Journal: Nature Communications

Article Title: Fibroblast-enriched endoplasmic reticulum protein TXNDC5 promotes pulmonary fibrosis by augmenting TGFβ signaling through TGFBR1 stabilization

doi: 10.1038/s41467-020-18047-x

Figure Lengend Snippet: a Transcript analysis showed significant upregulation of TXNDC5 mRNA in human IPF ( n = 26, central and lateral lung from 13 biologically independent samples), compared with control ( n = 18, central and lateral lung from 9 biologically independent samples), lung tissues. b Immunoblot analysis showed a marked increase in TXNDC5 and αSMA protein levels in human IPF ( n = 5 biologically independent samples), compared with control ( n = 3 biologically independent samples), lung tissues. c Protein expression level of TXNDC5 and αSMA were also increased in IPF, compared with control, lung fibroblasts following TGFβ1 treatment ( n = 5 biologically independent samples per group). d Re-analyses of microarray data from IPF human lung fibroblasts (GSE40839) revealed strong positive correlation between the expression level of TXNDC5 and that of genes encoding fibrogenic proteins ( COL1A1, ELN, ACTA2 ) in human lung fibroblasts (n = 13 biologically independent samples per group) (Data are presented as mean ± SEM, P value determined using two-tailed Mann–Whitney U test. Source data are provided as a Source Data file).

Article Snippet: Primary adult human pulmonary fibroblasts (ScienCell, CA, USA, 3310) were cultured in Fibroblast Medium (ScienCell, CA, USA, 2301) containing 2% FBS, 1% Fibroblast Growth Supplement (FGS) and 1%penicillin/streptomycin solution (P/S) in a humidified atmosphere of room air supplemented with 95% O 2 /5% CO 2 at 37 °C.

Techniques: Control, Western Blot, Expressing, Microarray, Two Tailed Test, MANN-WHITNEY

Journal: Nature Communications

Article Title: Fibroblast-enriched endoplasmic reticulum protein TXNDC5 promotes pulmonary fibrosis by augmenting TGFβ signaling through TGFBR1 stabilization

doi: 10.1038/s41467-020-18047-x

Figure Lengend Snippet: a Transcript expression levels of Txndc5 and fibrogenic protein genes ( Col1a1, Col3a1, Eln, Fn and Ctgf ) were markedly increased in the lung tissues from BLM-( n = 4 biologically independent animals per group), compared with PBS- (Sham, n = 3 biologically independent animals), treated WT mice on Day 21. b Immunohistochemical (IHC) staining on the serial sections of mouse lungs showed a strong upregulation of αSMA and TXNDC5 in the lung tissues from BLM-, compared with PBS- (Sham), treated WT mice on Day 21 ( n = 9 fields examined over 3 biologically independent animals per group). c IF staining for TXNDC5 on lung sections from sham-operated and BLM-treated Col1a1-GFP Tg mice. BLM treatment significantly increased TXNDC5 expression and the number of GFP-positive cells in the mouse lungs on Day 21 ( n = 3 biologically independent animals). There was a high degree of co-localization of TXNDC5 with GFP-positive, collagen producing lung fibroblasts (white arrows and inset) (data are presented as mean SEM, P value determined using two-tailed Mann–Whitney U test. Source data are provided as a Source Data file. BLM bleomycin).

Article Snippet: Primary adult human pulmonary fibroblasts (ScienCell, CA, USA, 3310) were cultured in Fibroblast Medium (ScienCell, CA, USA, 2301) containing 2% FBS, 1% Fibroblast Growth Supplement (FGS) and 1%penicillin/streptomycin solution (P/S) in a humidified atmosphere of room air supplemented with 95% O 2 /5% CO 2 at 37 °C.

Techniques: Expressing, Immunohistochemical staining, Immunohistochemistry, Staining, Two Tailed Test, MANN-WHITNEY

Journal: Nature Communications

Article Title: Fibroblast-enriched endoplasmic reticulum protein TXNDC5 promotes pulmonary fibrosis by augmenting TGFβ signaling through TGFBR1 stabilization

doi: 10.1038/s41467-020-18047-x

Figure Lengend Snippet: Protein ( n = 6 biologically independent samples per group) ( a ) and transcript ( n = 6 biologically independent samples per group) ( b ) expression levels of COL1A1, fibronectin, elastin, periostin, and αSMA/ ACTA2 were markedly increased in control (shScr) human pulmonary fibroblasts (HPF) following TGFβ1 (10 ng/ml) treatment. TGFβ1-induced upregulation of these fibrogenic proteins/genes was significantly attenuated in HPF with TXNDC5 depletion (sh TXNDC5 ). c TGFβ1 treatment significantly increased the cellular proliferation activity in shScr-, but not in sh TXNDC5 -, transduced HPF ( n = 24 biologically independent samples per group). ECM proteins (COL1A1 and fibronectin), markers for fibroblast activation (periostin and αSMA) ( n = 9 biologically independent samples per group) ( d ) and fibroblast proliferation activity ( n = 9 biologically independent samples per group) ( e ) were markedly increased in HPF transduced with TXNDC5 (TXNDC5 OE), compared with empty, expression vector (Data are presented as mean ± SEM, P value determined using two-tailed Mann–Whitney U test. Source data are provided as a Source Data file. n.s. non-significant, ctrl control, KD knockdown, OE overexpress).

Article Snippet: Primary adult human pulmonary fibroblasts (ScienCell, CA, USA, 3310) were cultured in Fibroblast Medium (ScienCell, CA, USA, 2301) containing 2% FBS, 1% Fibroblast Growth Supplement (FGS) and 1%penicillin/streptomycin solution (P/S) in a humidified atmosphere of room air supplemented with 95% O 2 /5% CO 2 at 37 °C.

Techniques: Expressing, Control, Activity Assay, Activation Assay, Transduction, Plasmid Preparation, Two Tailed Test, MANN-WHITNEY, Knockdown

Journal: Nature Communications

Article Title: Fibroblast-enriched endoplasmic reticulum protein TXNDC5 promotes pulmonary fibrosis by augmenting TGFβ signaling through TGFBR1 stabilization

doi: 10.1038/s41467-020-18047-x

Figure Lengend Snippet: a Immunoblots showed that TGFBR1, but not TGFBR2, was markedly upregulated in control HPF (shScr) following TGFβ1 treatment. TXNDC5 knockdown (sh TXNDC5 ) prevented TGFBR1 upregulation induced by TGFβ1 treatment completely ( n = 6 biologically independent samples per group). b Forced TXNDC5 expression led to marked upregulation of TGFBR1, but not TGFBR2, protein in HPF ( n = 12 biologically independent samples per group). c TGFBR1 and COL1A1 proteins were both markedly upregulated in the lung tissues from WT, but not Txndc5 −/− , mice 21 days following BLM treatment (WT sham n = 4, WT BLM n = 5, Txndc5 −/− sham n = 3, Txndc5 −/− BLM n = 5 biologically independent animals). d Representative IF staining and quantification ( e ) of TGFBR1 in Col1a1-GFP Tg and Col1a1-GFP Tg * Txndc5 −/− mouse lungs with PBS (Sham) or BLM treatment on day 21 ( n = 9 fields examined over 3 biologically independent animals per group). TGFBR1 was marked increased and showed strong co-localization with GFP-positive lung fibroblasts in BLM-treated mouse lungs. Global deletion of TXNDC5 prevented the upregulation of TGFBR1 in mouse lungs following BLM treatment (Data are presented as mean ± SEM, P value determined using two-tailed Mann–Whitney U test. Source data are provided as a Source Data file. n.s. non-significant, KD knockdown, OE overexpress, BLM bleomycin, TGFBR1 TGFβ receptor type 1, TGFBR2 TGFβ receptor type 2).

Article Snippet: Primary adult human pulmonary fibroblasts (ScienCell, CA, USA, 3310) were cultured in Fibroblast Medium (ScienCell, CA, USA, 2301) containing 2% FBS, 1% Fibroblast Growth Supplement (FGS) and 1%penicillin/streptomycin solution (P/S) in a humidified atmosphere of room air supplemented with 95% O 2 /5% CO 2 at 37 °C.

Techniques: Western Blot, Control, Knockdown, Expressing, Staining, Two Tailed Test, MANN-WHITNEY

Journal: Nature Communications

Article Title: Fibroblast-enriched endoplasmic reticulum protein TXNDC5 promotes pulmonary fibrosis by augmenting TGFβ signaling through TGFBR1 stabilization

doi: 10.1038/s41467-020-18047-x

Figure Lengend Snippet: a Forced expression of TXNDC5 in HPF led to increased SMAD3 phosphorylation, fibroblast activation (as evidenced by increased periostin levels) and ECM (COL1A1 and fibronectin) production, all of which were abolished completely by the treatment of LY364947 (10 μM for 48 h), a TGFBR1 kinase inhibitor (n = 6 biologically independent samples per group). b Knockdown of TGFBR1 reversed TXNDC5 overexpression-induced COL1A1, periostin, and αSMA expression ( n = 4 biologically independent samples per group) (Data are presented as mean ± SEM, P value determined using two-tailed Mann–Whitney U test. Source data are provided as a Source Data file. n.s. non-significant, KD knockdown, OE overexpress, shTGFBR1 TGFBR1 knockdown with shRNA).

Article Snippet: Primary adult human pulmonary fibroblasts (ScienCell, CA, USA, 3310) were cultured in Fibroblast Medium (ScienCell, CA, USA, 2301) containing 2% FBS, 1% Fibroblast Growth Supplement (FGS) and 1%penicillin/streptomycin solution (P/S) in a humidified atmosphere of room air supplemented with 95% O 2 /5% CO 2 at 37 °C.

Techniques: Expressing, Phospho-proteomics, Activation Assay, Knockdown, Over Expression, Two Tailed Test, MANN-WHITNEY, shRNA

Journal: Nature Communications

Article Title: Fibroblast-enriched endoplasmic reticulum protein TXNDC5 promotes pulmonary fibrosis by augmenting TGFβ signaling through TGFBR1 stabilization

doi: 10.1038/s41467-020-18047-x

Figure Lengend Snippet: a A cycloheximide chase assay performed in HPF with TXNDC5 knockdown (sh TXNDC5 ) showed accelerated degradation of TGFBR1 protein, comparing to control (shScr) ( n = 4 biologically independent samples per group). b Overexpression of TXNDC5 slowed down TGFBR1 protein degradation significantly in HFP ( n = 7 biologically independent samples per group). c TXNDC5 depletion-induced downregulation of TGFBR1 protein was partially reversed by the treatment of proteasome inhibitor MG132 (20μM for 48 h) ( n = 7 biologically independent samples per group). d , e Overexpression of WT, but not AAA mutant (see text for details), TXNDC5 protein in HPF led to significant upregulation of TGFBR1, ECM (fibronectin and COL1A1) proteins and fibroblast activation markers (αSMA and periostin) ( n = 8 biologically independent samples per group) (Data are presented as mean ± SEM, P value determined using two-tailed unpaired t tests. Source data are provided as a Source Data file. OE overexpress, TXNDC5 AAA TXNDC5 mutant lacking its PDI enzyme activity).

Article Snippet: Primary adult human pulmonary fibroblasts (ScienCell, CA, USA, 3310) were cultured in Fibroblast Medium (ScienCell, CA, USA, 2301) containing 2% FBS, 1% Fibroblast Growth Supplement (FGS) and 1%penicillin/streptomycin solution (P/S) in a humidified atmosphere of room air supplemented with 95% O 2 /5% CO 2 at 37 °C.

Techniques: Knockdown, Control, Over Expression, Mutagenesis, Activation Assay, Two Tailed Test, Activity Assay

Journal: Nature Communications

Article Title: Fibroblast-enriched endoplasmic reticulum protein TXNDC5 promotes pulmonary fibrosis by augmenting TGFβ signaling through TGFBR1 stabilization

doi: 10.1038/s41467-020-18047-x

Figure Lengend Snippet: a Illustration of experimental design for deletion of Txndc5 in pulmonary fibroblasts. Tamoxifen (80 mg/kg i.p. every other day) was administered between 7–21 days after BLM treatment. b Picrosirius red staining (left panels) and second harmonic generation (SHG) images (right panels) of lung sections from Col1a2-cre/ERT2 ( Col1a2-cre ) and Col1a2-Cre/ERT2*Txndc5 fl/fl ( Txndc5 cKO ) mice 7 day and 21 day after intra-tracheal administration of BLM. The quantitative results of picrosirius red-( Col1a2-cre , n = 3 in D0 and D7, n = 5 in D21; Txndc5 cKO , n = 3 in D0 and D7, n = 6 in D21 biologically independent animals) ( c ) and SHG- ( Col1a2-cre , n = 3; Txndc5 cKO , n = 3 in D0 and D7, n = 5 in D21 biologically independent animals) ( d ) positive areas showed rapid progression of PF in Col1a2-cre , but not in Txndc5 cKO , lungs after BLM instillation. For each of the lung sections scanned for SHG, additional two-photon-excited fluorescence (TPEF) imaging was obtained to show the outline of the imaged tissue. e Hydroxyproline content was similarly increased in Col1a2-cre and Txndc5 cKO on Day 7 (before Tamoxifen injection) following BLM treatment. Hydroxyproline content continued to increase in the mouse lungs of Col1a2-cre mice between 7 and 21 days post-BLM treatment ( Col1a2-cre , n = 3 in D0, n = 4 in D7, n = 5 in D21; Txndc5 cKO , n = 3 in D0 and D7, n = 7 in D21 biologically independent animals). The hydroxyproline content remained stable in BLM-treated Txndc5 cKO mouse lungs after tamoxifen-induced Txndc5 deletion. Representative lung function parameters ( Col1a2-cre , n = 5 in D0, n = 3 in D7, n = 6 in D21; Txndc5 cKO , n = 4 in D0, n = 3 in D7, n = 7 in D21 biologically independent animals) ( f ) and pressure-volume curves (on Day 21, sham: n = 3, BLM: n = 6 biologically independent animals) ( g ) determined using flexiVent FX system in Col1a2-cre and Txndc5 cKO mice following sham procedure or BLM treatment. h Schematic summary of the proposed profibrotic mechanisms by which TXNDC5 contributes to the pathogenesis of pulmonary fibrosis (Data are presented as mean ± SEM, P value determined using two-tailed Mann–Whitney U test. Source data are provided as a Source Data file. BLM bleomycin). Illustrations in a and h were created by T.H.L.

Article Snippet: Primary adult human pulmonary fibroblasts (ScienCell, CA, USA, 3310) were cultured in Fibroblast Medium (ScienCell, CA, USA, 2301) containing 2% FBS, 1% Fibroblast Growth Supplement (FGS) and 1%penicillin/streptomycin solution (P/S) in a humidified atmosphere of room air supplemented with 95% O 2 /5% CO 2 at 37 °C.

Techniques: Staining, Fluorescence, Imaging, Injection, Two Tailed Test, MANN-WHITNEY