Fig 1: Loss of BRCA1 transcriptionally induces ß-hCG expression in different breast cancer cells. (a) Immunoflourescence analysis of stable knockdown of BRCA1 in MDAMB-231 cells. (b) Relative Luciferase activity of CGB5 and CGB7 upon stable knockdown of BRCA1 in MDAMB-231. Stable clones of control shRNA transfected MDAMB-231 cells served as control. Relative luciferase units were expressed as percentage of control. (c) qRT-PCR analysis of CGB5 and CGB7 expression upon stable knockdown of BRCA1 in MDAMB-231. Expression was normalized to control shRNA transfected stable MDAMB-231. qRT-PCR analysis of CGB5 and CGB7 expression in (d) SUM149 (e) MX1 and (f) MCF7 upon silencing BRCA1. Expression was normalized to control siRNA transfected SUM149, MX1 and MCF7, respectively.
Fig 2: In vitro chronic, 10-day, hCG exposure to immature cortical and hippocampal neurons is not toxic and protects them from NMDA-dependent excitotoxic injury. (A,B) MAP2 fluorescence following immunocytochemical staining of dissociated cortical and hippocampal neurons exposed to control vs. 2 IU/mL of hCG for 9 days prior and 24 h following IBO (50 µM; IBO) exposure. Prolonged hCG did not appear to affect neuronal survival. Note also the relative preservation of neurites in hCG-exposed neurons after IBO exposure (most noticeable in cortical neurons) compared with control (CSS; controlled salt solution). (C) Quantitative bar graph analysis of the effects of hCG on neuronal survival as a measure of LDH activity in the neuronal media or MAP2 IR in control and hCG-treated neuronal cultures 24 h after injury with IBO. Cortical and hippocampal neurons exposed to hCG demonstrated a reduction in IBO-mediated increases in LDH activity and a relative preservation of neurite staining compared with non-hCG-treated cells. Scale bars = 165 µm. Error bars show SE; *p < 0.05 and **p < 0.01. hCG, human chorionic gonadotropin; IBO, ibotenic acid; IR, immunoreactivity; LDH, lactase dehydrogenase.
Fig 3: Trophoblast-like tissue contains trophoblast subtypes STB and EVT. a) Fluorescence image of cavity-like structures (white asterisks) in 3D tissue at days 33 which are characterized by the expression of F-actin and CGB (an STB marker). The white square highlights multinucleated STB (white arrowheads) shown in the enlarged image. Multinucleated STB indicated the cell fusion of TS tissue. b,c) Representative pictures of trophoblast-like tissue (TS tissue) (day 18 [b]; day 33 [c]) and first-trimester placenta (Placenta) (n = 3; 6–8 weeks gestation) showing CGB, E-cad, and ENDOU. White arrowheads indicate multinucleated STB and white arrows. d) Representative electron transmission microscopy images of the TS tissue. The square areas were enlarged as shown on the right. Red arrowheads indicate microvilli on the surface of the TS tissue. nu: nucleus. e) ELISA for hCG-ß secreted by TS tissue. The amount of hCG-ß (pg mL-1) produced by TS tissue (n = 5 time points during the differentiation of 3D tissue from day 10 to day 25) at 48 h is shown. Supernatants from TS tissue cultures at different time points were derived independently from two experiments. f) Representative pictures of TS tissue showing the EVT marker HLA-G at days 33. White arrow heads indicate sporadic HLA-G+ cells. Scale bars: 100 µm. Scale bars: 50 µm (a-[Enlarged]) and 10 µm (b-[Enlarged]) in TS tissue.
Fig 4: ß-hCG induces migration, invasion and EMT. (a) Wound healing assay in HCC1937 and HCC1937/wt BRCA1 upon silencing the endogenous ß-hCG expression by transfecting with ß-hCG siRNA. “Csi” represents scrambled siRNA and “ß-hCG si” represents ß-hCG siRNA. Graph represents the distance migrated by cells in µm. (b) Invasion assay in HCC1937 and HCC1937/wt BRCA1 upon silencing the endogenous ß-hCG expression. Graph represents the total number of invaded cells. (c) Immunoflourescence of Vimentin in HCC1937 control vector transfected (HCC1937 C.vec), HCC1937 ß-hCG transfected (HCC1937 ß), HCC1937/wt BRCA1 control vector transfected (HCC1937/wt BRCA1 C.vec) and HCC1937/wt BRCA1 ß-hCG transfected (HCC1937/wt BRCA1 ß) cells. DAPI was used as nuclear stain. (d) Immunoflourescence of E-cadherin and P-cadherin in HCC1937 C.vec, HCC1937 ß, HCC1937/wt BRCA1 C.vec and HCC1937/wt BRCA1 ß. (e) Western blot analysis of the Vimentin and E-cadherin upon overexpression of ß-hCG in HCC1937 and HCC1937/wt BRCA1 and (f) upon silencing the endogenous ß-hCG expression in HCC1937 and HCC1937/wt BRCA1.
Fig 5: ß-hCG induces stemness in HCC1937. (a) Sphere formation upon ß-hCG overexpression in HCC1937 cells. (b) Arrows indicate the formation of spheres from adherent cells. (c) Immunoflourescence analysis of OCT4, Slug, E-cadherin and Vimentin in spheres derived from HCC1937 ß. (d) qRT-PCR analysis of EMT markers (Slug, Zeb, Vimentin, Twist, Snail, E-cadherin and fibronectin) in the spheres and adherent cells of ß-hCG overexpressed HCC1937. (e) qRT-PCR analysis of stem cell markers (Nanog, SOX2 and OCT4) in the spheres and adherent cells of ß-hCG overexpressed HCC1937. (f) Mammosphere forming ability in non-adherent plates on day 4 in HCC1937, HCC1937/wt BRCA1, HCC1937 ß and HCC1937/wt BRCA1 ß. (g) Mammosphere with a minimum size of 50 µm from HCC1937, HCC1937/wt BRCA1, HCC1937 ß and HCC1937/wt BRCA1 ß was counted manually. All the experiments were done in triplicates. (h) Number of spheres (>50 µm in size) and number of adherent colonies (>around 50 cells/colonies) were counted manually in ß-hCG overexpressing HCC1937 after treating with indicated doses of drug, 17AAG for 72 h. (i) Representative images of colony forming and sphere forming ability of ß-hCG overexpressing HCC1937 (HCC1937 ß) after treating with indicated doses of 17AAG for 9 days. All error bars in the graphs represent s.d.
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