Aortic carboxypeptidase-like protein potentiates β1 integrin signaling in mesenchymal progenitors

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  Figure S1.. Validation of recombinant aortic carboxypeptidase-like protein (ACLP) and hydrogel system. (A, B) Total protein (10 μg) was separated on 4–12% SDS–PAGE gels followed by Coomassie staining of purified recombinant ACLP generated from stably transfected AD293 (B) and Western blot analysis using an anti-Myc antibody. (C) Cell-free collagen polymerization assay of type 1 collagen polymerized with ACLP for 45 min. Turbidity was read at 410 nm. (D) Incorporation of recombinant myc-tagged ACLP into collagen I–coated hydrogels was confirmed using an ELISA-based detection assay. Absorbance was measured at 450 nm. Gelatin substrate was used as a negative control to assess collagen specificity. Matrix-ACLP refers to ACLP incorporated into the substrate during polymerization, whereas soluble ACLP was added in solution after substrate polymerization. Data represent the mean ± SD from triplicate wells. Statistical significance was assessed using Welch’s t test; P < 0.05.

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  Figure 1.. Collagen-bound aortic carboxypeptidase-like protein (ACLP) induces early cell spreading through TGFβR1-independent mechanisms. (A) Schematic of the cell stimulation by the ECM assay. 10T1/2 mesenchymal progenitors were serum-starved in suspension for 1 h, then seeded onto 12 kPa polyacrylamide hydrogels coated with type I collagen polymerized ± 30 nM recombinant ACLP. (B) Cells were fixed at 30, 60, and 90 min and stained with phalloidin (Alexa Fluor 555) to visualize F-actin. Phalloidin staining was segmented in CellProfiler using minimum cross-entropy thresholding (diameter 100–100,000 px), and cell area was measured from the resulting single-cell objects. (C) Cell spreading at 90 min on col1 hydrogels treated with 30 nM soluble ACLP. (D) Cell spreading at 90 min ± 5 μM SB431542 (TGFβR/ALK5 inhibitor). Data represent the mean ± SD from three independent experiments (n = 3). At least 30 cells per condition per replicate were analyzed (≥100 cells total). Statistical comparisons used Welch’s t tests; P < 0.05.

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  Figure S2.. Collagen-bound aortic carboxypeptidase-like protein sustained increased cell spreading, independent of cell adhesion. (A) 10T1/2 mesenchymal progenitors were seeded on col1 or col1-ACLP hydrogels for 48 h as described in Fig 1A. Cells were stained with phalloidin (Alexa Fluor 555) to visualize F-actin. Phalloidin staining was segmented in CellProfiler using minimum cross-entropy thresholding (diameter 100–100,000 pixels), and cell area was measured from the resulting single-cell objects. (B) Cell adhesion was quantified by the crystal violet assay at the indicated time points; absorbance was measured at 550 nm. Data represent the mean ± SD from three independent experiments (n = 3). At least 30 cells per condition per replicate were analyzed (≥100 cells in total). Statistical comparisons used Welch’s t tests; P < 0.05.

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  Figure 2.. Collagen-bound aortic carboxypeptidase-like protein (ACLP) enhances β1 integrin activation and focal adhesion maturation. 10T1/2 mesenchymal progenitors were seeded on col1 or col1-ACLP hydrogels as described in Fig 1A and fixed after 90 min. (A, B, C) Cells were stained for (left) (A) active β1 integrin (HUTS-4), (B) paxillin, or (C) pFAK (Tyr397). Representative images are shown; scale bars, 10 μm. Quantification (right) of integrated fluorescence edge intensity in CellProfiler. Nuclei were segmented (minimum cross-entropy), used to define single-cell boundaries, and signal intensities were measured from the corresponding fluorescence channels. Per-cell values represent integrated edge intensity measurements. Data represent the mean ± SD from three independent experiments (n = 3). At least 30 cells per condition per replicate were analyzed (≥100 cells in total). Statistical comparisons were performed using Welch’s t tests; P < 0.05.

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  Figure 3.. Collagen-bound aortic carboxypeptidase-like protein (ACLP) enhanced RhoA and Rac1 GTPase activity. 10T1/2 mesenchymal progenitors were seeded on col1, col1-ACLP, or col1-ACLP+SB hydrogels for 30 min as described in Fig 1A. (A, B, C) Activation of (A) Rac1, (B) RhoA, and (C) Cdc42 was measured using G-LISA activation assays (Cytoskeleton, Inc.). Absorbance was measured at 490 nm. Data represent the mean ± SD from three independent experiments (3 × 105 cells per n; n = 3). Statistical comparisons were performed using Welch’s t tests or one-way ANOVA, as appropriate; P < 0.05.

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  Figure 4.. Collagen-bound aortic carboxypeptidase-like protein (ACLP) increases F-actin assembly and MRTFA nuclear accumulation. 10T1/2 mesenchymal progenitors were seeded on col1, col1-ACLP, or col1-ACLP+SB hydrogels and fixed after 90 min. (A) Immunofluorescence staining of F-actin with phalloidin (Alexa Fluor 555). Representative images are shown; scale bars, 10 μm. (B) (left) Immunoblot of G-actin and F-actin fractions separated using a G/F-actin assay kit (Cytoskeleton, BK037). (B) (Right) Quantification of the G-actin/F-actin ratio from three independent experiments (3 × 105 cells per n; n = 3). (C) Immunofluorescence staining for MRTFA (Alexa Fluor 488) with phalloidin and DAPI nuclear counterstain. Representative images are shown; scale bars, 10 μm. (D) Quantification of MRTFA localization in CellProfiler. Nuclei and phalloidin were segmented (minimum cross-entropy), used to define nuclear and cytoplasmic boundaries, and signal intensities were measured from the corresponding fluorescence channels. Data represent the mean ± SD from three independent experiments. At least 30 cells per condition per replicate were analyzed (≥100 cells in total). Statistical comparisons were performed…

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  Figure 5.. Aortic carboxypeptidase-like protein (ACLP) activates a transcriptional program revealing enriched for focal adhesions, ECM– receptor interactions, and actin cytoskeleton pathways. (A) Principal component analysis of bulk RNA-seq data from 10T1/2 mesenchymal progenitors cultured for 18 h on col1, col1-ACLP, or col1-ACLP+SB hydrogels (n = 4 biological replicates per group). (B) Volcano plot showing differentially expressed genes (DEGs) between col1 and col1-ACLP. Significantly up-regulated genes (log2FC > 0.58, adj P < 0.05) are shown in red and significantly down-regulated genes (log2FC < −0.58, adj P < 0.05) in blue; nonsignificant genes appear in gray. The dashed horizontal line denotes −log10 (adj P = 0.05). (C) KEGG GSEA of genes up-regulated in col1-ACLP relative to col1. Pathways associated with focal adhesion, regulation of actin cytoskeleton, and ECM–receptor interaction were significantly enriched (FDR < 0.05). (C, D) Heatmaps of core gene enrichments from selected KEGG pathways shown in (C), displayed as log2FC relative to col1. (E) Volcano plot comparing col1 and col1-ACLP+SB. (B) Significantly up-regulated and down-regulated genes are colored as in (B). (F)…

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  Figure S3.. Validation of select DEGs from RNA sequencing. RT–qPCR of select differentially expressed genes. CT values were normalized to Ppia. Statistical significance was assessed using Welch’s t test or one-way ANOVA, as appropriate; P < 0.05 (ns, nonsignificant, **P < 0.01, ***P < 0.001, ****P < 0.0001).

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  Figure 6.. Collagen-bound aortic carboxypeptidase-like protein (ACLP) elicits conserved transcriptional responses in primary stromal progenitors. (A) Gonadal white adipose tissue (gWAT) was digested to isolate the stromal vascular fraction (SVF). Cells were plated for 48 h on 12 kPa hydrogels coated with type I collagen polymerized ± 30 nM ACLP. (B) Principal component analysis of bulk RNA-seq data (n = 3 biological replicates per condition). (C) Volcano plot showing differentially expressed genes (DEGs) between col1 and col1-ACLP. Significantly up-regulated genes (log2FC > 0.58, adj P < 0.05) are shown in red and significantly down-regulated genes (log2FC < −0.58, adj P < 0.05) in blue; nonsignificant genes appear in gray. Dashed horizontal line denotes −log10 (adj P = 0.05). (D) KEGG GSEA of genes up-regulated in col1-ACLP relative to col1. Pathways associated with focal adhesion, regulation of actin cytoskeleton, and ECM–receptor interaction were significantly enriched (FDR < 0.05).