Journal: Breast cancer research : BCR

Article Title: Metastatic breast cancer cells overexpress and secrete miR-218 to regulate type I collagen deposition by osteoblasts.

doi: 10.1186/s13058-018-1059-y

Figure Lengend Snippet: Fig. 3 miR-218 directly targets INHBB and YY1, and increases INHBA expression. a Relative RNA level of INHBB and INHBA normalized to 18S in MDA-231 transfected with miR-218 or control miRNA mimic at 24 h and 72 h, respectively. ***P < 0.001. b Western blot analyses of inhibin βB and inhibin βA in mimic-transfected MDA-231 cells at 48 h. c Relative RNA level of INHBB and INHBA normalized to 18S in MDA-231-bone transfected with anti-miR-218 or control antagomiR at 24 h and 48 h, respectively.. **P < 0.01; ***P < 0.001. d Sequence alignment of miR-218 and its predicted targets INHBB and YY1. e psiCheck2 reporter plasmids and miR-218 mimic or its corresponding control were transfected into indicated cell lines. Renilla and firefly luciferase activity was measured at 48 h. *P < 0.05; n.s. not significant. f Relative RNA level of YY1 normalized to 18S in mimic- transfected MDA-231 cells at 24 h. ***P < 0.001. g Western blot analyses of YY1 and inhibin βA in MDA-231 cells co-transfected with miR-218 mimic and YY1-overexpressing plasmid, or the corresponding controls at 72 h. h psiCheck2 reporter plasmids and miR-218 mimic or its corresponding control vector were transfected into MDA-231 cells. Renilla and firefly luciferase activity was measured at 48 h. **P < 0.01; n.s. not significant

Article Snippet: Protein detection was performed using the following antibodies: Collagen alpha-1(I) chain carboxy-telopeptide (LF68) (Kerafast; Boston, MA, USA, ENH018), Inhibin βA (Novus, NBP1–30928), Inhibin βB (Thermo Fisher Scientific, PA5–28814), Inhibin α (Santa Cruz Biotechnology; Dallas, TX, USA, SC-22048), YY1 (Abcam; Cambridge, MA, USA, ab109228), Phospho-Smad2 (Ser465/467) (Cell Signaling Technology; Danvers, MA, USA, 3108S), SMAD2 (Cell Signaling Technology, 3122S), Phospho-Smad3 (Ser423/425) (Cell Signaling Technology, 9520S), SMAD3 (Cell Signaling Technology, 9523S), tissue inhibitor of metalloproteinases 3 (TIMP3) (Abcam, ab155749), β-actin (Sigma-Aldrich, A1978), as well as anti-rabbit, anti-mouse, and anti-goat HRP-conjugated secondary antibodies (Santa Cruz Biotechnology).

Techniques: Expressing, Transfection, Control, Western Blot, Sequencing, Luciferase, Activity Assay, Plasmid Preparation

Journal: Breast cancer research : BCR

Article Title: Metastatic breast cancer cells overexpress and secrete miR-218 to regulate type I collagen deposition by osteoblasts.

doi: 10.1186/s13058-018-1059-y

Figure Lengend Snippet: Fig. 4 miR-218-regulated inhibin βA affects SMAD2/3 signaling in a cell-dependent manner. a and b Western blot analyses of phospho-SMAD2/3 in MDA-231 (a) or MC3T3 (b) that was serum-starved overnight and treated with EV-depleted CM collected from indicated cells for 30 min or 2 h. CM producing cell lines were transfected, PBS washed 48 h after transfection and then incubated with serum-free medium overnight before CM collection and EV depletion by ultracentrifugation. c Western blot analyses of inhibin βA, inhibin βB, and inhibin α in CM from indicated cell lines that was concentrated with medium concentrator columns. Arrows indicate the position of antigen. Control mimic-transfected MDA-231 whole cell lysate (WCL) was used as positive control for inhibin βA and inhibin βB. d Western blot analyses of inhibin βA monomer or dimer (activin A) in CM from indicated cell lines under non-reducing condition and without boiling the samples. Bottom and top arrows indicate inhibin βA monomer and dimer, respectively. e Western blot analyses of inhibin α in MC3T3 CM concentrated from 20 ml CM. WCL of MCF10A was used as a positive control. f and g Western blot analyses of phospho-SMAD2/3 in MDA-231 (f) or MC3T3 (g) that was serum-starved overnight and treated with CM collected from indicated cells for 30 min or 2 h. For MDA-231 cells (f), recombinant inhibin α protein was added to CM as indicated. For MC3T3 cells (g), anti-inhibin α antibody was added to CM as indicated

Article Snippet: Protein detection was performed using the following antibodies: Collagen alpha-1(I) chain carboxy-telopeptide (LF68) (Kerafast; Boston, MA, USA, ENH018), Inhibin βA (Novus, NBP1–30928), Inhibin βB (Thermo Fisher Scientific, PA5–28814), Inhibin α (Santa Cruz Biotechnology; Dallas, TX, USA, SC-22048), YY1 (Abcam; Cambridge, MA, USA, ab109228), Phospho-Smad2 (Ser465/467) (Cell Signaling Technology; Danvers, MA, USA, 3108S), SMAD2 (Cell Signaling Technology, 3122S), Phospho-Smad3 (Ser423/425) (Cell Signaling Technology, 9520S), SMAD3 (Cell Signaling Technology, 9523S), tissue inhibitor of metalloproteinases 3 (TIMP3) (Abcam, ab155749), β-actin (Sigma-Aldrich, A1978), as well as anti-rabbit, anti-mouse, and anti-goat HRP-conjugated secondary antibodies (Santa Cruz Biotechnology).

Techniques: Western Blot, Transfection, Incubation, Control, Positive Control, Recombinant

Journal: Breast cancer research : BCR

Article Title: Metastatic breast cancer cells overexpress and secrete miR-218 to regulate type I collagen deposition by osteoblasts.

doi: 10.1186/s13058-018-1059-y

Figure Lengend Snippet: Fig. 5 CM from miR-218-overexpressing cells inhibits type I collagen processing in differentiated MC3T3. a Western blot analyses of type I collagen and Timp3 in the CM from differentiated MC3T3 treated with indicated EV-depleted CM for 16 days. CM producing cell lines were transfected, PBS washed 48 h after transfection and then incubated with serum-free medium overnight before CM collection and EV depletion by ultracentrifugation. Osteoblast differentiation factors were then added into collected CM, which was used to treated MC3T3 cells and replaced every 3–4 days. Cellular β- actin was used as control. b Relative RNA level of Timp3 normalized to 18S in CM-treated differentiated MC3T3 at day 18. ***P < 0.001. c Relative RNA level of Timp3 in CM-treated differentiated MC3T3 at day 18. Anti-inhibin βA (1 ng/ml) antibody was added to CM as indicated. ***P < 0.001. d Western blot analyses of Timp3 in the CM from differentiated MC3T3 treated with indicated CM with or without anti-inhibin α antibody for 16 days. Cellular β-actin was used as control. e Proposed model of bone niche adaptation mediated by miR-218 through direct secretion and targeting of type I procollagen in osteoblasts (1) or regulation of inhibin βA whose secretion in turn blocks procollagen processing by osteoblasts (2)

Article Snippet: Protein detection was performed using the following antibodies: Collagen alpha-1(I) chain carboxy-telopeptide (LF68) (Kerafast; Boston, MA, USA, ENH018), Inhibin βA (Novus, NBP1–30928), Inhibin βB (Thermo Fisher Scientific, PA5–28814), Inhibin α (Santa Cruz Biotechnology; Dallas, TX, USA, SC-22048), YY1 (Abcam; Cambridge, MA, USA, ab109228), Phospho-Smad2 (Ser465/467) (Cell Signaling Technology; Danvers, MA, USA, 3108S), SMAD2 (Cell Signaling Technology, 3122S), Phospho-Smad3 (Ser423/425) (Cell Signaling Technology, 9520S), SMAD3 (Cell Signaling Technology, 9523S), tissue inhibitor of metalloproteinases 3 (TIMP3) (Abcam, ab155749), β-actin (Sigma-Aldrich, A1978), as well as anti-rabbit, anti-mouse, and anti-goat HRP-conjugated secondary antibodies (Santa Cruz Biotechnology).

Techniques: Western Blot, Transfection, Incubation, Control