periostin mouse monoclonal antibody ab  (Proteintech)

Journal: bioRxiv

Article Title: Aging-enhanced accumulation of fibroblasts excludes oligodendrocytes in demyelinated lesions

doi: 10.1101/2025.07.27.667059

Figure Lengend Snippet: Immunofluorescence images of day 7 LPC lesions labeled with: (a) DAPI, MBP, NFH and GFAP in wildtype mice; (b) DAPI, Iba1 and TdTomato (TdTom) in PDGFRβ-TdTom mice; (c) DAPI, TdTom and PDGFRβ in PDGFRβ-TdTom mice; (d) DAPI, PDGFRβ, COL1A1 and SMA in wildtype mice. Scale bar = 100 μm, inset = 25 μm. e-j. Graphs comparing day 7 and day 21 (e, h) lesion volume, (f, i) TdTom positive volume, (g, j) proportion of LPC lesion that is TdTom positive. k. Representative images of day 7 and 21 LPC lesions labeling for PDGFRβ-TdTom and (left) periostin (POSTN), (middle) fibronectin (FN1), and (right) SMA. Scale bar = 100 μm. l-n. Graphs comparing the proportion of day 7 and 21 lesions positive for (l) POSTN, (m) FN1, (n) SMA. Data was acquired from two experiments; n=8 per experimental group. Significance is indicated as *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001; unpaired t -test, comparing day 7 and day 21 LPC lesions. Data presented as the mean.

Article Snippet: Antibodies used: anti-mouse myelin basic protein (MBP, BioLegend, PA1-10008), anti-mouse POSTN (R&D Systems, AF2955), anti-mouse αSMA-Cy3 (Millipore, C6198), anti-mouse FN1 (Millipore, AB2033), anti-CD45 (Thermofisher, MA5-17687), rabbit anti-mouse pan-laminin antibody (a gift from L. Sorokin, University of Münster, Münster, Germany; 1:1000), anti-mouse/human PDGFRβ (R&D Systems, AF1042), anti-mouse Iba1 (Wako, 019-19741), anti-mouse CD45-AF488 (Biolegend, 103122), Col1a1 (Invitrogen, PA1-26204), anti-mouse GFAP (Biolegend, PCK-591P), anti-mouse NFH (RPCA-NF-H), anti-mouse PDGFRα (R&D Systems, AF1062), anti-mouse Olig2 (Millipore, ab9610), GFP (Aveslab, GFP-1020), anti-mouse sulfatide O4 (R&D Systems, MAB1326), anti-mouse Arg1 (BioLegend, 678802), and anti-mouse MHCII (Thermofisher, 13-5321-82).

Techniques: Immunofluorescence, Labeling

Journal: Frontiers in Immunology

Article Title: Inflammation-driven periostin in ECRS has contrasting effects on tissue structural integrity and osteitis

doi: 10.3389/fimmu.2025.1596746

Figure Lengend Snippet: Correlation between periostin expression and osteitis severity in patients with CRS. Patients were categorized into non-ECRS and ECRS groups based on their JECSREC scores, with ECRS confirmed by eosinophil counts exceeding 70 cells per high-power field (HPF). (A) Representative photomicrographs of immunohistochemical staining for periostin (brown) and Sirius Red staining for eosinophil infiltration (pink) in the human nasal mucosa (scale bars = 100 μm). (B) Quantitative analysis of periostin levels in the nasal ethmoid mucosa, measured using ELISAs, in the control (n = 3), non-ECRS (n = 13), and ECRS groups (n = 10). (C) Osteitis severity was evaluated using the Global Osteitis Scoring Scale based on computed tomography images in the same group. (D) Correlation between periostin concentration and osteitis score in all groups. A moderate positive correlation was observed (r = 0.4171, P = 0.034; Spearman’s rank correlation). Results represent the mean ± SEM. Exact P values are shown in the graph. Statistical significance was assessed using the Kruskal–Wallis test for (B) and (C) . CRS, chronic rhinosinusitis; ELISA, enzyme-linked immunosorbent assay; ECRS, eosinophilic chronic rhinosinusitis; HPF, high-power field; JECSREC, Japanese Epidemiological Survey of Refractory Eosinophilic Chronic Rhinosinusitis; non-ECRS, non-eosinophilic chronic rhinosinusitis; SEM, standard error of the mean.

Article Snippet: Blocking was performed using 5% bovine serum albumin for 1 h. The sections were then incubated overnight at 4°C with primary antibodies against fibronectin (Abcam, ab2413), periostin (R&D Systems, AF2955), osteopontin (Abcam, ab218237), and α-SMA (Abcam, ab5694; all 1:200).

Techniques: Expressing, Immunohistochemical staining, Staining, Control, Computed Tomography, Concentration Assay, Enzyme-linked Immunosorbent Assay

Journal: Frontiers in Immunology

Article Title: Inflammation-driven periostin in ECRS has contrasting effects on tissue structural integrity and osteitis

doi: 10.3389/fimmu.2025.1596746

Figure Lengend Snippet: Th2 cytokine-induced periostin expression in HNECs and HNFs. HNECs and HNFs were treated with 5 ng/mL of IL-4, IL-13, IL-1β, TNF-α, and IFN-α and subsequently harvested on days 0, 1, and 2. (A) Western blot analysis of periostin expression levels in HNECs and HNFs treated with each cytokine for 0–2 days. β-actin used as a loading control for normalization. Band intensities were quantified from three independent experiments, normalized to the control group (set as 1), and expressed as relative intensities. (B) Quantitative RT-PCR analysis of POSTN mRNA levels in HNECs and HNFs following cytokine treatment, with normalized expression values on day 0. (C) Measurement of periostin concentrations in the culture supernatants by ELISA. (D) Comparative analysis of periostin secretion per equivalent number of cells quantified by ELISA. All experiments were independently repeated three times. Results represent the mean ± SEM. Exact P values are shown in the graph. Statistical analysis was performed using two-way ANOVA with Dunnett’s multiple comparisons test for (A–C) and Sidak’s multiple comparisons test for (D) . ELISA, enzyme-linked immunosorbent assay; HNEC, human nasal epithelial cell; HNF, human nasal fibroblast; IFN, interferon; IL, interleukin; POSTN , periostin; SEM, standard error of the mean; TNF, tumor necrosis factor.

Article Snippet: Blocking was performed using 5% bovine serum albumin for 1 h. The sections were then incubated overnight at 4°C with primary antibodies against fibronectin (Abcam, ab2413), periostin (R&D Systems, AF2955), osteopontin (Abcam, ab218237), and α-SMA (Abcam, ab5694; all 1:200).

Techniques: Expressing, Western Blot, Control, Quantitative RT-PCR, Enzyme-linked Immunosorbent Assay

Journal: Frontiers in Immunology

Article Title: Inflammation-driven periostin in ECRS has contrasting effects on tissue structural integrity and osteitis

doi: 10.3389/fimmu.2025.1596746

Figure Lengend Snippet: Synergistic effects of co-cultured HNECs and HNFs on periostin secretion. (A) Schematic representation of the non-contact co-culture system using Transwell inserts. After differentiation of HNECs by air-liquid interface (ALI) culture on Transwell inserts, the inserts were placed into plates containing HNFs in the bottom chamber. (B) Western blot analysis of periostin expression levels in separately cultured HNECs, separately cultured HNFs, and non-contact co-cultured HNECs and HNFs over a period of 0–2 days. β-actin was used as a loading control for normalization. Band intensities were quantified from three independent experiments, normalized to the control group (set as 1), and expressed as relative intensities. (C) Quantitative RT-PCR analysis of POSTN mRNA levels in HNECs and HNFs under different culture conditions, with values normalized to the level on day 0. (D) Comparative analysis of periostin levels in supernatants collected with PBS from the apical side of the Transwell in HNECs and co-cultured HNECs, quantified by ELISA. (E) Comparative analysis of periostin levels in supernatants collected from the basolateral side of the Transwell in HNECs, HNFs, and the co-culture condition, quantified by ELISA. All experiments were independently repeated three times. Results represent the mean ± SEM. Exact P values are shown in the graph. Statistical analysis was performed using two-way ANOVA with Sidak’s multiple comparisons test (B–E) . ALI, air-liquid interface; ELISA, enzyme-linked immunosorbent assay; HNEC, human nasal epithelial cell; HNF, human nasal fibroblast; SEM, standard error of the mean.

Article Snippet: Blocking was performed using 5% bovine serum albumin for 1 h. The sections were then incubated overnight at 4°C with primary antibodies against fibronectin (Abcam, ab2413), periostin (R&D Systems, AF2955), osteopontin (Abcam, ab218237), and α-SMA (Abcam, ab5694; all 1:200).

Techniques: Cell Culture, Co-Culture Assay, Western Blot, Expressing, Control, Quantitative RT-PCR, Enzyme-linked Immunosorbent Assay

Journal: Frontiers in Immunology

Article Title: Inflammation-driven periostin in ECRS has contrasting effects on tissue structural integrity and osteitis

doi: 10.3389/fimmu.2025.1596746

Figure Lengend Snippet: Periostin secreted by IL-4 treated nasal fibroblasts mediates the osteogenesis of MG63 cells. (A) Schematic overview of the conditioned medium preparation and MG63 osteogenic differentiation assay. HNFs were stimulated with IL-4 (5 ng/mL) for 2 days, and the conditioned medium was concentrated using a 30 kDa filter. Periostin concentrations were quantified by ELISA, and the periostin-containing medium was designated as CM-HNF. MG63 cells were cultured in osteogenic medium supplemented with CM-HNF, CM-HNF pretreated with a periostin-neutralizing antibody (CM-HNF + Ab), or recombinant human periostin (rhPOSTN, 200 ng/mL) for 10 days to induce osteogenic differentiation. (B) Representative photomicrographs of ALP staining showing osteoblast differentiation of MG63 cells induced by the osteogenic medium (scale bars = 500 μm). (C) Quantification of ALP-positive cells by counting the number of cells per high-power field in each well (n = 6, pooled from two independent experiments with three wells each). Statistical analysis was performed using one-way ANOVA with Tukey’s multiple comparisons test. (D) Quantitative RT-PCR analysis of ALP and OCN mRNA expression in MG63 cells under each treatment condition, with values normalized to those of the control group. Experiments were independently repeated three times. Statistical analysis was performed using two-way ANOVA with Tukey’s multiple comparisons test. Results represent the mean ± SEM. Exact P values are shown in the graph. Ab, antibody; ALP, alkaline phosphatase; rhPOSTN, recombinant human periostin; ELISA, enzyme-linked immunosorbent assay; HNF, human nasal fibroblast; IL, interleukin; OCN, osteocalcin; SEM, standard error of the mean.

Article Snippet: Blocking was performed using 5% bovine serum albumin for 1 h. The sections were then incubated overnight at 4°C with primary antibodies against fibronectin (Abcam, ab2413), periostin (R&D Systems, AF2955), osteopontin (Abcam, ab218237), and α-SMA (Abcam, ab5694; all 1:200).

Techniques: Differentiation Assay, Enzyme-linked Immunosorbent Assay, Cell Culture, Recombinant, Staining, Quantitative RT-PCR, Expressing, Control

Journal: Frontiers in Immunology

Article Title: Inflammation-driven periostin in ECRS has contrasting effects on tissue structural integrity and osteitis

doi: 10.3389/fimmu.2025.1596746

Figure Lengend Snippet: Effect of periostin deficiency on osteogenesis in a mouse model of ECRS. (A) Protocols for the development of eosinophilic rhinosinusitis in WT and KO mice. The ECRS condition was induced by exposing the mice to multiple allergens that stimulate Th2-type inflammation, mimicking clinical ECRS. (B) Representative photomicrographs of immunohistochemical RUNX2 staining in the ventral meatus area of the nasal cavity, showing RUNX2-positive cells along the nasal bone outline (scale bars = 50 μm). (C) Quantification of RUNX2-positive cells in the subepithelium of the nasal bone, with cell counts performed from sections of the ventral meatus area (n = 4). (D) Measurement of bone thickness in the ventral meatus of the nasal cavity across groups. Bone thickness was measured at five different locations in each mouse (n = 4). (E) Representative immunofluorescence images of periostin (green), osteopontin (red), and nuclei (blue) in the ventral meatus area of the nasal cavity (scale bars = 50 μm). Colocalization of periostin and osteopontin appeared as yellow signals resulting from the overlay of green and red fluorescence. (F) Quantification of periostin and osteopontin-positive areas and their colocalization in the nasal bone. Immunofluorescence analysis was performed on nasal tissue sections (n = 4), with paired regions (left and right) sampled from the same anatomical site of the ventral meatus. The positive signal areas for periostin and osteopontin, as well as the areas colocalized with periostin, were quantified using ImageJ. All values were expressed as a percentage of the total nasal bone area. Results represent the mean ± SEM. Exact P values are shown in the graph. Statistical analysis was performed by Kruskal–Wallis test with Dunn’s multiple comparisons test (C, D, F) . ECRS, eosinophilic chronic rhinosinusitis; KO, periostin knockout; PBS, phosphate-buffered saline; RUNX2, RUNX family transcription factor 2; SEM, standard error or the mean; WT, wild-type.

Article Snippet: Blocking was performed using 5% bovine serum albumin for 1 h. The sections were then incubated overnight at 4°C with primary antibodies against fibronectin (Abcam, ab2413), periostin (R&D Systems, AF2955), osteopontin (Abcam, ab218237), and α-SMA (Abcam, ab5694; all 1:200).

Techniques: Immunohistochemical staining, Staining, Immunofluorescence, Fluorescence, Knock-Out, Saline

Journal: Frontiers in Immunology

Article Title: Inflammation-driven periostin in ECRS has contrasting effects on tissue structural integrity and osteitis

doi: 10.3389/fimmu.2025.1596746

Figure Lengend Snippet: Effect of periostin deficiency on the maintenance of tissue structure in ECRS model mice. (A) Representative photomicrographs of Masson’s trichrome staining showing collagen (blue) deposition in the subepithelium of the nasal mucosa in different groups (scale bars = 50 μm). Representative photomicrographs showing epithelial collapse in the nasal cavity of the WT and KO ECRS groups, indicating a localized breakdown in the epithelial layer (scale bars = 50 μm). (B) Number of epithelial collapses in the nasal cavity. (C) Representative immunofluorescence images of periostin (green), α-SMA (red), and nuclei (blue) in the ventral meatus of the nasal cavity. Colocalization of periostin and α-SMA appeared as yellow signals resulting from the overlay of green and red fluorescence (scale bars = 50 μm). (D) Quantification of periostin, fibronectin-positive, and colocalization areas in the nasal subepithelium. (E) Representative immunofluorescence images of periostin (green), fibronectin (red), EMBP (white), and nuclei (blue) in the same anatomical region. (F) Quantification of periostin, fibronectin-positive, and colocalization areas in the nasal subepithelium. Immunofluorescence analysis was performed on nasal tissue sections (n = 4), with paired regions (left and right) sampled from the same anatomical site of the ventral meatus. The positive signal areas for periostin, α-SMA, and fibronectin, as well as the areas colocalized with periostin, were quantified using ImageJ (D, F) . All values were expressed as a percentage of the total nasal subepithelium. Results represent the mean ± SEM. Exact P values are shown in the graph. Statistical analysis was performed by Kruskal–Wallis test with Dunn’s multiple comparisons test (B, D, F) . α-SMA, alpha-smooth muscle actin; ECRS, eosinophilic chronic rhinosinusitis; KO, periostin knockout; PBS, phosphate-buffered saline; SEM, standard error or the mean; WT, wild-type.

Article Snippet: Blocking was performed using 5% bovine serum albumin for 1 h. The sections were then incubated overnight at 4°C with primary antibodies against fibronectin (Abcam, ab2413), periostin (R&D Systems, AF2955), osteopontin (Abcam, ab218237), and α-SMA (Abcam, ab5694; all 1:200).

Techniques: Staining, Immunofluorescence, Fluorescence, Knock-Out, Saline

Journal: The Journal of Clinical Investigation

Article Title: PIEZO1 mediates periostin + myofibroblast activation and pulmonary fibrosis in mice

doi: 10.1172/JCI184158

Figure Lengend Snippet: ( A ) Schematic representation showing the genetic strategy for the generation of the Postn-CreER T2 ; mT/mG mice for lineage tracing. ( B ) Schematic diagram of the experimental design. Postn-CreER T2 ; mT/mG mice were challenged to a single intratracheal inhalation of 1 U/kg BLM followed by injection with tamoxifen on 5 consecutive days. The control mice ( Postn-CreER T2 ; mT/mG ) without BLM injury (–BLM) were induced by 5 consecutive days tamoxifen and analyzed at day 21 after tamoxifen injection. ( C ) Representative images of Postn + lineage GFP + cells in Postn-CreER T2 ; mT/mG mice after tamoxifen treatment and BLM challenge. Immunofluorescent staining showing tdTomato and GFP single channels in addition to a merged image. Scale bar: 20 μm. ( D and E ) Western blot analysis ( D ) and quantification ( E ) of the indicated protein levels in the lung of mice at 21 days post bleomycin injury (d.p.i.). n = 4–8. ( F ) Representative images to identify the Postn + cells in the mouse lung sections. Antibodies against the stromal markers (α-SMA, Desmin, Pdgfr-β, and Pdgfr-α), endothelial marker CD31, and alveolar type 1 (AT1) cell marker Rage were costained in the lung sections of Postn-CreER T2 ; mT/mG mice at 21 d.p.i. Scale bar: 20 μm. ( G ) Quantification of colocalization of the GFP + cells with α-SMA + , Desmin + , Pdgfr-β + , Pdgfr-α + , CD31 + , or Rage + cells in the lung sections of Postn-CreER T2 ; mT/mG mice at 21 d.p.i. n = 10. ( H ) Representative images of Postn expression, which were costained with α-SMA in the lung sections of patients with IPF. Scale bar: 20 μm. n = 8. Shown are mean values ± SEM. Statistical significance was determined by unpaired Student’s t test or the Mann-Whitney U test. *** P < 0.001.

Article Snippet: After blocking in 5% skim milk for 1 hour, blots were probed using the below primary antibodies: Fibronectin1 (1:1000, Cat F3648, Sigma-Aldrich), Postn (1:1000, Cat AF2955, R&D Systems), α-SMA (1:1000, Cat AF1032, Affinity), GFP (1:1000, Cat 66002-1-IG, ProteinTech), YAP/TAZ (1:1000, Cat 8418S, CST), Phospho-YAP (1:1000, Cat 13008S, CST), Ki67 (1:1000, Cat ab16667, Abcam), P21 (1:1000, Cat 10355-1-AP, ProteinTech), and γH2AX (1:1000, Cat ab81299, Abcam) was used as the loading control.

Techniques: Injection, Control, Staining, Western Blot, Marker, Expressing, MANN-WHITNEY

Journal: The Journal of Clinical Investigation

Article Title: PIEZO1 mediates periostin + myofibroblast activation and pulmonary fibrosis in mice

doi: 10.1172/JCI184158

Figure Lengend Snippet: ( A ) Schematic representation showing the genetic strategy for generation of the Postn-CreER T2 ; R26-iDTR mice for ablation of Postn + cells. ( B ) Schematic diagram of the experimental design. Postn-CreER T2 ; R26-iDTR were challenged to a single intratracheal inhalation of BLM followed by injection with tamoxifen on 5 consecutive days. Then the mice were injected with DT or vehicle for 7 consecutive days. After 7 days, the control ( R26-iDTR ) and Postn-CreER T2 ; R26-iDTR mice were euthanized for subsequent analysis. ( C ) Real-time qPCR analysis of iDTR mRNA expression levels in the lung of Postn-CreER T2 ; R26-iDTR and control mice. n = 4–5. ( D and E ) Survival percentage ( D ) and body weight change ( E ) of Postn-CreER T2 ; R26-iDTR and control mice. n = 5. ( F ) ( left ) Representative images of H&E, Sirius Red, and Masson staining in the lung sections of Postn-CreER T2 ; R26-iDTR mice after Postn + cell ablation. ( right ) Quantification of the Ashcroft score, Sirius red fibrosis and Masson fibrosis area. (Scale bar: 1 mm, top; 100 μm, bottom). n = 5. ( G ) Representative images of Postn and α-SMA expression and quantification of α-SMA + and Postn + area in the lung sections of Postn-CreER T2 ; R26-iDTR mice after Postn + cells ablation. Scale bar:100 μm. n = 5. ( H ) Hydroxyproline content in the lung of ablated and control mice after BLM challenge. n = 5. ( I ) Real-time qPCR analysis of Acta2, Col1a1, Postn, Fn1, Col3a1, and Tgfb1 mRNA expression levels in the lungs of Postn-CreER T2 ; R26-iDTR mice after the Postn + cells ablation. n = 5. Shown are mean values ± SEM. Statistical significance was determined by the Mann-Whitney U test. ** P < 0.01.

Article Snippet: After blocking in 5% skim milk for 1 hour, blots were probed using the below primary antibodies: Fibronectin1 (1:1000, Cat F3648, Sigma-Aldrich), Postn (1:1000, Cat AF2955, R&D Systems), α-SMA (1:1000, Cat AF1032, Affinity), GFP (1:1000, Cat 66002-1-IG, ProteinTech), YAP/TAZ (1:1000, Cat 8418S, CST), Phospho-YAP (1:1000, Cat 13008S, CST), Ki67 (1:1000, Cat ab16667, Abcam), P21 (1:1000, Cat 10355-1-AP, ProteinTech), and γH2AX (1:1000, Cat ab81299, Abcam) was used as the loading control.

Techniques: Injection, Control, Expressing, Staining, MANN-WHITNEY

Journal: The Journal of Clinical Investigation

Article Title: PIEZO1 mediates periostin + myofibroblast activation and pulmonary fibrosis in mice

doi: 10.1172/JCI184158

Figure Lengend Snippet: ( A ) Schematic representation showing the genetic strategy for the generation of the Piezo1-CreER; R26-GFP mice. ( B ) Schematic diagram of the experimental design. Piezo1-CreER; R26-GFP mice were injected with tamoxifen for 5 consecutive days followed by a single intratracheal inhalation of bleomycin (+BLM) or vehicle (–BLM). ( C ) Representative images of PIEZO1 expression in the Postn + or α-SMA + area before and after BLM challenge of Piezo1-CreER;R26-GFP mice, respectively. Scale bar: 20 μm. ( D ) Quantification of GFP + in Postn + or α-SMA + cells. n = 6–7. ( E ) Representative images of PIEZO1 expression in the lung sections of patients with IPF. Scale bar: 20 μm. ( F ) Quantification of membrane tension before and after myofibroblast differentiation. n = 32. ( G ) Relative Piezo1 mRNA levels in myofibroblasts transfected with siCtrl or with siPiezo1. n = 6. ( H ) Representative cell-attached patch clamp traces of stretch-activated currents in myofibroblasts transfected with siCtrl or with siPiezo1. The holding potential was –70 mV and the membrane was stretched by pulses of negative pressure with a 10 mm Hg increment. ( I ) Statistical analysis of 4–5 independent recordings. ( J ) Representative cell-attached patch clamp traces of stretch-activated currents in myofibroblasts exposed to GdCl 3 or PBS. The holding potential was –70 mV and the membrane was stretched by pulses of negative pressure with a 10 mm Hg increment. ( K ) Statistical analysis of 4 independent recordings. ( L ) Fluo-4–loaded myofibroblasts transfected with siCtrl or with siPiezo1 were exposed to 1 μM Yoda1, and [Ca 2+ ] i was determined as fluorescence intensity (RFU, relative fluorescence units); line indicates the addition of Yoda1. Bar diagrams show the AUC of the Ca 2+ transient. n = 18–45. Shown are mean values ± SEM. Statistical significance was determined by unpaired Student’s t test or the Mann-Whitney U test. ** P < 0.01; *** P < 0.001.

Article Snippet: After blocking in 5% skim milk for 1 hour, blots were probed using the below primary antibodies: Fibronectin1 (1:1000, Cat F3648, Sigma-Aldrich), Postn (1:1000, Cat AF2955, R&D Systems), α-SMA (1:1000, Cat AF1032, Affinity), GFP (1:1000, Cat 66002-1-IG, ProteinTech), YAP/TAZ (1:1000, Cat 8418S, CST), Phospho-YAP (1:1000, Cat 13008S, CST), Ki67 (1:1000, Cat ab16667, Abcam), P21 (1:1000, Cat 10355-1-AP, ProteinTech), and γH2AX (1:1000, Cat ab81299, Abcam) was used as the loading control.

Techniques: Injection, Expressing, Membrane, Transfection, Patch Clamp, Fluorescence, MANN-WHITNEY

Journal: The Journal of Clinical Investigation

Article Title: PIEZO1 mediates periostin + myofibroblast activation and pulmonary fibrosis in mice

doi: 10.1172/JCI184158

Figure Lengend Snippet: ( A ) Schematic representation showing the genetic strategy for the generation of the Postn-CreER T2 ;Piezo1 fl/fl mice ( Pn-Piezo1 - KO ). And schematic diagram of the experimental design. ( B ) Representative images of H&E, Sirius Red, and Masson staining in the lung sections of Pn-Piezo1-KO and control ( Piezo1 fl/fl ) mice after BLM challenge. (scale bar: 1 mm, top; 100 μm, bottom). ( C ) Quantification of the Ashcroft score, Sirius red, and Masson fibrosis area. n = 10–12. ( D ) Representative images of Postn and α-SMA expression in the lung sections of Pn-Piezo1 – KO and control mice after BLM challenge. Scale bar: 20 μm, n = 10–12. ( E ) Hydroxyproline content in the lung of Pn-Piezo1 – KO and control mice after BLM challenge. n = 7. ( F and G ) Western blot analysis ( F ) and quantification ( G ) of Fn1, Col1a1, Acta2, and Postn protein levels in lung homogenates of Pn-Piezo1 – KO and control mice at 21 d.p.i. n = 4. ( H ) Relative Acta2, Postn, Col1a1, Col3a1, Fn1, and Tgfb1 mRNA levels in the lung homogenates of Pn-Piezo1 – KO and control mice at 21 d.p.i. n = 5–7. ( I ) Lung resistance and dynamic compliance (Cdyn) analysis of Pn-Piezo1 – KO and control mice at 21 d.p.i. n = 4–7. ( J ) Representative images of H&E, Sirius Red, and Masson staining in the lung of C57BL/6J mice receiving BLM followed by GsMTx4 treatment. (Scale bar: 1 mm, top; 100 μm, bottom). ( K ) Quantification of the Ashcroft score, Sirius red fibrosis, and Masson fibrosis area. n = 8–10. ( L ) ( left ) Representative images of Postn and α-SMA expression in the lung sections of C57BL/6 mice receiving BLM followed by GsMTx4 treatment. Scale bar: 50 μm. ( right ) Quantification of α-SMA + and Postn + area in the lung of C57BL/6J mice receiving BLM followed by GsMTx4 treatment. n = 8. Shown are mean values ± SEM. Statistical significance was determined by unpaired Student’s t test or the Mann-Whitney U test. ** P < 0.01; *** P < 0.001.

Article Snippet: After blocking in 5% skim milk for 1 hour, blots were probed using the below primary antibodies: Fibronectin1 (1:1000, Cat F3648, Sigma-Aldrich), Postn (1:1000, Cat AF2955, R&D Systems), α-SMA (1:1000, Cat AF1032, Affinity), GFP (1:1000, Cat 66002-1-IG, ProteinTech), YAP/TAZ (1:1000, Cat 8418S, CST), Phospho-YAP (1:1000, Cat 13008S, CST), Ki67 (1:1000, Cat ab16667, Abcam), P21 (1:1000, Cat 10355-1-AP, ProteinTech), and γH2AX (1:1000, Cat ab81299, Abcam) was used as the loading control.

Techniques: Staining, Control, Expressing, Western Blot, MANN-WHITNEY

Journal: The Journal of Clinical Investigation

Article Title: PIEZO1 mediates periostin + myofibroblast activation and pulmonary fibrosis in mice

doi: 10.1172/JCI184158

Figure Lengend Snippet: ( A ) Representative images ( left ) and quantification ( right ) of YAP/TAZ expression in the Postn + cells in the lung sections of Pn-Piezo1 - KO and control ( Piezo1 fl/fl ) mice after BLM challenge. High magnification images show nuclear (arrowheads) and extranuclear (arrows) YAP/TAZ. Percentage of nuclear YAP/TAZ in GFP-positive cells was quantified. Scale bar: 20 μm. n = 6–8. ( B ) Real-time qPCR analysis of Lats2 , Cyr61 , and AmotL2 mRNA expression levels in the lung homogenates of Pn-Piezo1 - KO and control mice at 21 d.p.i. n = 5–7. ( C ) Schematic representation showing the genetic strategy for the generation of the Postn-CreER T2 ;Yap fl/fl ;Taz fl/fl mice ( Pn-Yap/Taz - dKO ). ( D ) Representative images ( left ) and quantification ( right ) of YAP/TAZ expression in the Postn + cells in the lung sections of Pn-Yap/Taz -dKO and control ( Yap fl/fl ;Taz fl/fl ) mice after BLM challenge. High magnification images show nuclear (arrowheads) and extranuclear (arrows) YAP/TAZ. Scale bar: 20 μm. n = 6–8. ( E ) ( left ) Representative images of H&E, Sirius Red, and Masson staining in the lung sections of Pn-Yap/Taz - dKO and control mice after BLM challenge. (Scale bar: 1 mm, top; 100 μm, bottom). ( right ) Quantification of the Ashcroft score, Sirius red fibrosis, and Masson fibrosis area. n = 8–13. ( F ) Real-time qPCR analysis of Acta2, Col1a1 , Postn , Col3a1, Fn1, and Tgfb1 mRNA expression levels in the lung of Pn-Yap/Taz - dKO and control mice after BLM challenge. n = 6–10. ( G ) Hydroxyproline content in the lungs of Pn-Yap/Taz - dKO and control mice after BLM challenge. n = 7–9. ( H ) Lung resistance and Cdyn analysis of Pn-Yap/Taz - dKO and control mice at 21 d.p.i. n = 6–8. Shown are mean values ± SEM. Statistical significance was determined by unpaired Student’s t test or the Mann-Whitney U test. ** P < 0.01; *** P < 0.001.

Article Snippet: After blocking in 5% skim milk for 1 hour, blots were probed using the below primary antibodies: Fibronectin1 (1:1000, Cat F3648, Sigma-Aldrich), Postn (1:1000, Cat AF2955, R&D Systems), α-SMA (1:1000, Cat AF1032, Affinity), GFP (1:1000, Cat 66002-1-IG, ProteinTech), YAP/TAZ (1:1000, Cat 8418S, CST), Phospho-YAP (1:1000, Cat 13008S, CST), Ki67 (1:1000, Cat ab16667, Abcam), P21 (1:1000, Cat 10355-1-AP, ProteinTech), and γH2AX (1:1000, Cat ab81299, Abcam) was used as the loading control.

Techniques: Expressing, Control, Staining, MANN-WHITNEY

Journal: The Journal of Clinical Investigation

Article Title: PIEZO1 mediates periostin + myofibroblast activation and pulmonary fibrosis in mice

doi: 10.1172/JCI184158

Figure Lengend Snippet: ( A ) Schematic representation showing the genetic strategy for generation of the Postn-CreER T2 ; Piezo1 fl/fl ; mT/mG mice ( Pn-Piezo1 - mT/mG ). ( B – D ) Representative images of Ki67 ( B ), P21 ( C ), and γ-H2A.X ( D ) expression in the GFP + cells in the lung sections of Pn-Piezo1 – KO , mT/mG, and Pn-mT/mG mice after BLM challenge. Scale bar: 20 μm. ( E ) Quantification of Ki67 + , P21 + , and γ-H2A.X + in Postn + myofibroblasts in the lung sections of Pn-Piezo1 – KO , mT/mG, and Pn-mT/mG mice after BLM challenge. n = 6. The double-positive cells were defined by a GFP membrane around the Ki67, P21, or γ-H2A.X-positive nuclear. ( F – H ) Representative images of Ki67 ( F ), P21 ( G ), and γ-H2A.X ( H ) expression in the GFP + cells in the lung sections of Pn-Yap/Taz - dKO and control mice after BLM challenge. Scale bar: 20 μm. ( I ) Quantification of Ki67 + , P21 + , and γ-H2A.X + in Postn + myofibroblast in the lung sections of Pn-Yap/Taz - dKO and control mice after BLM challenge. n = 5–11. ( J and K ) Quantification of TUNEL + myofibroblast percentage in the lung sections of Pn-Piezo1 - KO , mT/mG ( J , n = 14–17) and Pn-Yap/Taz - dKO ( K , n = 17–28) mice after BLM challenge. Shown are mean values ± SEM. Statistical significance was determined by unpaired Student’s t test or the Mann-Whitney U test. ** P < 0.01; *** P < 0.001.

Article Snippet: After blocking in 5% skim milk for 1 hour, blots were probed using the below primary antibodies: Fibronectin1 (1:1000, Cat F3648, Sigma-Aldrich), Postn (1:1000, Cat AF2955, R&D Systems), α-SMA (1:1000, Cat AF1032, Affinity), GFP (1:1000, Cat 66002-1-IG, ProteinTech), YAP/TAZ (1:1000, Cat 8418S, CST), Phospho-YAP (1:1000, Cat 13008S, CST), Ki67 (1:1000, Cat ab16667, Abcam), P21 (1:1000, Cat 10355-1-AP, ProteinTech), and γH2AX (1:1000, Cat ab81299, Abcam) was used as the loading control.

Techniques: Expressing, Membrane, Control, TUNEL Assay, MANN-WHITNEY