGF2 (IGF-II gene) using siRNAs in MM231 cells (n = 4 biological replicates; one-sample t test; *P < 0.05 and ***P < 0.001). (D and E) Representative images (D) and quantification (E) of MM231 cell invasion in in- verted collagen/fibronectin matrices after IGF2 silencing. Scale bars, 50 μm. n = 3 biological replicates, performed in duplicate with three stacks per transwell; one-way ANOVA with Tukey correction; ***P < 0.001. (F and G) Representative images (F) and quantification (G) of MM231 cell invasion in inverted collagen/fibronectin matri- ces treated with PBS, IGF-II (10 ng/ml), IgG1κ (10 μg/ml), or anti–IGF-II (10 μg/ml). Scale bars, 50 μm. n = 3 biological replicates, performed in duplicate with three stacks per transwell; one- way ANOVA with Tukey correction; ***P < 0.001. (H and I) Representative IGF-II Western blot (H) and quantification (I) of TIF, MM231, MM468, and HCC1937 cells (n = 6 biological replicates; one- sample t test; ***P < 0.001). (J) ELISA for human IGF-II in conditioned media from TIF, MM231, MM468, and HCC1937 cells (n = 4 biological replicates; one-way ANOVA with Dunnett’s cor- rection; *P < 0.05 and ***P < 0.001). (K and L) Matrigel invasion assays for MM231 (K) and MM468 (L) cells treated with IgG1κ or anti–IGF-II (n = 3, eight fields of view (FOVs) per chamber, two to three invasion chambers per condition per replicate; two-tailed Student’s t test with Welch’s correction; **P < 0.01). Scale bars, 100 μm. (M) Schematic of the proposed mechanism for IGFBP2 inhibition of invasion through dis- ruption of breast cancer IGF-II autocrine signaling.

Article Snippet: For the exogenous protein inverse invasion screen, cells were treated with either PBS or one of the following recombinant proteins (5 μM): SPARC (R&D Systems, 941-SP), DKK1 (R&D Systems, 5439-DK), DKK3 (R&D Systems, 1118-DK), TFPI (R&D Systems, 2974-PI), TFPI2 (R&D Systems, 2545-PI), HHIP (R&D Systems, 9280-HP), PAI1 (R&D Systems, 1786-PI), IGFBP2 (R&D Systems, 674-B2), IGFBP7 (R&D Systems, 1334-B7), and GSN (Novus, H00002934-Q01).

Techniques: Liquid Chromatography with Mass Spectroscopy, Liquid Chromatography, Mass Spectrometry, Immunoprecipitation, Western Blot, Enzyme-linked Immunosorbent Assay, Two Tailed Test, Inhibition

Journal: Science advances

Article Title: IGFBP2 secretion by mammary adipocytes limits breast cancer invasion.

doi: 10.1126/sciadv.adg1840

Figure Lengend Snippet: Fig. 5. IGFBP2 expression during breast cancer progression. (A) Representative H&E-stained breast tissue samples from healthy patients, patients with DCIS, or pa- tients with IDC. Scale bars, 200 μm. (B) Quantification of adipocytes per section from healthy (n = 8 patients, three to eight sections per patient), DCIS (n = 6 patients, one to four sections per patient), and IDC (n = 3 patients, one to two sections per patient). Kruskal-Wallis test using Dunn’s test to correct for multiple comparisons (**P < 0.01 and ***P < 0.001). (C) Representative image from a patient sample stained for IGFBP2 (magenta) and counterstained with DAPI (cyan) and keratin-8/keratin-14 (KRT8/14; red). Scale bar, 100 μm. n = 8 normal reduction mammoplasty patient samples. Autofluorescence is given in green. (D) Representative image of human DCIS breast cancer tissue stained for IGFBP2 (magenta) and counterstained with DAPI (cyan) and KRT8/14 (red). Scale bar, 100 μm (n = 4 DCIS patient samples). Autofluorescence is given in green. (E) Representative image of human IDC breast cancer tissue stained for IGFBP2 (magenta) and counterstained with DAPI (cyan) and KRT8/14 (red; n = 3 IDC patient samples). Scale bar, 100 μm. Autofluorescence is given in green. The brightness of IGFBP2 staining was increased for display purposes only. (F) Quantification of IGFBP2 per adipocyte from healthy (n = 8 patients, 143 to 280 adipocytes per patient), DCIS (n = 6 patients, 39 to 410 adipocytes per patient), and IDC (n = 3 patients, 75 to 283 adipocytes per patient). Kruskal-Wallis test using Dunn’s test to correct for multiple comparisons (***P < 0.001).

Article Snippet: For the exogenous protein inverse invasion screen, cells were treated with either PBS or one of the following recombinant proteins (5 μM): SPARC (R&D Systems, 941-SP), DKK1 (R&D Systems, 5439-DK), DKK3 (R&D Systems, 1118-DK), TFPI (R&D Systems, 2974-PI), TFPI2 (R&D Systems, 2545-PI), HHIP (R&D Systems, 9280-HP), PAI1 (R&D Systems, 1786-PI), IGFBP2 (R&D Systems, 674-B2), IGFBP7 (R&D Systems, 1334-B7), and GSN (Novus, H00002934-Q01).

Techniques: Expressing, Staining

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