Fig 2: HER2 deficiency rescued the dysregulated gene profiles in IPF lung fibroblasts. (A–C) HER2 knockdown efficiency was confirmed by qRT-PCR (A) and Western blotting (B and C; A, n = 8 per group; C, n = 5 per group). (D) Cell-surface protein level of HER2 and SEMA7A in HER2 knockdown IPF lung fibroblasts. (E and F) Representative images (E) and index quantification (F; n = 9 per group) of fibroblast invasion after HER2 knockdown. (G) Protein levels of p-HER2, total HER2, SEMA7A, and FOXF1 in IPF lung fibroblasts after treatment of HER2 inhibitor, Lapatinib, at increasing concentrations. (H) Downregulation of cell-surface expression of SEMA7A, F3, and ITGA6 in Lapatinib-treated fibroblasts was determined by flow cytometry analysis. (I) Transcription levels of other representative genes in IPF lung fibroblasts after Lapatinib treatment were determined by qRT-PCR (n = 3 per group). Scale bar: 1 mm (E). CTL, control; KD, knockdown. Three or four independent experiments were performed on fibroblasts from different patients (A, C, F, and I). Data are the mean ± SEM. *, P < 0.05; **, P < 0.01; ****, P < 0.0001 by Student’s t test (A, C, and F) or one-way ANOVA (I). Source data are available for this figure: SourceData F7.

Fig 3: ERBB2 (HER2) was the top inhibited upstream regulator in noninvasive fibroblasts. (A) Flow cytometry analysis confirmed the overexpression of SEMA7A in lung fibroblasts. (B) Cell proliferation rates of fibroblasts with SEMA7A overexpression or control fibroblasts were determined by EdU assays. (C) Cell-surface expression of SEMA7A was determined by flow cytometry on single-cell homogenate of CD31-, CD45-, EPCAM- cells from IPF and healthy samples. (D) Single-cell Western blot confirmed the downregulation of FOXF1 in invasive fibroblasts. (E and F) Dot plot visualization of the -Log10(FDR) (E) and bar plot visualization of the Activation Z-score (F) of the top 30 activated and inhibited upstream regulators of noninvasive fibroblasts by IPA analysis. ERBB2 was the top inhibited regulators of noninvasive fibroblasts. ERBB2 was highlighted as the most inhibited regulator. (G) The regulating network of invasive fibroblasts combining canonical signaling pathways and upstream regulators showed that the core signaling pathway was the invasion of tumor cell lines, suggesting that invasive lung fibroblasts had metastatic cancer-related signatures.

Fig 4: Dose-response characterization of the influence of thalidomide, lenalidomide, pomalidomide, atRA, and SB431542 on LPM differentiation of XCL-1 clones. High content imaging analysis of XCL-1 hiPSC clones that were differentiated for 2 days in APEL2 + GSKi in the presence of compound and subsequently stained for FOXF1 and NANOG (only FOXF1 data shown here) and counter-stained with DAPI. Untreated DMSO control is represented as the lowest concentration in each graph. Data represent the mean +/- SEM %FOXF1+/DAPI+ cells from at least 4 independent experiments. Nonlinear regression analysis was performed using the 4-parameter [inhibitor] vs response variable slope plotting tool in GraphPad Prism. (A–E) Dose-response characterization of thalidomide (A), pomalidomide (B), lenalidomide (C), atRA (D), and SB431542 (E) in CRBN mock (hollow circles) or CRBN KO/KI (red filled circles) clones. (F–J) Dose-response characterization of thalidomide (F), pomalidomide (G), lenalidomide (H), atRA (I), and SB431542 (J) in SALL4 mock (hollow circles) and SALL4G416A (blue filled circles) clones.

Fig 5: Characterization of Gibco and XCL-1 hiPSCs Undergoing LPM Differentiation by High Content Imaging and qRT-PCR. (A,B) High content imaging-based 5-color assay of Gibco hiPSCs cultured in mTeSR1 (A) versus APEL2 + GSKi (B) for 2 days. hiPSCs were co-stained for DAPI (panel 1), FOXF1 (panel 2), SALL4 (panel 3), OCT4 (panel 4), and T (panel 5). Panel 6 contains a false-colored merged image of OCT4 (red), T (green), FOXF1 (white) and SALL4 (blue) expression. (C) Quantification of marker expression in Gibco hiPSCs by high content imaging, presented as the mean ± SEM aggregated from 3 independent experiments of the % nuclei expressing each marker. Thresholding for each marker was performed relative to a non-expressing control cell type (human endothelial cells) for SALL4 and OCT4 or relative to undifferentiated hiPSCs for FOXF1 and T. (D,E) Confocal laser scanning microscopy images of Gibco hiPSCs cultured in mTeSR1 (D) versus APEL2 + GSKi (E) conditions for 2d and co-stained for DAPI (panel 1), FOXF1 (panel 2), and NANOG (panel 3). (F–J) Scatter plot of FOXF1 and NANOG intensity of DAPI + objects tabulated from cultures of Gibco hiPSCs (F), XCL-1 CRBN Mock hiPSCs (G), XCL-1 CRBN KO/KI hiPSCs (H), XCL-1 SALL4 Mock hiPSCs (I), and XCL-1 SALL4G416A hiPSCs (J) that were cultured either in mTeSR1 (black circles) or APEL2 + GSKi (red circles) and co-stained for DAPI, FOXF1 (x-axis), and NANOG (y-axis). (K) qRT-PCR characterization of Gibco hiPSCs and XCL-1 hiPSCs cultured in APEL2 + GSKi for 2 days relative to the undifferentiated control for each cell type cultured in mTeSR1. Data are presented as the log2 fold change relative to ACTB and undifferentiated control for pluripotency markers NANOG, POU5F1, and SALL4, primitive streak genes TBXT and SOX17, LPM genes FOXF1, GATA4, and KDR, and limb mesoderm genes PITX1 and TBX5. Color scales denote increased expression (red) or decreased expression (blue) relative to undifferentiated control.