Journal: Cancer research

Article Title: Osteoblast-secreted factors mediate dormancy of metastatic prostate cancer in the bone via activation of the TGFβRIII-p38MAPK-pS249/T252RB pathway

doi: 10.1158/0008-5472.CAN-17-1051

Figure Lengend Snippet: (A) RNAs from undifferentiated (Day 0) or differentiated (Day 30) osteoblasts were used in a whole-genome microarray analysis. 42 transcripts encoding secreted factors upregulated in Day 30 osteoblasts were subdivided into three groups, based on p values and false discovery rate (FDR). Fold increase is shown on right. (B) RNA levels of select TGFβ and GDF family genes during osteoblast differentiation. (C) TGFβ2 or GDF10 expression by qRT-PCR (upper) or western blot (lower). Undifferentiated osteoblast precursor MC3T3-E1 cells were used as a control. (D) TGFβ1 RNA levels. *p < 0.01 by t test. (E) TGFβ2 and GDF10 are two of many factors upregulated and secreted by osteoblasts over 30 days of differentiation in culture.

Article Snippet: Antibodies: phospho-p38MAPK, p38MAPK, RB, Smad2/3, phospho-Smad1/5, p27Kip1, GAPDH, β-actin (Cell Signaling); phospho(S249/T252)-RB ( 8 ); Ki-67 (Dako); TGFβ2 and TGFβRIII (Proteintech); GDF10, p21 and TGFβRIII (R&D); β-tubulin, α-tubulin (GeneTex).

Techniques: Microarray, Expressing, Quantitative RT-PCR, Western Blot, Control

Journal: Cancer research

Article Title: Osteoblast-secreted factors mediate dormancy of metastatic prostate cancer in the bone via activation of the TGFβRIII-p38MAPK-pS249/T252RB pathway

doi: 10.1158/0008-5472.CAN-17-1051

Figure Lengend Snippet: (A) C4-2B4 cells treated with TGFβ2, GDF10 or TGFβ1 for 72 h were analyzed by live-cell imaging. Quiescent cells that did not divide in 60–72 h relative to total cells counted were quantified (mean ± s.e.m). n cells were analyzed in multiple experiments: TGFβ2 (N=5–8), GDF10 (N=4), and TGFβ1 (N=5), except for TGFβ2 or GDF10 dose response (N=1). P values were by t test. Live-cell images (h:min) of cells treated with TGFβ2 (B) or GDF10 (C), followed by co-immunostaining for Ki-67 and p27. Colored asterisks, control cell progenies. Red box, enlarged. Cells a-d, quiescent cells following TGFβ2 or GDF10 treatment. All bars, 10 μm. (D) Cellular quiescence of several other PCa cells treated and analyzed as in (A) in multiple experiments: C4-2b (N=3–5), PC3-mm2 (N=2–3), 22Rv1 (N=2–3), BPH-1 (N=2–3).

Article Snippet: Antibodies: phospho-p38MAPK, p38MAPK, RB, Smad2/3, phospho-Smad1/5, p27Kip1, GAPDH, β-actin (Cell Signaling); phospho(S249/T252)-RB ( 8 ); Ki-67 (Dako); TGFβ2 and TGFβRIII (Proteintech); GDF10, p21 and TGFβRIII (R&D); β-tubulin, α-tubulin (GeneTex).

Techniques: Live Cell Imaging, Immunostaining, Control

Journal: Cancer research

Article Title: Osteoblast-secreted factors mediate dormancy of metastatic prostate cancer in the bone via activation of the TGFβRIII-p38MAPK-pS249/T252RB pathway

doi: 10.1158/0008-5472.CAN-17-1051

Figure Lengend Snippet: (A) TGFβRIII levels in knockdown clones C4-2B4-shTβRIII-#2 and #3 were analyzed by immunoprecipitation followed by immunoblotting, quantified against pGIPZ-sh-Vector cells, and signals normalized against actin controls. Bar, TGFβRIII 100-kDa core protein and glycosylated forms. (B) Cells in (A) were treated with 50 ng/ml TGFβ2 or GDF10 for 72 h. Cellular quiescence was determined by live-cell imaging. n cells were monitored in N=3–5 experiments, except for shTβRIII-#3 cells (N=2). P values are by t test. (C) Left, C4-2B4 cells were treated with TGFβ2, immunostained for p38MAPK, and counterstained with DAPI. Images were captured by confocal microscopy. Box, enlarged. Right, phospho-p38MAPK (p-p38) immunostaining following TGFβ2 or GDF10 time course. (D) C4-2b and PC3-mm2 cells and (E) C4-2B4-sh-Vector, C4-2B4-shTβRIII-#2 and C4-2B4-shTβRIII-#3 cells were immunostained for p-p38 after treatment with TGFβ2 or GDF10 for 60 min. All bars, 10 μm.

Article Snippet: Antibodies: phospho-p38MAPK, p38MAPK, RB, Smad2/3, phospho-Smad1/5, p27Kip1, GAPDH, β-actin (Cell Signaling); phospho(S249/T252)-RB ( 8 ); Ki-67 (Dako); TGFβ2 and TGFβRIII (Proteintech); GDF10, p21 and TGFβRIII (R&D); β-tubulin, α-tubulin (GeneTex).

Techniques: Knockdown, Clone Assay, Immunoprecipitation, Western Blot, Plasmid Preparation, Live Cell Imaging, Confocal Microscopy, Immunostaining

Journal: Cancer research

Article Title: Osteoblast-secreted factors mediate dormancy of metastatic prostate cancer in the bone via activation of the TGFβRIII-p38MAPK-pS249/T252RB pathway

doi: 10.1158/0008-5472.CAN-17-1051

Figure Lengend Snippet: (A) C4-2B4 cells were treated with 50 ng/ml TGFβ2 or GDF10 and co-immunostained for p-p38 and phospho-S249/T252-RB (8). (B) Cells treated as in (A) for 180 min were co-immunostained as in (A), followed with proximity ligation assay. Cells incubated with either primary antibodies alone or no antibodies served as negative controls. Boxes, enlarged. All bars, 10 μm. PLA spots/nucleus were quantified in n nuclei. P values are by t test. #, no spots detected.

Article Snippet: Antibodies: phospho-p38MAPK, p38MAPK, RB, Smad2/3, phospho-Smad1/5, p27Kip1, GAPDH, β-actin (Cell Signaling); phospho(S249/T252)-RB ( 8 ); Ki-67 (Dako); TGFβ2 and TGFβRIII (Proteintech); GDF10, p21 and TGFβRIII (R&D); β-tubulin, α-tubulin (GeneTex).

Techniques: Proximity Ligation Assay, Incubation

Journal: Cancer research

Article Title: Osteoblast-secreted factors mediate dormancy of metastatic prostate cancer in the bone via activation of the TGFβRIII-p38MAPK-pS249/T252RB pathway

doi: 10.1158/0008-5472.CAN-17-1051

Figure Lengend Snippet: (A) C4-2B4 cells stably-expressing empty vector or p38DN were examined by western blot for p-p38 followed by p38MAPK. β-tubulin, loading control. (B) Cells in (A) were treated with 50 ng/ml TGFβ2 or GDF10 for 60 min and immunostained for p-p38. (C) Cells in (A) were treated with TGFβ2 or GDF10 for 72 h. Cellular quiescence was determined by live-cell imaging. n cells were monitored in multiple experiments: C4-2B4-Vector (N=5–7), C4-2B4-p38DN (N=4–5). P values are by t test. (D) C4-2b-Vector or C4-2b-p38DN cells were generated and examined by western blot as in (A). (E) Cells in (D) were treated as in (B) and immunostained for p-p38. All bars, 10 μm. (F) Cellular quiescence in C4-2b-Vector or C4-2b-p38DN cells after TGFβ2 or GDF10 incubation was determined as in (C). n cells were followed in multiple experiments: C4-2b-Vector (N=4–5), C4-2b-p38DN (N=3–5).

Article Snippet: Antibodies: phospho-p38MAPK, p38MAPK, RB, Smad2/3, phospho-Smad1/5, p27Kip1, GAPDH, β-actin (Cell Signaling); phospho(S249/T252)-RB ( 8 ); Ki-67 (Dako); TGFβ2 and TGFβRIII (Proteintech); GDF10, p21 and TGFβRIII (R&D); β-tubulin, α-tubulin (GeneTex).

Techniques: Stable Transfection, Expressing, Plasmid Preparation, Western Blot, Control, Live Cell Imaging, Generated, Incubation