Journal: bioRxiv

Article Title: 3D-bioprinted, phototunable hydrogel models for studying adventitial fibroblast activation in pulmonary arterial hypertension

doi: 10.1101/2022.11.11.516188

Figure Lengend Snippet: Fibroblast activation in 3D-bioprinted models of pulmonary arterial adventitia. A) Fibrotic activation in soft and stiffened 3D hydrogels measured by αSMA expression. HPAAFs in stiffened constructs were significantly more positive for αSMA than cells in soft constructs. Columns represent mean ± SEM, n = 3. *, p < 0.05, Mann-Whitney U test. B) Representative confocal images of immunostaining for αSMA, actin, and DAPI in soft and stiffened 3D hydrogels. HPAAFs in stiffened constructs showed more prevalent αSMA immunofluorescence than cells in soft constructs. Scale bar = 250 µm. C) Fibroblast proliferation in soft and stiffened 3D bioprinted constructs measured by EdU positivity. HPAAFs in stiffened constructs were significantly more positive for EdU than cells in soft constructs. Columns represent mean ± SEM, n = 3. *, p < 0.05, Mann-Whitney U test. D) Representative confocal images of immunostaining for EdU and Hoechst dye in soft and stiffened 3D hydrogels. HPAAFs in stiffened constructs showed more prevalent EdU immunofluorescence than cells in soft constructs. Scale bar = 300 µm.

Article Snippet: [ ] Human pulmonary artery adventitia fibroblasts (HPAAFs; Accegen, Fairfield, NJ) from a 2-year-old male patient were cultured in complete HPAAF culture media according to manufacturer instructions.

Techniques: Activation Assay, Expressing, Construct, MANN-WHITNEY, Immunostaining, Immunofluorescence

Journal: iScience

Article Title: Changes in nascent chromatin structure regulate activation of the pro-fibrotic transcriptome and myofibroblast emergence in organ fibrosis

doi: 10.1016/j.isci.2023.106570

Figure Lengend Snippet: The chromatin-based mechanism facilitated by UTX KDM activity is paralleled in primary lung fibroblasts (A–D, G) Representative CAA images of primary HLF. Cells were labeled with EdU (green) for 20min and counterstained with DAPI (blue). PLA is shown in red and in split channel (black and white). Graphs depict the number of PLA per EdU labeled nuclei. Scale bar: 10μm. (A) Representative CAA images of H3K27me3 (red) at 0h and 5h post-treatment with 5 ng/mL TGF-β in primary lung fibroblasts that were pretreated for 12h with GSK-J4 prior to the addition of TGF-β. (B) Representative CAA images of UTX (red) at 0h and 5h post-stimulation with 5 ng/ml TGF-β in primary HLF. (C) Representative CAA images for KDM6B (red) at 0h and 5h post-treatment with 5 ng/ml TGFβ in primary lung fibroblasts. (D) Representative CAA images of MRTF-A (red) at 0h and 24h post-stimulation with 5 ng/ml TGF-β in primary HLF that were pretreated for 12h with GSK-J4 prior to addition of TGF-β. (E and F) Chromatin was immunoprecipitated with MRTF-A antibody and IgG isotype control +/− GSK-J4. qPCR was performed for MRTF-A-containing genes: ACTA2 and COL1A1 . (G) Representative CAA images of MRTF-A (red) at 0h and 4h from thymidine release following 24h thymidine treatment in HLF. (H) Representative images of EdU (green) labeling at 0h and 4h after thymidine release following 24h thymidine treatment in HLF. Cultures were counterstained with DAPI (blue). Scale bar: 10 μm. Representative data are expressed as average ±SEM. ∗∗∗∗p < 0.0001 ∗p < 0.05 by one-way ANOVA with multiple comparisons (A, D) and unpaired t-test (B, C, E, F, G).

Article Snippet: Human: pulmonary fibroblasts , ScienCell Research Laboratories , Cat# 3300.

Techniques: Activity Assay, Labeling, Immunoprecipitation, Control

Journal: iScience

Article Title: Changes in nascent chromatin structure regulate activation of the pro-fibrotic transcriptome and myofibroblast emergence in organ fibrosis

doi: 10.1016/j.isci.2023.106570

Figure Lengend Snippet:

Article Snippet: Human: pulmonary fibroblasts , ScienCell Research Laboratories , Cat# 3300.

Techniques: Plasmid Preparation, Recombinant, Isolation, Reverse Transcription, SYBR Green Assay, Staining, Imaging, Software, Microscopy

Journal: Cell & Bioscience

Article Title: A missing piece of the puzzle in pulmonary fibrosis: anoikis resistance promotes fibroblast activation

doi: 10.1186/s13578-022-00761-2

Figure Lengend Snippet: SiO 2 -induced pulmonary inflammation induced anoikis resistance only in HPF-a cells. A The CCK-8 assay suggested that a 6-h CM treatment significantly increased the viability of HPF-a cells (n = 5); *P < 0.05 compared with the control group. CM from macrophages treated with SiO 2 increased the viability of HPF-a cells ( B ), BEAS-2B cells ( C ) and HPMECs ( D ) (n = 5); *P < 0.05 compared with the control group. Representative western blot and densitometry analyses of five separate experiments showing the effects of CM on the expression of the anoikis resistance marker NTRK2 in HPF-a ( E ), BEAS-2B ( F ) and HPMECs ( G ); (n = 5); *P < 0.05 compared with the 0-h group, **P < 0.01 compared with the 0-h group, and ***P < 0.001 compared with the 0-h group. H Sirius Red staining and Masson’s trichrome staining showed that SiO 2 induced more collagen deposition and incomplete pulmonary alveoli in the lung tissues, indicating that the mouse silicosis model was successfully established. Scale bar = 100 μm. I Immunofluorescence staining shows the expression of the fibroblast marker Vimentin and the anoikis protein NTRK2 in mouse lung tissue. Scale bar = 100 μm. (J) Spatial transcriptomics showing the coexpression of Ntrk2 and Vimentin

Article Snippet: Human pulmonary fibroblasts from adults (HPF-a), mouse pulmonary fibroblasts (MLG) and RAW264.7 cells were purchased from ScienCell and maintained in DMEM containing 10% FBS with 5% CO 2 and 37 °C in an incubator (Thermo Heracell 150i CO 2 incubator, Thermo Fisher Scientific, Inc., Germany).

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

Journal: Cell & Bioscience

Article Title: A missing piece of the puzzle in pulmonary fibrosis: anoikis resistance promotes fibroblast activation

doi: 10.1186/s13578-022-00761-2

Figure Lengend Snippet: Anoikis resistance-mediated fibroblast activation in response to SiO 2 . A HPF-a cells were treated with Con-CM and SiO 2 -CM for 24 h and double stained with Annexin V and PI. The sum of the counts in Q2-1, Q2-2 and Q2-4 was defined as the number of apoptotic cells. B Statistical analysis of three independent experiments of HPF-a cell apoptosis using FCM. C The CCK-8 assay detected the viability of HPF-a cells after treatment with Con-CM and SiO 2 -CM for 24 h (n = 5). ***P < 0.001 compared with the control group. D HPF-a cells were cultured in wells pretreated with poly-HEMA for 48 h and collected for counting (n = 5). ***P < 0.001 compared with the control group. E HPF-a cells were suspended for 48 h, and cell viability was detected using the CCK-8 assay (n = 5). ***P < 0.001 compared with the control group. F HPF-a cells were suspended for 48 h, and the expression level of TrkB was detected (n = 5). ***P < 0.001 compared with the control group. G HPF-a cells were suspended for 48 h before they were treated with Con-CM and SiO 2 -CM for 24 h and double stained with Annexin V and PI. The sum of the counts in Q2-1, Q2-2 and Q2-4 was defined as the number of apoptotic cells. H Statistical analysis of three independent experiments of HPF-a cell apoptosis using FCM; ***P < 0.001 compared with the attached group. I Statistical analysis of five independent experiments of HPF-a cell viability using the CCK-8 assay (n = 5); ***P < 0.001 compared with the attached group. J Spatial transcriptomics showed the coexpression of apoptosis markers ( Bim1) and Vimentin . K Spatial transcriptomics showing the coexpression of proliferation markers ( Cdk1 ) and Vimentin

Article Snippet: Human pulmonary fibroblasts from adults (HPF-a), mouse pulmonary fibroblasts (MLG) and RAW264.7 cells were purchased from ScienCell and maintained in DMEM containing 10% FBS with 5% CO 2 and 37 °C in an incubator (Thermo Heracell 150i CO 2 incubator, Thermo Fisher Scientific, Inc., Germany).

Techniques: Activation Assay, Staining, CCK-8 Assay, Control, Cell Culture, Expressing

Journal: Cell & Bioscience

Article Title: A missing piece of the puzzle in pulmonary fibrosis: anoikis resistance promotes fibroblast activation

doi: 10.1186/s13578-022-00761-2

Figure Lengend Snippet: SiO 2 -CM promotes fibrosis and anoikis resistance in HPF-a cells growing in ECM. A Schematic diagram of fibroblasts transplanted to ECM after decellularization. B Cell counting showed the effect of SiO 2 -CM on MLG cell proliferation cultured in ECM (n = 5). *P < 0.05 compared with the control group. C In the 3D migration experiment, the migration of fibroblasts increased after 24 h of exposure to SiO 2 -CM. Scale bar = 275 μm. D Statistical results of the number of migrating fibroblasts in the 3D migration experiment and the maximum migration distance after 24 h of exposure to SiO 2 -CM (n = 5). *P < 0.05 compared with the control group and **P < 0.01 compared with the control group. Immunofluorescence staining showing the expression of the fibroblast markers Vimentin ( E ) and α-SMA ( F ) and the anoikis protein NTRK2 to detect the effect of SiO 2 -CM on MLG cells. Scale bar = 200 μm

Article Snippet: Human pulmonary fibroblasts from adults (HPF-a), mouse pulmonary fibroblasts (MLG) and RAW264.7 cells were purchased from ScienCell and maintained in DMEM containing 10% FBS with 5% CO 2 and 37 °C in an incubator (Thermo Heracell 150i CO 2 incubator, Thermo Fisher Scientific, Inc., Germany).

Techniques: Cell Counting, Cell Culture, Control, Migration, Immunofluorescence, Staining, Expressing

Journal: Cell & Bioscience

Article Title: A missing piece of the puzzle in pulmonary fibrosis: anoikis resistance promotes fibroblast activation

doi: 10.1186/s13578-022-00761-2

Figure Lengend Snippet: ZC3H4 is involved in anoikis resistance-mediated fibroblast activation in response to SiO 2 . A Representative western blot showing that macrophage CM upregulated ZC3H4 expression in HPF-a cells. B Densitometry analyses of five separate experiments suggested that macrophage CM upregulated ZC3H4 expression in HPF-a cells (n = 5); *P < 0.05 and **P < 0.001 compared with 0 h. C Representative western blot analysis suggesting that poly-HEMA attenuated CM-induced increases in ZC3H4 expression in HPF-a cells. D Densitometry analyses of five separate experiments (n = 5); *P < 0.05 compared with the control group. E Representative western blot showing the effects of ZC3H4-NIC transfection on ZC3H4 and NTRK2 expression in HPF-a cells. F Densitometry analyses of three separate experiments suggested that ZC3H4 NIC transfection affected CM-induced increases in ZC3H4 and TrkB expression in HPF-a cells (n = 3); *P < 0.05 and **P < 0.001 compared with the corresponding control group. G Spatial transcriptomics showing the coexpression of Zc3h4 and Vimentin

Article Snippet: Human pulmonary fibroblasts from adults (HPF-a), mouse pulmonary fibroblasts (MLG) and RAW264.7 cells were purchased from ScienCell and maintained in DMEM containing 10% FBS with 5% CO 2 and 37 °C in an incubator (Thermo Heracell 150i CO 2 incubator, Thermo Fisher Scientific, Inc., Germany).

Techniques: Activation Assay, Western Blot, Expressing, Control, Transfection

Journal: Cell & Bioscience

Article Title: A missing piece of the puzzle in pulmonary fibrosis: anoikis resistance promotes fibroblast activation

doi: 10.1186/s13578-022-00761-2

Figure Lengend Snippet: SiO 2 -induced pulmonary inflammation activates ERS, but not autophagy, in HPF-a cells. A Representative western blot showing that macrophage CM upregulated ERS-related protein expression in HPF-a cells. B Densitometry analyses of five separate experiments (n = 5); *P < 0.05, **P < 0.01, and ***P < 0.001 compared with 0 h. C HPF-a cells were treated with tunicamycin (an ERS agonist), and ZC3H4 and NTRK2 expression was upregulated in response to tunicamycin treatment. D Densitometry analyses of five separate experiments (n = 5); *P < 0.05 compared with the control. E HPF-a cells were treated with salubrinal (an ERS inhibitor) for 6 h, and western blot analysis showed that ZC3H4 and NTRK2 levels were downregulated in response to salubrinal treatment. F Densitometry analyses of five separate experiments (n = 5); *P < 0.05 compared with the control

Article Snippet: Human pulmonary fibroblasts from adults (HPF-a), mouse pulmonary fibroblasts (MLG) and RAW264.7 cells were purchased from ScienCell and maintained in DMEM containing 10% FBS with 5% CO 2 and 37 °C in an incubator (Thermo Heracell 150i CO 2 incubator, Thermo Fisher Scientific, Inc., Germany).

Techniques: Western Blot, Expressing, Control

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