Journal: Evidence-based Complementary and Alternative Medicine : eCAM

Article Title: Triptolide Transcriptionally Represses HER2 in Ovarian Cancer Cells by Targeting NF- κ B

doi: 10.1155/2012/350239

Figure Lengend Snippet: Downregulation of HER2 expression by TPL in oral, breast, and ovarian cancer cell lines. (a) KB and OEC-M1 human oral cancer cell lines; (b) BT-474, MCF-7/HER, and MCF-7 human breast cancer cell lines; and (c) SKOV-3, OVCAR-3, and TOV-21G human ovarian cancer cell lines were treated with 50 nM of TPL or vehicle (0.01% DMSO) control for 24 h. Cell lysates were immunoblotted with antibodies specific for HER2/neu or actin. SKOV-3 cells were treated with the indicated dose of TPL for 72 h. Morphological variations were recorded (d), and growth inhibition by TPL was quantified (e). The experiment was repeated three times.

Article Snippet: The promoter fragments were cloned into the pGL4 vector (Promega) and sequenced, resulting in the promoter reporter vectors pGL4-HER2-F1-Luc, pGL4-HER2-F2-Luc, pGL4-HER2-F3-Luc, and pGL4-HER2-F4-Luc, respectively.

Techniques: Expressing, Inhibition

Journal: Evidence-based Complementary and Alternative Medicine : eCAM

Article Title: Triptolide Transcriptionally Represses HER2 in Ovarian Cancer Cells by Targeting NF- κ B

doi: 10.1155/2012/350239

Figure Lengend Snippet: TPL downregulates HER2 expression and PI3K/Akt activity in SKOV-3 cells. (a) SKOV-3 cells were treated with various doses of TPL or PBS for 48 h (left panel) and 50 nM of TPL for the indicated time (right panel). HER2 and actin protein levels were determined by western blotting. Densitometric analysis of the percentage of decrease was determined using Adobe Photoshop software (b). HER2 expression was downregulated by TPL in SKOV-3 cell line in a dose- and time-dependent manner. (c) Downregulation of HER2 and phospho-PI3K/Akt by TPL. SKOV-3 cells were treated with the indicated concentration of TPL for 48 h. Western blot analysis of cell lysates was performed with antibodies recognizing HER2, phospho-HER2-Y1248 (p-HER2), p85 PI3K, phospho-Y p85 PI3K (p-PI3K), Akt, phospho-Akt-S473 (p-Akt), extracellular signal-regulated kinase (ERK), phospho-extracellular signal-regulated kinase-T202/Y204 (p-ERK), and actin. The densitometric analysis was performed as previously mentioned and is shown in (d). The data are presented as the mean ± standard error from three independent experiments. * P < 0.05 and ** P < 0.01 versus the vehicle-treated control group.

Article Snippet: The promoter fragments were cloned into the pGL4 vector (Promega) and sequenced, resulting in the promoter reporter vectors pGL4-HER2-F1-Luc, pGL4-HER2-F2-Luc, pGL4-HER2-F3-Luc, and pGL4-HER2-F4-Luc, respectively.

Techniques: Expressing, Activity Assay, Western Blot, Software, Concentration Assay

Journal: Evidence-based Complementary and Alternative Medicine : eCAM

Article Title: Triptolide Transcriptionally Represses HER2 in Ovarian Cancer Cells by Targeting NF- κ B

doi: 10.1155/2012/350239

Figure Lengend Snippet: Transcriptional repression of HER2 by TPL. (a) SKOV-3 cells were treated with 50 nM of TPL and harvested at the indicated time. The quantitative PCR was performed as described in . (b) TPL inhibits neu promoter activity. For the luciferase assay, NIH3T3 cells were cotransfected with neu promoter luciferase (0.8 μ g) and pCMV- β -gal (0.2 μ g) plasmid DNA for 6 h and then treated with various concentrations of TPL for 24 h. The activity of luciferase in relative light units (RLUs) was normalized against β -gal activity. (c) The pGL4-HER2-F1-Luc through pGL4-HER2-F4-Luc constructs were transfected into NIH3T3 cells for 6 h, and then 50 nM TPL was added for 24 h. The RLUs were determined as above. TPL downregulated reporter expression at the HER2 F4 (from −207 to −103) region.

Article Snippet: The promoter fragments were cloned into the pGL4 vector (Promega) and sequenced, resulting in the promoter reporter vectors pGL4-HER2-F1-Luc, pGL4-HER2-F2-Luc, pGL4-HER2-F3-Luc, and pGL4-HER2-F4-Luc, respectively.

Techniques: Real-time Polymerase Chain Reaction, Activity Assay, Luciferase, Plasmid Preparation, Construct, Transfection, Expressing

Journal: Evidence-based Complementary and Alternative Medicine : eCAM

Article Title: Triptolide Transcriptionally Represses HER2 in Ovarian Cancer Cells by Targeting NF- κ B

doi: 10.1155/2012/350239

Figure Lengend Snippet: NF- κ B prevents the reduction of HER2 expression caused by treatment with TPL. NIH3T3 cells were transfected with NF- κ B-p65 or -p50 (2 μ g) or vector only. After 6 h, TPL (100 nM) or vehicle was added to the medium for 24 h. Luciferase assays were performed to detect HER2-F1 (a) and HER2-F4 (b) promoter activities. (c) SKOV-3 (d), TOV-21G (e), and OVCAR-3 cells were transfected with NF- κ B-p65 or -p50 (2 μ g) or vector only for 24 h and then treated with TPL or vehicle for another 24 h. Cell lysates were immunoblotted with antibodies specific for HER2/neu, p-HER2, NF- κ B-p65, NF- κ B-p50, or actin.

Article Snippet: The promoter fragments were cloned into the pGL4 vector (Promega) and sequenced, resulting in the promoter reporter vectors pGL4-HER2-F1-Luc, pGL4-HER2-F2-Luc, pGL4-HER2-F3-Luc, and pGL4-HER2-F4-Luc, respectively.

Techniques: Expressing, Transfection, Plasmid Preparation, Luciferase

Journal: Evidence-based Complementary and Alternative Medicine : eCAM

Article Title: Triptolide Transcriptionally Represses HER2 in Ovarian Cancer Cells by Targeting NF- κ B

doi: 10.1155/2012/350239

Figure Lengend Snippet: A nude mouse model showing inhibition of SKOV-3 xenograft tumor growth as well as HER2 and Ki-67 expression by TPL. (a) Daily treatment with TPL (0.15 mg/kg) after SKOV-3 tumor xenograft transplantation significantly reduced tumor size compared with vehicle. (b) Body weight in mice was not significantly different between the TPL-treated and PBS-treated groups. (c) Downregulation of HER2 and (d) Ki-67 expression by TPL in SKOV-3-induced xenograft solid tumors in nude mice. The IHC analyses of SKOV-3-xenografted tumors were taken from the inoculated nude mice. TPL significantly reduced HER2 expression (brown color) in tumor sections from HercepTest scores of 3+ to 2+ ( n = 5) (c). In addition, TPL-treated mice had significantly less Ki-67 protein (red color) than vehicle controls (d) ( n = 4).

Article Snippet: The promoter fragments were cloned into the pGL4 vector (Promega) and sequenced, resulting in the promoter reporter vectors pGL4-HER2-F1-Luc, pGL4-HER2-F2-Luc, pGL4-HER2-F3-Luc, and pGL4-HER2-F4-Luc, respectively.

Techniques: Inhibition, Expressing, Transplantation Assay

Journal: Evidence-based Complementary and Alternative Medicine : eCAM

Article Title: Triptolide Transcriptionally Represses HER2 in Ovarian Cancer Cells by Targeting NF- κ B

doi: 10.1155/2012/350239

Figure Lengend Snippet: A schematic model demonstrating the effects of TPL on the regulation of NF- κ B and HER2. TPL inhibits tumor proliferation via downregulation of NF- κ B and HER2.

Article Snippet: The promoter fragments were cloned into the pGL4 vector (Promega) and sequenced, resulting in the promoter reporter vectors pGL4-HER2-F1-Luc, pGL4-HER2-F2-Luc, pGL4-HER2-F3-Luc, and pGL4-HER2-F4-Luc, respectively.

Techniques:

Journal: The Journal of Experimental Medicine

Article Title: HER2 drives lung fibrosis by activating a metastatic cancer signature in invasive lung fibroblasts

doi: 10.1084/jem.20220126

Figure Lengend Snippet: Cell-surface markers were used to identify invasive fibroblasts. (A) Violin plot comparisons of cell-surface marker gene expression in invasive and noninvasive fibroblasts. (B) Relative expression of cell-surface marker genes in bulk RNA-seq on invasive and noninvasive fibroblasts ( n = 9 per group). (C) Relative expression of SEMA7A , F3 , and ITGA6 in invasive and noninvasive fibroblasts by qRT-PCR ( n = 6 for SEMA7A and F3 , and n = 4 for ITGA6 ). (D) Cell-surface expression of SEMA7A, F3, and ITGA6 in invasive and noninvasive fibroblasts by flow cytometry. (E) Western blot analysis of SEMA7A, F3, and ITGA6 expression in invasive and noninvasive fibroblasts. GAPDH served as loading control. (F) Cell-surface expression of CD274, F3, and ITGA6 in SEMA7A negative and high fibroblasts by flow cytometry. (G) Heatmap of consistent genes in SEMA7A high and invasive, SEMA7A negative and noninvasive fibroblasts, respectively, by bulk RNA-seq. non, noninvasive fibroblasts; in, invasive fibroblasts; neg, negative. Three independent experiments were performed on fibroblasts from different patients (B and C). Data are the mean ± SEM. *, P < 0.05; ****, P < 0.0001 by student’s t test (B and C). Source data are available for this figure: .

Article Snippet: Directly conjugated antibodies used were anti-SEMA7A-PE and anti-SEMA7A-BV480 (clone KS-2; BD Biosciences), anti-CD274-PE (clone 29E.2A3; Biolegend), anti-F3-PE (clone HTF-1; BD Biosciences), anti-ITGA6-PE (clone GoH3; BD Biosciences), APC anti-CD340 (erbB2/HER2, Clone 24D2; Biosciences), FITC anti-human CD326 (EpCAM, Clone 9C4; Biosciences), PE/Cy7 anti-human CD31 (Clone WM59; Biosciences), and PE/Cy7 anti-human CD45 (Clone HI30; Biosciences).

Techniques: Marker, Expressing, RNA Sequencing Assay, Quantitative RT-PCR, Flow Cytometry, Western Blot

Journal: The Journal of Experimental Medicine

Article Title: HER2 drives lung fibrosis by activating a metastatic cancer signature in invasive lung fibroblasts

doi: 10.1084/jem.20220126

Figure Lengend Snippet: Specific cell-surface marker genes of invasive fibroblasts promoted invasion and fibrosis. (A) Cell sorting strategy of F3, SEMA7A, and ITGA6 negative and high fibroblasts for following experiments. (B and C) Relative mRNA levels (B; n = 4 per group) and total protein levels (C) of F3, SEMA7A, and ITGA6 expression in sorted F3, SEMA7A, and ITGA6 negative and high fibroblasts. (D and E) Representative images (D) and index (E; SEMA7A Neg/High migration/invasion, n = 4 per group; F3 Neg/High migration, n = 12 per group, invasion, n = 9 per group; ITGA6 Neg/High migration, n = 9 per group, invasion, n = 12 per group) of migration and invasion of SEMA7A, F3, and ITGA6 negative and high fibroblasts. (F) Cell adhesion of SEMA7A and ITGA6 high and negative fibroblasts was quantified ( n = 4 per group). (G) Overexpression of SEMA7A was confirmed by Western blotting. (H) Cell-surface expression of SEMA7A in SEMA7A overexpression and control fibroblasts. (I and J) Representative images (I) and index (J; n = 3 per group) of migration and invasion of SEMA7A overexpression fibroblasts. (K) Quantification of percentage of SEMA7A + fibroblasts by flow cytometry on freshly isolated normal and IPF human lungs (Normal, n = 4; IPF, n = 7). (L and M) Trichrome staining (L) and hydroxyproline (M; n = 10 per group) of mice lungs receiving SEMA7A high and negative fibroblasts, and age-matched mice were treated with culture medium only. Dash-boxed regions were shown at higher magnification. CTL, control; OE, overexpression. Scale bar: 1 mm (D and I), 500 μm (L). Three or four independent experiments were performed on fibroblasts from different patients (B, E, F, J, K, and M). Data are the mean ± SEM. *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001 by Student’s t test (B, E, F, and I–K) and two-way ANOVA (M). Source data are available for this figure: .

Article Snippet: Directly conjugated antibodies used were anti-SEMA7A-PE and anti-SEMA7A-BV480 (clone KS-2; BD Biosciences), anti-CD274-PE (clone 29E.2A3; Biolegend), anti-F3-PE (clone HTF-1; BD Biosciences), anti-ITGA6-PE (clone GoH3; BD Biosciences), APC anti-CD340 (erbB2/HER2, Clone 24D2; Biosciences), FITC anti-human CD326 (EpCAM, Clone 9C4; Biosciences), PE/Cy7 anti-human CD31 (Clone WM59; Biosciences), and PE/Cy7 anti-human CD45 (Clone HI30; Biosciences).

Techniques: Marker, FACS, Expressing, Migration, Over Expression, Western Blot, Flow Cytometry, Isolation, Staining

Journal: The Journal of Experimental Medicine

Article Title: HER2 drives lung fibrosis by activating a metastatic cancer signature in invasive lung fibroblasts

doi: 10.1084/jem.20220126

Figure Lengend Snippet: 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: .

Article Snippet: Directly conjugated antibodies used were anti-SEMA7A-PE and anti-SEMA7A-BV480 (clone KS-2; BD Biosciences), anti-CD274-PE (clone 29E.2A3; Biolegend), anti-F3-PE (clone HTF-1; BD Biosciences), anti-ITGA6-PE (clone GoH3; BD Biosciences), APC anti-CD340 (erbB2/HER2, Clone 24D2; Biosciences), FITC anti-human CD326 (EpCAM, Clone 9C4; Biosciences), PE/Cy7 anti-human CD31 (Clone WM59; Biosciences), and PE/Cy7 anti-human CD45 (Clone HI30; Biosciences).

Techniques: Quantitative RT-PCR, Western Blot, Expressing, Flow Cytometry

Journal: The Journal of Experimental Medicine

Article Title: HER2 drives lung fibrosis by activating a metastatic cancer signature in invasive lung fibroblasts

doi: 10.1084/jem.20220126

Figure Lengend Snippet: Cell-surface markers were used to identify invasive fibroblasts. (A) Violin plot comparisons of cell-surface marker gene expression in invasive and noninvasive fibroblasts. (B) Relative expression of cell-surface marker genes in bulk RNA-seq on invasive and noninvasive fibroblasts ( n = 9 per group). (C) Relative expression of SEMA7A , F3 , and ITGA6 in invasive and noninvasive fibroblasts by qRT-PCR ( n = 6 for SEMA7A and F3 , and n = 4 for ITGA6 ). (D) Cell-surface expression of SEMA7A, F3, and ITGA6 in invasive and noninvasive fibroblasts by flow cytometry. (E) Western blot analysis of SEMA7A, F3, and ITGA6 expression in invasive and noninvasive fibroblasts. GAPDH served as loading control. (F) Cell-surface expression of CD274, F3, and ITGA6 in SEMA7A negative and high fibroblasts by flow cytometry. (G) Heatmap of consistent genes in SEMA7A high and invasive, SEMA7A negative and noninvasive fibroblasts, respectively, by bulk RNA-seq. non, noninvasive fibroblasts; in, invasive fibroblasts; neg, negative. Three independent experiments were performed on fibroblasts from different patients (B and C). Data are the mean ± SEM. *, P < 0.05; ****, P < 0.0001 by student’s t test (B and C). Source data are available for this figure: .

Article Snippet: Directly conjugated antibodies used were anti-SEMA7A-PE and anti-SEMA7A-BV480 (clone KS-2; BD Biosciences), anti-CD274-PE (clone 29E.2A3; Biolegend), anti-F3-PE (clone HTF-1; BD Biosciences), anti-ITGA6-PE (clone GoH3; BD Biosciences), APC anti-CD340 (erbB2/HER2, Clone 24D2; Biosciences), FITC anti-human CD326 (EpCAM, Clone 9C4; Biosciences), PE/Cy7 anti-human CD31 (Clone WM59; Biosciences), and PE/Cy7 anti-human CD45 (Clone HI30; Biosciences).

Techniques: Marker, Expressing, RNA Sequencing Assay, Quantitative RT-PCR, Flow Cytometry, Western Blot

Journal: The Journal of Experimental Medicine

Article Title: HER2 drives lung fibrosis by activating a metastatic cancer signature in invasive lung fibroblasts

doi: 10.1084/jem.20220126

Figure Lengend Snippet: Specific cell-surface marker genes of invasive fibroblasts promoted invasion and fibrosis. (A) Cell sorting strategy of F3, SEMA7A, and ITGA6 negative and high fibroblasts for following experiments. (B and C) Relative mRNA levels (B; n = 4 per group) and total protein levels (C) of F3, SEMA7A, and ITGA6 expression in sorted F3, SEMA7A, and ITGA6 negative and high fibroblasts. (D and E) Representative images (D) and index (E; SEMA7A Neg/High migration/invasion, n = 4 per group; F3 Neg/High migration, n = 12 per group, invasion, n = 9 per group; ITGA6 Neg/High migration, n = 9 per group, invasion, n = 12 per group) of migration and invasion of SEMA7A, F3, and ITGA6 negative and high fibroblasts. (F) Cell adhesion of SEMA7A and ITGA6 high and negative fibroblasts was quantified ( n = 4 per group). (G) Overexpression of SEMA7A was confirmed by Western blotting. (H) Cell-surface expression of SEMA7A in SEMA7A overexpression and control fibroblasts. (I and J) Representative images (I) and index (J; n = 3 per group) of migration and invasion of SEMA7A overexpression fibroblasts. (K) Quantification of percentage of SEMA7A + fibroblasts by flow cytometry on freshly isolated normal and IPF human lungs (Normal, n = 4; IPF, n = 7). (L and M) Trichrome staining (L) and hydroxyproline (M; n = 10 per group) of mice lungs receiving SEMA7A high and negative fibroblasts, and age-matched mice were treated with culture medium only. Dash-boxed regions were shown at higher magnification. CTL, control; OE, overexpression. Scale bar: 1 mm (D and I), 500 μm (L). Three or four independent experiments were performed on fibroblasts from different patients (B, E, F, J, K, and M). Data are the mean ± SEM. *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001 by Student’s t test (B, E, F, and I–K) and two-way ANOVA (M). Source data are available for this figure: .

Article Snippet: Directly conjugated antibodies used were anti-SEMA7A-PE and anti-SEMA7A-BV480 (clone KS-2; BD Biosciences), anti-CD274-PE (clone 29E.2A3; Biolegend), anti-F3-PE (clone HTF-1; BD Biosciences), anti-ITGA6-PE (clone GoH3; BD Biosciences), APC anti-CD340 (erbB2/HER2, Clone 24D2; Biosciences), FITC anti-human CD326 (EpCAM, Clone 9C4; Biosciences), PE/Cy7 anti-human CD31 (Clone WM59; Biosciences), and PE/Cy7 anti-human CD45 (Clone HI30; Biosciences).

Techniques: Marker, FACS, Expressing, Migration, Over Expression, Western Blot, Flow Cytometry, Isolation, Staining

Journal: The Journal of Experimental Medicine

Article Title: HER2 drives lung fibrosis by activating a metastatic cancer signature in invasive lung fibroblasts

doi: 10.1084/jem.20220126

Figure Lengend Snippet: 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 −Log 10 (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.

Article Snippet: Directly conjugated antibodies used were anti-SEMA7A-PE and anti-SEMA7A-BV480 (clone KS-2; BD Biosciences), anti-CD274-PE (clone 29E.2A3; Biolegend), anti-F3-PE (clone HTF-1; BD Biosciences), anti-ITGA6-PE (clone GoH3; BD Biosciences), APC anti-CD340 (erbB2/HER2, Clone 24D2; Biosciences), FITC anti-human CD326 (EpCAM, Clone 9C4; Biosciences), PE/Cy7 anti-human CD31 (Clone WM59; Biosciences), and PE/Cy7 anti-human CD45 (Clone HI30; Biosciences).

Techniques: Flow Cytometry, Over Expression, Expressing, Western Blot, Activation Assay

Journal: The Journal of Experimental Medicine

Article Title: HER2 drives lung fibrosis by activating a metastatic cancer signature in invasive lung fibroblasts

doi: 10.1084/jem.20220126

Figure Lengend Snippet: Transcription factors regulated lung fibroblast invasion. (A–D) Knockdown of transcriptional factors was confirmed by qRT-PCR (A and B) and Western blotting (C and D). A, n = 9 for FOXF1 , n = 4 for CREBRF , TSC22D1, and KLF9 , n = 6 for MXI1 , n = 3 for NFE2L2 ; B, n = 4 for HMGA2 , n = 5 for DPF3 . (E) FOXF1 and SEMA7A expressions showed negative correlation in scRNA-seq by SeqGeq. (F and G) Relative mRNA levels of FOXF1 , SEMA7A , and collagen-related protein gene, ACTA2 and COL1A1 (F, n = 5 per group) and cell-surface expression of SEMA7A (G) after FOXF1 knockdown. (H–J) Representative images (H) and index quantification (I and J) of migration and invasion of fibroblasts after knockdown assay. I, FOXF1, CREBRF, TSC22D1, and MXI1, n = 6 for migration and invasion, KLF9, n = 3 for migration and invasion, NFE2L2, n = 3 for migration and n = 11 for invasion; J, HMGA2, n = 6 for migration and invasion, DPF3, n = 3 for migration and invasion. CTL, control; non, noninvasive; in, invasive. Three or four independent experiments were performed on fibroblasts from different patients (A, B, F, I, and J). Data are the mean ± SEM. Scale bar: 1 mm. *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001 by Student’s t test. Source data are available for this figure: .

Article Snippet: Directly conjugated antibodies used were anti-SEMA7A-PE and anti-SEMA7A-BV480 (clone KS-2; BD Biosciences), anti-CD274-PE (clone 29E.2A3; Biolegend), anti-F3-PE (clone HTF-1; BD Biosciences), anti-ITGA6-PE (clone GoH3; BD Biosciences), APC anti-CD340 (erbB2/HER2, Clone 24D2; Biosciences), FITC anti-human CD326 (EpCAM, Clone 9C4; Biosciences), PE/Cy7 anti-human CD31 (Clone WM59; Biosciences), and PE/Cy7 anti-human CD45 (Clone HI30; Biosciences).

Techniques: Quantitative RT-PCR, Western Blot, Expressing, Migration

Journal: The Journal of Experimental Medicine

Article Title: HER2 drives lung fibrosis by activating a metastatic cancer signature in invasive lung fibroblasts

doi: 10.1084/jem.20220126

Figure Lengend Snippet: Significantly activated ERBB2 (HER2) signaling pathway in invasive fibroblasts. (A and B) Dot plot visualization of the −Log 10 (FDR) (A) and bar plot visualization of the activation Z-score (B) of the top 30 activated and inhibited upstream regulators of invasive fibroblasts by IPA analysis. ERBB2 was the most activated regulators of invasive fibroblasts. ERBB2 was highlighted as the most activated regulator. (C) IPA analysis revealed the upstream regulators of metastatic lung adenocarcinoma cancer cells compared to primary cancer cells retrieved from GSE131907 . Most of the top activated/inhibited (listed in red/green texts, respectively) upstream regulators of invasive fibroblasts were these of metastatic cancer cells. (D) Pearson correlation analysis of activation z-score of shared upstream regulators ( n = 129) of invasive fibroblasts versus metastatic lung adenocarcinoma cancer cell. Linear regression analysis was performed and visualized in red line. (E) p-HER2, total HER2, and SEMA7A protein levels in sorted SEMA7A high and negative fibroblasts in nine IPF fibroblast lines were determined by Western blot. GAPDH served as loading control. (F) Quantification of the Western blot was used to determine the relative protein levels of p-HER2, total HER2, and SEMA7A in E ( n = 9 per group). (G and H) p-HER2 and total HER2 in sorted fibroblasts from three normal and four IPF lung were determined by Western blot and quantification was performed (H; n = 4). Neg, megative; P, proximal lung regions; D, distal lung regions. Two independent experiments were performed on fibroblasts from different patients (F and H). Data are the mean ± SEM. *, P < 0.05; **, P < 0.01; ****, P < 0.0001 by Student’s t test (F and H). Source data are available for this figure: .

Article Snippet: Directly conjugated antibodies used were anti-SEMA7A-PE and anti-SEMA7A-BV480 (clone KS-2; BD Biosciences), anti-CD274-PE (clone 29E.2A3; Biolegend), anti-F3-PE (clone HTF-1; BD Biosciences), anti-ITGA6-PE (clone GoH3; BD Biosciences), APC anti-CD340 (erbB2/HER2, Clone 24D2; Biosciences), FITC anti-human CD326 (EpCAM, Clone 9C4; Biosciences), PE/Cy7 anti-human CD31 (Clone WM59; Biosciences), and PE/Cy7 anti-human CD45 (Clone HI30; Biosciences).

Techniques: Activation Assay, Western Blot

Journal: The Journal of Experimental Medicine

Article Title: HER2 drives lung fibrosis by activating a metastatic cancer signature in invasive lung fibroblasts

doi: 10.1084/jem.20220126

Figure Lengend Snippet: HER2 signaling activation increased fibroblast invasion and fibrosis. (A) The expression of invasive and noninvasive specific genes in HER2 overexpression normal fibroblasts were detected by qRT-PCR ( n = 3 per group). (B) Heatmap of the differentially expressed genes of control and HER2 overexpressing normal human lung fibroblasts by bulk RNA-seq. (C) Volcano plot of the top differentially expressed genes between control and HER2 overexpressed normal human lung fibroblasts by bulk RNA-seq. Red dots indicated the genes at Fold_change >0.5 and black dots indicated the genes at Fold_change ≤0.5. (D) Relative expression of invasive and noninvasive specific genes in HER2 overexpression normal fibroblasts detected by bulk RNA-seq ( n = 5 per group). (E) Upregulated cell-surface expression of SEMA7A in HER2 overexpression normal lung fibroblasts was confirmed by flow cytometry analysis. (F and G) Western blotting confirmation of the expression of p-HER2, HER2, SEMA7A, and FOXF1 in HER2 overexpression normal human lung fibroblasts (F) and quantification of the densitometry (G; n = 3 per group). GAPDH served as loading control. (H and I) Representative images (H) and index quantification (I; n = 6 per group) of normal lung fibroblast invasion after HER2 overexpression. (J and K) Trichrome staining (J) and hydroxyproline (K; n = 10 per group) of mice lungs receiving HER2 overexpressing and control normal human lung fibroblasts, and age-matched mice were treated with culture medium only. Dash-boxed regions were shown at higher magnification. CTL, control; OE, overexpression. Scale bar: 1 mm (H) and 500 μm (J). Three or four independent experiments were performed on fibroblasts from different patients (A, D, G, I, and K). Data are the mean ± SEM. *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001 by Student’s t test (A, D, G, and I) and two-way ANOVA (K). Source data are available for this figure: .

Article Snippet: Directly conjugated antibodies used were anti-SEMA7A-PE and anti-SEMA7A-BV480 (clone KS-2; BD Biosciences), anti-CD274-PE (clone 29E.2A3; Biolegend), anti-F3-PE (clone HTF-1; BD Biosciences), anti-ITGA6-PE (clone GoH3; BD Biosciences), APC anti-CD340 (erbB2/HER2, Clone 24D2; Biosciences), FITC anti-human CD326 (EpCAM, Clone 9C4; Biosciences), PE/Cy7 anti-human CD31 (Clone WM59; Biosciences), and PE/Cy7 anti-human CD45 (Clone HI30; Biosciences).

Techniques: Activation Assay, Expressing, Over Expression, Quantitative RT-PCR, RNA Sequencing Assay, Flow Cytometry, Western Blot, Staining

Journal: The Journal of Experimental Medicine

Article Title: HER2 drives lung fibrosis by activating a metastatic cancer signature in invasive lung fibroblasts

doi: 10.1084/jem.20220126

Figure Lengend Snippet: 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: .

Article Snippet: Directly conjugated antibodies used were anti-SEMA7A-PE and anti-SEMA7A-BV480 (clone KS-2; BD Biosciences), anti-CD274-PE (clone 29E.2A3; Biolegend), anti-F3-PE (clone HTF-1; BD Biosciences), anti-ITGA6-PE (clone GoH3; BD Biosciences), APC anti-CD340 (erbB2/HER2, Clone 24D2; Biosciences), FITC anti-human CD326 (EpCAM, Clone 9C4; Biosciences), PE/Cy7 anti-human CD31 (Clone WM59; Biosciences), and PE/Cy7 anti-human CD45 (Clone HI30; Biosciences).

Techniques: Quantitative RT-PCR, Western Blot, Expressing, Flow Cytometry

Journal: The Journal of Experimental Medicine

Article Title: HER2 drives lung fibrosis by activating a metastatic cancer signature in invasive lung fibroblasts

doi: 10.1084/jem.20220126

Figure Lengend Snippet: Blocking HER2 signaling inhibited IPF lung fibroblast invasion and attenuated fibrosis. (A–C) Representative images (A) and index quantification (B and C) of migration and invasion of fibroblasts treated with increasing doses of Lapatinib or DMSO ( n = 3 per group). (D–F) Representative images (D) and index quantification (E and F) of migration and invasion of fibroblasts treated with Pertuzumab or IgG1 ( n = 3 per group). (G and H) Masson’s trichrome staining of collagen in lung sections (G) and hydroxyproline content in lung tissues (H) from NSG mice injected with SEMA7A high IPF fibroblasts and treated with Lapatinib, vehicle control, Pertuzumab, or IgG1 control ( n = 10 per group). Dash-boxed regions were shown at higher magnification. Three independent experiments were performed on fibroblasts from different patients (B, C, E, and F). Data are the mean ± SEM. Scale bar: 1 mm (A and D) and 500 μm (G). *, P < 0.05; **, P < 0.01; ***, P < 0.001; and ****, P < 0.0001 by one-way ANOVA (B, C, E, and F) and two-way ANOVA (H).

Article Snippet: Directly conjugated antibodies used were anti-SEMA7A-PE and anti-SEMA7A-BV480 (clone KS-2; BD Biosciences), anti-CD274-PE (clone 29E.2A3; Biolegend), anti-F3-PE (clone HTF-1; BD Biosciences), anti-ITGA6-PE (clone GoH3; BD Biosciences), APC anti-CD340 (erbB2/HER2, Clone 24D2; Biosciences), FITC anti-human CD326 (EpCAM, Clone 9C4; Biosciences), PE/Cy7 anti-human CD31 (Clone WM59; Biosciences), and PE/Cy7 anti-human CD45 (Clone HI30; Biosciences).

Techniques: Blocking Assay, Migration, Staining, Injection

Journal: The Journal of Experimental Medicine

Article Title: HER2 drives lung fibrosis by activating a metastatic cancer signature in invasive lung fibroblasts

doi: 10.1084/jem.20220126

Figure Lengend Snippet: Cell-surface markers were used to identify invasive fibroblasts. (A) Violin plot comparisons of cell-surface marker gene expression in invasive and noninvasive fibroblasts. (B) Relative expression of cell-surface marker genes in bulk RNA-seq on invasive and noninvasive fibroblasts ( n = 9 per group). (C) Relative expression of SEMA7A , F3 , and ITGA6 in invasive and noninvasive fibroblasts by qRT-PCR ( n = 6 for SEMA7A and F3 , and n = 4 for ITGA6 ). (D) Cell-surface expression of SEMA7A, F3, and ITGA6 in invasive and noninvasive fibroblasts by flow cytometry. (E) Western blot analysis of SEMA7A, F3, and ITGA6 expression in invasive and noninvasive fibroblasts. GAPDH served as loading control. (F) Cell-surface expression of CD274, F3, and ITGA6 in SEMA7A negative and high fibroblasts by flow cytometry. (G) Heatmap of consistent genes in SEMA7A high and invasive, SEMA7A negative and noninvasive fibroblasts, respectively, by bulk RNA-seq. non, noninvasive fibroblasts; in, invasive fibroblasts; neg, negative. Three independent experiments were performed on fibroblasts from different patients (B and C). Data are the mean ± SEM. *, P < 0.05; ****, P < 0.0001 by student’s t test (B and C). Source data are available for this figure: .

Article Snippet: Directly conjugated antibodies used were anti-SEMA7A-PE and anti-SEMA7A-BV480 (clone KS-2; BD Biosciences), anti-CD274-PE (clone 29E.2A3; Biolegend), anti-F3-PE (clone HTF-1; BD Biosciences), anti-ITGA6-PE (clone GoH3; BD Biosciences), APC anti-CD340 (erbB2/HER2, Clone 24D2; Biosciences), FITC anti-human CD326 (EpCAM, Clone 9C4; Biosciences), PE/Cy7 anti-human CD31 (Clone WM59; Biosciences), and PE/Cy7 anti-human CD45 (Clone HI30; Biosciences).

Techniques: Marker, Expressing, RNA Sequencing Assay, Quantitative RT-PCR, Flow Cytometry, Western Blot

Journal: The Journal of Experimental Medicine

Article Title: HER2 drives lung fibrosis by activating a metastatic cancer signature in invasive lung fibroblasts

doi: 10.1084/jem.20220126

Figure Lengend Snippet: Specific cell-surface marker genes of invasive fibroblasts promoted invasion and fibrosis. (A) Cell sorting strategy of F3, SEMA7A, and ITGA6 negative and high fibroblasts for following experiments. (B and C) Relative mRNA levels (B; n = 4 per group) and total protein levels (C) of F3, SEMA7A, and ITGA6 expression in sorted F3, SEMA7A, and ITGA6 negative and high fibroblasts. (D and E) Representative images (D) and index (E; SEMA7A Neg/High migration/invasion, n = 4 per group; F3 Neg/High migration, n = 12 per group, invasion, n = 9 per group; ITGA6 Neg/High migration, n = 9 per group, invasion, n = 12 per group) of migration and invasion of SEMA7A, F3, and ITGA6 negative and high fibroblasts. (F) Cell adhesion of SEMA7A and ITGA6 high and negative fibroblasts was quantified ( n = 4 per group). (G) Overexpression of SEMA7A was confirmed by Western blotting. (H) Cell-surface expression of SEMA7A in SEMA7A overexpression and control fibroblasts. (I and J) Representative images (I) and index (J; n = 3 per group) of migration and invasion of SEMA7A overexpression fibroblasts. (K) Quantification of percentage of SEMA7A + fibroblasts by flow cytometry on freshly isolated normal and IPF human lungs (Normal, n = 4; IPF, n = 7). (L and M) Trichrome staining (L) and hydroxyproline (M; n = 10 per group) of mice lungs receiving SEMA7A high and negative fibroblasts, and age-matched mice were treated with culture medium only. Dash-boxed regions were shown at higher magnification. CTL, control; OE, overexpression. Scale bar: 1 mm (D and I), 500 μm (L). Three or four independent experiments were performed on fibroblasts from different patients (B, E, F, J, K, and M). Data are the mean ± SEM. *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001 by Student’s t test (B, E, F, and I–K) and two-way ANOVA (M). Source data are available for this figure: .

Article Snippet: Directly conjugated antibodies used were anti-SEMA7A-PE and anti-SEMA7A-BV480 (clone KS-2; BD Biosciences), anti-CD274-PE (clone 29E.2A3; Biolegend), anti-F3-PE (clone HTF-1; BD Biosciences), anti-ITGA6-PE (clone GoH3; BD Biosciences), APC anti-CD340 (erbB2/HER2, Clone 24D2; Biosciences), FITC anti-human CD326 (EpCAM, Clone 9C4; Biosciences), PE/Cy7 anti-human CD31 (Clone WM59; Biosciences), and PE/Cy7 anti-human CD45 (Clone HI30; Biosciences).

Techniques: Marker, FACS, Expressing, Migration, Over Expression, Western Blot, Flow Cytometry, Isolation, Staining

Journal: The Journal of Experimental Medicine

Article Title: HER2 drives lung fibrosis by activating a metastatic cancer signature in invasive lung fibroblasts

doi: 10.1084/jem.20220126

Figure Lengend Snippet: 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: .

Article Snippet: Directly conjugated antibodies used were anti-SEMA7A-PE and anti-SEMA7A-BV480 (clone KS-2; BD Biosciences), anti-CD274-PE (clone 29E.2A3; Biolegend), anti-F3-PE (clone HTF-1; BD Biosciences), anti-ITGA6-PE (clone GoH3; BD Biosciences), APC anti-CD340 (erbB2/HER2, Clone 24D2; Biosciences), FITC anti-human CD326 (EpCAM, Clone 9C4; Biosciences), PE/Cy7 anti-human CD31 (Clone WM59; Biosciences), and PE/Cy7 anti-human CD45 (Clone HI30; Biosciences).

Techniques: Quantitative RT-PCR, Western Blot, Expressing, Flow Cytometry

Journal: The Journal of Experimental Medicine

Article Title: HER2 drives lung fibrosis by activating a metastatic cancer signature in invasive lung fibroblasts

doi: 10.1084/jem.20220126

Figure Lengend Snippet: Cell-surface markers were used to identify invasive fibroblasts. (A) Violin plot comparisons of cell-surface marker gene expression in invasive and noninvasive fibroblasts. (B) Relative expression of cell-surface marker genes in bulk RNA-seq on invasive and noninvasive fibroblasts ( n = 9 per group). (C) Relative expression of SEMA7A , F3 , and ITGA6 in invasive and noninvasive fibroblasts by qRT-PCR ( n = 6 for SEMA7A and F3 , and n = 4 for ITGA6 ). (D) Cell-surface expression of SEMA7A, F3, and ITGA6 in invasive and noninvasive fibroblasts by flow cytometry. (E) Western blot analysis of SEMA7A, F3, and ITGA6 expression in invasive and noninvasive fibroblasts. GAPDH served as loading control. (F) Cell-surface expression of CD274, F3, and ITGA6 in SEMA7A negative and high fibroblasts by flow cytometry. (G) Heatmap of consistent genes in SEMA7A high and invasive, SEMA7A negative and noninvasive fibroblasts, respectively, by bulk RNA-seq. non, noninvasive fibroblasts; in, invasive fibroblasts; neg, negative. Three independent experiments were performed on fibroblasts from different patients (B and C). Data are the mean ± SEM. *, P < 0.05; ****, P < 0.0001 by student’s t test (B and C). Source data are available for this figure: .

Article Snippet: Directly conjugated antibodies used were anti-SEMA7A-PE and anti-SEMA7A-BV480 (clone KS-2; BD Biosciences), anti-CD274-PE (clone 29E.2A3; Biolegend), anti-F3-PE (clone HTF-1; BD Biosciences), anti-ITGA6-PE (clone GoH3; BD Biosciences), APC anti-CD340 (erbB2/HER2, Clone 24D2; Biosciences), FITC anti-human CD326 (EpCAM, Clone 9C4; Biosciences), PE/Cy7 anti-human CD31 (Clone WM59; Biosciences), and PE/Cy7 anti-human CD45 (Clone HI30; Biosciences).

Techniques: Marker, Expressing, RNA Sequencing Assay, Quantitative RT-PCR, Flow Cytometry, Western Blot

Journal: The Journal of Experimental Medicine

Article Title: HER2 drives lung fibrosis by activating a metastatic cancer signature in invasive lung fibroblasts

doi: 10.1084/jem.20220126

Figure Lengend Snippet: Specific cell-surface marker genes of invasive fibroblasts promoted invasion and fibrosis. (A) Cell sorting strategy of F3, SEMA7A, and ITGA6 negative and high fibroblasts for following experiments. (B and C) Relative mRNA levels (B; n = 4 per group) and total protein levels (C) of F3, SEMA7A, and ITGA6 expression in sorted F3, SEMA7A, and ITGA6 negative and high fibroblasts. (D and E) Representative images (D) and index (E; SEMA7A Neg/High migration/invasion, n = 4 per group; F3 Neg/High migration, n = 12 per group, invasion, n = 9 per group; ITGA6 Neg/High migration, n = 9 per group, invasion, n = 12 per group) of migration and invasion of SEMA7A, F3, and ITGA6 negative and high fibroblasts. (F) Cell adhesion of SEMA7A and ITGA6 high and negative fibroblasts was quantified ( n = 4 per group). (G) Overexpression of SEMA7A was confirmed by Western blotting. (H) Cell-surface expression of SEMA7A in SEMA7A overexpression and control fibroblasts. (I and J) Representative images (I) and index (J; n = 3 per group) of migration and invasion of SEMA7A overexpression fibroblasts. (K) Quantification of percentage of SEMA7A + fibroblasts by flow cytometry on freshly isolated normal and IPF human lungs (Normal, n = 4; IPF, n = 7). (L and M) Trichrome staining (L) and hydroxyproline (M; n = 10 per group) of mice lungs receiving SEMA7A high and negative fibroblasts, and age-matched mice were treated with culture medium only. Dash-boxed regions were shown at higher magnification. CTL, control; OE, overexpression. Scale bar: 1 mm (D and I), 500 μm (L). Three or four independent experiments were performed on fibroblasts from different patients (B, E, F, J, K, and M). Data are the mean ± SEM. *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001 by Student’s t test (B, E, F, and I–K) and two-way ANOVA (M). Source data are available for this figure: .

Article Snippet: Directly conjugated antibodies used were anti-SEMA7A-PE and anti-SEMA7A-BV480 (clone KS-2; BD Biosciences), anti-CD274-PE (clone 29E.2A3; Biolegend), anti-F3-PE (clone HTF-1; BD Biosciences), anti-ITGA6-PE (clone GoH3; BD Biosciences), APC anti-CD340 (erbB2/HER2, Clone 24D2; Biosciences), FITC anti-human CD326 (EpCAM, Clone 9C4; Biosciences), PE/Cy7 anti-human CD31 (Clone WM59; Biosciences), and PE/Cy7 anti-human CD45 (Clone HI30; Biosciences).

Techniques: Marker, FACS, Expressing, Migration, Over Expression, Western Blot, Flow Cytometry, Isolation, Staining

Journal: The Journal of Experimental Medicine

Article Title: HER2 drives lung fibrosis by activating a metastatic cancer signature in invasive lung fibroblasts

doi: 10.1084/jem.20220126

Figure Lengend Snippet: 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 −Log 10 (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.

Article Snippet: Directly conjugated antibodies used were anti-SEMA7A-PE and anti-SEMA7A-BV480 (clone KS-2; BD Biosciences), anti-CD274-PE (clone 29E.2A3; Biolegend), anti-F3-PE (clone HTF-1; BD Biosciences), anti-ITGA6-PE (clone GoH3; BD Biosciences), APC anti-CD340 (erbB2/HER2, Clone 24D2; Biosciences), FITC anti-human CD326 (EpCAM, Clone 9C4; Biosciences), PE/Cy7 anti-human CD31 (Clone WM59; Biosciences), and PE/Cy7 anti-human CD45 (Clone HI30; Biosciences).

Techniques: Flow Cytometry, Over Expression, Expressing, Western Blot, Activation Assay

Journal: The Journal of Experimental Medicine

Article Title: HER2 drives lung fibrosis by activating a metastatic cancer signature in invasive lung fibroblasts

doi: 10.1084/jem.20220126

Figure Lengend Snippet: Transcription factors regulated lung fibroblast invasion. (A–D) Knockdown of transcriptional factors was confirmed by qRT-PCR (A and B) and Western blotting (C and D). A, n = 9 for FOXF1 , n = 4 for CREBRF , TSC22D1, and KLF9 , n = 6 for MXI1 , n = 3 for NFE2L2 ; B, n = 4 for HMGA2 , n = 5 for DPF3 . (E) FOXF1 and SEMA7A expressions showed negative correlation in scRNA-seq by SeqGeq. (F and G) Relative mRNA levels of FOXF1 , SEMA7A , and collagen-related protein gene, ACTA2 and COL1A1 (F, n = 5 per group) and cell-surface expression of SEMA7A (G) after FOXF1 knockdown. (H–J) Representative images (H) and index quantification (I and J) of migration and invasion of fibroblasts after knockdown assay. I, FOXF1, CREBRF, TSC22D1, and MXI1, n = 6 for migration and invasion, KLF9, n = 3 for migration and invasion, NFE2L2, n = 3 for migration and n = 11 for invasion; J, HMGA2, n = 6 for migration and invasion, DPF3, n = 3 for migration and invasion. CTL, control; non, noninvasive; in, invasive. Three or four independent experiments were performed on fibroblasts from different patients (A, B, F, I, and J). Data are the mean ± SEM. Scale bar: 1 mm. *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001 by Student’s t test. Source data are available for this figure: .

Article Snippet: Directly conjugated antibodies used were anti-SEMA7A-PE and anti-SEMA7A-BV480 (clone KS-2; BD Biosciences), anti-CD274-PE (clone 29E.2A3; Biolegend), anti-F3-PE (clone HTF-1; BD Biosciences), anti-ITGA6-PE (clone GoH3; BD Biosciences), APC anti-CD340 (erbB2/HER2, Clone 24D2; Biosciences), FITC anti-human CD326 (EpCAM, Clone 9C4; Biosciences), PE/Cy7 anti-human CD31 (Clone WM59; Biosciences), and PE/Cy7 anti-human CD45 (Clone HI30; Biosciences).

Techniques: Quantitative RT-PCR, Western Blot, Expressing, Migration

Journal: The Journal of Experimental Medicine

Article Title: HER2 drives lung fibrosis by activating a metastatic cancer signature in invasive lung fibroblasts

doi: 10.1084/jem.20220126

Figure Lengend Snippet: Significantly activated ERBB2 (HER2) signaling pathway in invasive fibroblasts. (A and B) Dot plot visualization of the −Log 10 (FDR) (A) and bar plot visualization of the activation Z-score (B) of the top 30 activated and inhibited upstream regulators of invasive fibroblasts by IPA analysis. ERBB2 was the most activated regulators of invasive fibroblasts. ERBB2 was highlighted as the most activated regulator. (C) IPA analysis revealed the upstream regulators of metastatic lung adenocarcinoma cancer cells compared to primary cancer cells retrieved from GSE131907 . Most of the top activated/inhibited (listed in red/green texts, respectively) upstream regulators of invasive fibroblasts were these of metastatic cancer cells. (D) Pearson correlation analysis of activation z-score of shared upstream regulators ( n = 129) of invasive fibroblasts versus metastatic lung adenocarcinoma cancer cell. Linear regression analysis was performed and visualized in red line. (E) p-HER2, total HER2, and SEMA7A protein levels in sorted SEMA7A high and negative fibroblasts in nine IPF fibroblast lines were determined by Western blot. GAPDH served as loading control. (F) Quantification of the Western blot was used to determine the relative protein levels of p-HER2, total HER2, and SEMA7A in E ( n = 9 per group). (G and H) p-HER2 and total HER2 in sorted fibroblasts from three normal and four IPF lung were determined by Western blot and quantification was performed (H; n = 4). Neg, megative; P, proximal lung regions; D, distal lung regions. Two independent experiments were performed on fibroblasts from different patients (F and H). Data are the mean ± SEM. *, P < 0.05; **, P < 0.01; ****, P < 0.0001 by Student’s t test (F and H). Source data are available for this figure: .

Article Snippet: Directly conjugated antibodies used were anti-SEMA7A-PE and anti-SEMA7A-BV480 (clone KS-2; BD Biosciences), anti-CD274-PE (clone 29E.2A3; Biolegend), anti-F3-PE (clone HTF-1; BD Biosciences), anti-ITGA6-PE (clone GoH3; BD Biosciences), APC anti-CD340 (erbB2/HER2, Clone 24D2; Biosciences), FITC anti-human CD326 (EpCAM, Clone 9C4; Biosciences), PE/Cy7 anti-human CD31 (Clone WM59; Biosciences), and PE/Cy7 anti-human CD45 (Clone HI30; Biosciences).

Techniques: Activation Assay, Western Blot

Journal: The Journal of Experimental Medicine

Article Title: HER2 drives lung fibrosis by activating a metastatic cancer signature in invasive lung fibroblasts

doi: 10.1084/jem.20220126

Figure Lengend Snippet: HER2 signaling activation increased fibroblast invasion and fibrosis. (A) The expression of invasive and noninvasive specific genes in HER2 overexpression normal fibroblasts were detected by qRT-PCR ( n = 3 per group). (B) Heatmap of the differentially expressed genes of control and HER2 overexpressing normal human lung fibroblasts by bulk RNA-seq. (C) Volcano plot of the top differentially expressed genes between control and HER2 overexpressed normal human lung fibroblasts by bulk RNA-seq. Red dots indicated the genes at Fold_change >0.5 and black dots indicated the genes at Fold_change ≤0.5. (D) Relative expression of invasive and noninvasive specific genes in HER2 overexpression normal fibroblasts detected by bulk RNA-seq ( n = 5 per group). (E) Upregulated cell-surface expression of SEMA7A in HER2 overexpression normal lung fibroblasts was confirmed by flow cytometry analysis. (F and G) Western blotting confirmation of the expression of p-HER2, HER2, SEMA7A, and FOXF1 in HER2 overexpression normal human lung fibroblasts (F) and quantification of the densitometry (G; n = 3 per group). GAPDH served as loading control. (H and I) Representative images (H) and index quantification (I; n = 6 per group) of normal lung fibroblast invasion after HER2 overexpression. (J and K) Trichrome staining (J) and hydroxyproline (K; n = 10 per group) of mice lungs receiving HER2 overexpressing and control normal human lung fibroblasts, and age-matched mice were treated with culture medium only. Dash-boxed regions were shown at higher magnification. CTL, control; OE, overexpression. Scale bar: 1 mm (H) and 500 μm (J). Three or four independent experiments were performed on fibroblasts from different patients (A, D, G, I, and K). Data are the mean ± SEM. *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001 by Student’s t test (A, D, G, and I) and two-way ANOVA (K). Source data are available for this figure: .

Article Snippet: Directly conjugated antibodies used were anti-SEMA7A-PE and anti-SEMA7A-BV480 (clone KS-2; BD Biosciences), anti-CD274-PE (clone 29E.2A3; Biolegend), anti-F3-PE (clone HTF-1; BD Biosciences), anti-ITGA6-PE (clone GoH3; BD Biosciences), APC anti-CD340 (erbB2/HER2, Clone 24D2; Biosciences), FITC anti-human CD326 (EpCAM, Clone 9C4; Biosciences), PE/Cy7 anti-human CD31 (Clone WM59; Biosciences), and PE/Cy7 anti-human CD45 (Clone HI30; Biosciences).

Techniques: Activation Assay, Expressing, Over Expression, Quantitative RT-PCR, RNA Sequencing Assay, Flow Cytometry, Western Blot, Staining

Journal: The Journal of Experimental Medicine

Article Title: HER2 drives lung fibrosis by activating a metastatic cancer signature in invasive lung fibroblasts

doi: 10.1084/jem.20220126

Figure Lengend Snippet: 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: .

Article Snippet: Directly conjugated antibodies used were anti-SEMA7A-PE and anti-SEMA7A-BV480 (clone KS-2; BD Biosciences), anti-CD274-PE (clone 29E.2A3; Biolegend), anti-F3-PE (clone HTF-1; BD Biosciences), anti-ITGA6-PE (clone GoH3; BD Biosciences), APC anti-CD340 (erbB2/HER2, Clone 24D2; Biosciences), FITC anti-human CD326 (EpCAM, Clone 9C4; Biosciences), PE/Cy7 anti-human CD31 (Clone WM59; Biosciences), and PE/Cy7 anti-human CD45 (Clone HI30; Biosciences).

Techniques: Quantitative RT-PCR, Western Blot, Expressing, Flow Cytometry

Journal: The Journal of Experimental Medicine

Article Title: HER2 drives lung fibrosis by activating a metastatic cancer signature in invasive lung fibroblasts

doi: 10.1084/jem.20220126

Figure Lengend Snippet: Blocking HER2 signaling inhibited IPF lung fibroblast invasion and attenuated fibrosis. (A–C) Representative images (A) and index quantification (B and C) of migration and invasion of fibroblasts treated with increasing doses of Lapatinib or DMSO ( n = 3 per group). (D–F) Representative images (D) and index quantification (E and F) of migration and invasion of fibroblasts treated with Pertuzumab or IgG1 ( n = 3 per group). (G and H) Masson’s trichrome staining of collagen in lung sections (G) and hydroxyproline content in lung tissues (H) from NSG mice injected with SEMA7A high IPF fibroblasts and treated with Lapatinib, vehicle control, Pertuzumab, or IgG1 control ( n = 10 per group). Dash-boxed regions were shown at higher magnification. Three independent experiments were performed on fibroblasts from different patients (B, C, E, and F). Data are the mean ± SEM. Scale bar: 1 mm (A and D) and 500 μm (G). *, P < 0.05; **, P < 0.01; ***, P < 0.001; and ****, P < 0.0001 by one-way ANOVA (B, C, E, and F) and two-way ANOVA (H).

Article Snippet: Directly conjugated antibodies used were anti-SEMA7A-PE and anti-SEMA7A-BV480 (clone KS-2; BD Biosciences), anti-CD274-PE (clone 29E.2A3; Biolegend), anti-F3-PE (clone HTF-1; BD Biosciences), anti-ITGA6-PE (clone GoH3; BD Biosciences), APC anti-CD340 (erbB2/HER2, Clone 24D2; Biosciences), FITC anti-human CD326 (EpCAM, Clone 9C4; Biosciences), PE/Cy7 anti-human CD31 (Clone WM59; Biosciences), and PE/Cy7 anti-human CD45 (Clone HI30; Biosciences).

Techniques: Blocking Assay, Migration, Staining, Injection