Fig 1: TPI1 modulates IPF in vitro and in vivo. (A and B) TPI1 knockdown in MRC-5 and WI-38 cells pretreated with 5 ng/ml transforming growth factor-beta1 (TGF-β1) for 24 h. Western blot analysis of TPI1, α-SMA, and COL1A1 expression in the indicated cells; β-actin served as an internal control. *P < 0.05; **P < 0.01; ***P < 0.001. (C to E) A 5-ethynyl-2′-deoxyuridine (EdU) assay was performed to evaluate the proliferation of TPI1-knockdown cells pretreated with 5 ng/ml TGF-β1 for 24 h. The percentage of EdU-positive cells was calculated by using ImageJ. The scale bar represents 50 μm; n = 3 biological replicates for each analysis. *P < 0.05; ns, no significance. (F and G) A wound healing assay was performed to evaluate cell migration. The extent of wound healing was recorded and quantified by using ImageJ. The scale bar represents 100 μm; n = 3 biological replicates for each analysis. *P < 0.05; ns, no significance. (H to J) Left panel: representative images for micro-CT, HE staining, Masson staining, and IHC staining in mice. The mice were divided into groups as indicated; the minimal number of mice was used (n = 6 per group). The scale bar represents 150 μm. Right panel: analysis of the IHC scores of TPI1 expression and Ashcroft scores of collagen expression in mouse lungs. **P < 0.01. (K) Western blot analysis of TPI1, α-SMA, and COL1A1 expression in the lung tissue of mice; α-tubulin served as an internal control. AAV, adeno-associated virus.
Fig 2: Biophysical Characterization of TPIR5G. (A) Analytical size-exclusion chromatography of WT and R5G TPI variants. Standards are indicated on the top axis. (B) Specific TPI activity for WT and R5G mutant TPI. Data points are the measurements for specific activity from assays containing 1, 5, or 10 ng of the indicated TPI protein. Unpaired t-test was used to determine significance, n = 8, error bars indicate Std. Dev. ns, not significant.
Fig 3: Triosephosphate isomerase 1 (TPI1) is upregulated in idiopathic pulmonary fibrosis (IPF). (A) The GSE47460 dataset from the Gene Expression Omnibus (GEO) database was used to analyze the differences in the messenger RNA (mRNA) levels of TPI1 between the normal control group and the IPF group. ***P < 0.001. (B) Left panel: slides from normal lung and IPF tissues were subjected to multiplex immunofluorescence (multi-IF) staining. TPI1 fluorescence is shown in green, and nuclear fluorescence is shown in blue. Right panel: relative quantitative analysis of the fluorescence intensity of TPI1. The scale bar represents 200 μm; n = 3 biological replicates for each analysis. *P < 0.05. (C) Western blot analysis of TPI1, alpha-smooth muscle actin (α-SMA), and collagen type I alpha 1 (COL1A1) expression in the lung tissue of mice; β-actin served as an internal control. (D to F) Left panel: representative images for micro-computed tomography (micro-CT), hematoxylin and eosin (HE) staining, Masson staining, and immunohistochemical (IHC) staining in mice (D). The mice were divided into groups as indicated; the minimal number of mice was used (n = 6 per group). The scale bar represents 200 μm. Right panel: analysis of the Ashcroft scores of collagen expression (E) and IHC scores of TPI1 expression (F) in mouse lungs. ***P < 0.001. NC, normal control; IntDen, integrated density; DAPI, 4′,6-diamidino-2-phenylindole; BLM, bleomycin.
Fig 4: Western blot of TPI from whole protein extracts. FB909 patient-derived fibroblast cells (R5G/f.s) exhibit a dramatic ~90% reduction in TPI protein compared to control fibroblast cells FB827. Two-tailed Mann–Whitney test was used to determine significance, n = 8 for each cell line, error is Std. Dev.
Fig 5: Compound 846 increases TPI protein levels in human fibroblasts. (A) Western blot analysis demonstrates significant ~190% average increase with 10 μM 846 treatment compared to DMSO-treated controls. (B) Representative blot used in 10 μM 846 treatment analysis. Two-tailed Mann–Whitney test was used to determine significance. p-values are shown, n = 8 for each group, error is Std. Dev.
Supplier Page from Abcam for Triose Phosphate Isomerase (TPI) Activity Assay Kit (Colorimetric)