Journal: Molecular Oncology

Article Title: Hepsin promotes breast tumor growth signaling via the TGFβ‐EGFR axis

doi: 10.1002/1878-0261.13545

Figure Lengend Snippet: Hepsin regulates EGFR signaling in a TGFβ‐dependent manner. (A) Western blot comparison of total‐EGFR (tEGFR) levels in control MCF10A pL6‐Ctrl ( N = 3) and MCF10A pL6‐TGFβ1V5, V5‐tagged TGFβ1 overexpressing cells ( N = 3), which secrete around 80–100 pg·mL −1 of TGFβ1. Data are presented as mean ± SD. Significance was tested using the student's t‐test. (B) Western blot analysis of tEGFR in control doxycycline‐ (DOX − ) ( N = 3) and hepsin overexpressing (DOX + ; N = 3) MCF10A‐ pIND20‐HPN pL6‐ TGFβ1V5 cells. Data are presented as mean ± SD. Significance was tested with the student's t ‐test. (C) Western blot analysis of tEGFR, phospho‐SMAD2/3 (pSMAD2/3), and total‐SMAD2 (tSMAD2) in MCF10A‐pIND20‐HPN pL6‐TGFβ1V5 cells. The cells were treated with ALK5 inhibitor (i) (10 μ m RepSox, N = 4) and ALK4/5/7 inhibitor (5 μ m A‐83‐01, N = 3) or DMSO (control, N = 4) for 48 h with (DOX + ; 1 μg·mL −1 ) or without (DOX − , control) hepsin overexpression (the numbers below the tEGFR blot indicate loading normalized values for tEGFR band intensity fold leftmost control lane). The histogram data are presented as mean ± SD. Significance was tested using unpaired t ‐test. (D) Western blot analysis of phospho‐EGFR (pEGFR), tEGFR, total‐MAPK (tMAPK) and phospho‐MAPK (pMAPK), and vinculin (loading control) in MCF10A‐pIND20‐HPN pL6‐TGFβ1V5 cells. The cells were treated with the ALK1/2/3/6 inhibitor (i) (10 μ m K02288), EGFR inhibitor (i) (10 μ m Erlotinib), and DMSO (control) for 48 h. Data are presented as mean ± SD. Significance was tested with the student's t ‐test ( N = 3). (E) Phase‐contrast microscopy images of MCF10A‐pIND20‐HPN cells and MCF10A‐pIND20‐HPN pL6‐TGFβ1V5 (overexpressing TGFβ1) cells grown in 3D culture for 2 weeks with (DOX + ; 1 μg·mL −1 ) or without (DOX − ) induction of hepsin overexpression. Cells were treated with 10 μ m EGFR or ALK1/2/3/6 inhibitor for 2 weeks as indicated in the figure. Scale bar represents 100 μm. (F) Phase‐contrast microscopy images of MCF10A‐pIND20‐HPN pL6‐TGFβ1V5 cells grown in 3D culture for 2 weeks in the presence of ALK1 inhibitory antibody (25 μg·mL −1 ). Scale bar represents 50 μm. For E and F, the experiments were repeated three times, with at least 100 epithelial structures counted per group in each repeat (one dot represents one structure), and black lines denote the mean values of each group. Significance was tested using the student's t ‐test. (G) Western blot analysis of the mitotic marker phospho histone H3 (pH3 S10) MCF10A‐pIND20‐HPN pL6‐TGFβ1V5 cell line grown in 3D culture in the presence of the EGFRi (1 μ m , 24 h) or DMSO. The quantification of blots of three independent experiments is shown in the graph (on the right), where the black lines denote the mean of each group. Significance was tested using the student's t ‐test. ns, not significant.

Article Snippet: The inhibitors used in this study were K02288 (Selleckchem, S7359, Houston, TX, USA), Galunisertib/LY2157299 (Selleckchem, S2230), RepSox (Sigma‐Aldrich/Merck, R0158), A‐83‐01 (MedChem Express, HY‐10432, Monmouth Junction, NJ, USA), Erlotinib (EGFRi, Selleckchem, S1023), and human ALK1 inhibitory antibody (R&D Systems, MAB3701).

Techniques: Western Blot, Comparison, Control, Over Expression, Microscopy, Marker

Journal: Journal of Rheumatic Diseases

Article Title: Elevated BMPR2 expression amplifies osteoblast differentiation in ankylosing spondylitis

doi: 10.4078/jrd.2023.0024

Figure Lengend Snippet: BMPR2 is highly expressed in patients with AS. (A) Two healthy control (HC)-OPs and two AS-OPs conducted RNA sequencing. Heatmap with the BMP-related genes were selected and shown. (B) BMPR1A, BMPR1B, BMPR2, ALK1, and ALK2 expressions were validated by RT-qPCR (HC-OPs: n=8; AS-OPs: n=8). BMPR2 and ALK1 expressions were validated by (C) immunoblotting (HC-OPs: n=2, AS-OPs: n=6) and (D) immunohistochemistry (HC: n=6; AS: n=6). BMPR2-positive cells in bone-lining cells were counted. Representative images are shown from two individual samples per group. (D) The scale bar showed is 200 μm. AS: ankylosing spondylitis, OP: osteoprogenitor. Values are the mean±standard error of the mean. Statistical significance was determined with *p<0.05, **p<0.01, by Mann–Whitney U test.

Article Snippet: The antibodies used for immunoblotting and immunofluorescence were as follows: RUNX2 (12556; Cell Signaling Technology), BMPR2 (sc-393304; Santa Cruz Biotechnology, Dallas, TX, USA), ALK1 (AF370; R&D Systems, Minneapolis, MN, USA), phos-smad1/5/8 (sc-12353; Santa Cruz Biotechnology), total-smad1/5/8 (sc-6031-R; Santa Cruz Biotechnology), OPG (sc-390518; Santa Cruz Biotechnology), phos-ERK (5683; Cell Signaling Technology), phos-p38 (9215; Cell Signaling Technology), and GAPDH (2118; Cell Signaling Technology).

Techniques: Control, RNA Sequencing, Quantitative RT-PCR, Western Blot, Immunohistochemistry, MANN-WHITNEY