Hypoxic conditions upregulate CNN3 in BeWo cells.

<p>BeWo cells were cultured either under normoxic or under hypoxic conditions for 24(A) Total protein lysates were examined with the Western Blot technique to detect CNN3 protein levels. For normalization, HPRT was stained on the same membrane. As hypoxia marker, HIF-1 alpha was detected as well. (B) Protein bands of CNN3 and HPRT were densitometrically measured on the Western Blot membrane and CNN3/HPRT levels are plotted in the graph (white column: normoxia; black column: hypoxia). n = 3. (C) BeWo cells were serum starved for 16 h and then treated with either PBS as control or 200 µM CoCl₂ for 6 h in serum free medium. Then total protein was isolated and a Western Blot was performed. The HIF-1 alpha, CNN3 and HPRT protein was detected on the membrane with specific antibodies. (D) A densitometric analysis was performed to determine CNN3/HPRT levels (white column: normoxia; black column: hypoxia). n = 3.</p

CNN3 activates ERK1/2 and p38.

<p>BeWo cells were transfected either with control or with CNN3-siRNA for 48 h, then serum starved for 24 h and finally harvested. Proteins were extracted from the cells and examined on a Western Blot. (A) Total ERK1/2, phospho-ERK1/2 (pERK); total p38, phospho-p38 (p38); total JNK and phospho-JNK (pJNK) levels were detected. To verify CNN3 knockdown, the protein was detected on the membrane using a specific antibody. For normalization, the membrane was stained for HPRT. (B) Densitometric analysis was performed and pERK/ERK, pp38/p38 and pJNK/JNK levels in either control or CNN3 knockdown cells are plotted in the graph. n = 3.</p

CNN3 mRNA expression is not altered in human placenta samples of preeclamptic mothers and of mothers whose fetus suffered from IUGR.

<p>Placentas were dissected to remove the amniotic membranes and the maternal decidua. The residual layer, containing the villous trees, was washed in PBS to remove the blood and a sample from the middle region was excised. Total RNA was purified and reverse transcribed into cDNA. Then, CNN3 mRNA expression was quantified in a Real-Time-qPCR analysis. For normalization, the expression level of several housekeeping genes was determined. (A) The placenta collection used for the study contained 17 preeclampsia samples (gestational week 32.2<u>+</u>2.8 (SD)) and 9 control samples (gestational week 35.8<u>+</u>2.9 (SD)) as control. For normalization, the genes HPRT, b2MG, b-actin and GAPDH were measured. (B) Placenta samples from a tissue collection of a previous study <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0103216#pone.0103216-Tzschoppe1" target="_blank">[24]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0103216#pone.0103216-Struwe1" target="_blank">[25]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0103216#pone.0103216-Tzschoppe2" target="_blank">[26]</a> were used for the analysis of CNN3 expression in 39 IUGR samples (gestational week 35.3<u>+</u>2.6 (SD)) and 31 AGA samples (gestational week 36.6<u>+</u>2.8 (SD)). For normalization, the mRNA levels of the housekeeping genes HPRT and b2MG were detected. (C) A cross section of a human placenta sample (from a healthy patient) containing villous trees was stained for CNN3 (anti-CNN3 antibody, red fluorescence), actin (phalloidin, green fluorescence) and nuclei (DAPI, blue fluorescence). Scale bar, 5 µm.</p

CNN3 regulates BeWo cell invasion.

<p>BeWo cells were transfected with siRNA against CNN3 or with control siRNA for 48(A) The cells were plated in serum free medium on transwell inserts coated with 100 µg matrigel at a density of 1×10⁵ cells/insert. In the lower chamber, medium containing 10% FCS was added as invasion stimulus. After 24 h, invaded cells were fixed and stained with H&E. (B) Five pictures from every membrane at 10x magnification were taken and cells were counted. Invaded cells/mm² are displayed in the graph, whereas the white column represents control and the black column CNN3 knockdown cells. n = 3. (C) An aliquot of the cells for the invasion assay was kept for Western Blot studies to verify CNN3 knockdown. Total protein lysates were extracted from the cells and examined via Western Blot. CNN3 was detected on the membrane using a specific antibody. For normalization, tubulin-alpha was detected on the same membrane. (D) For the MTT Proliferation assay, cells were transfected with siRNA-CNN3 for 48 h and then cultured in a 96-well plate for 24 h. Then the proliferation rate was determined using the MTT proliferation kit (ATCC). Relative proliferation rates for CNN3-siRNA transfected cells (white column) are plotted compared to the control-siRNA transfected cells (set as 1). n = 3.</p

Hypoxia stimulates BeWo cell invasion and p38 activity.

<p>(A) BeWo cells were plated in serum free medium on transwell inserts coated with matrigel. In the lower chamber, medium containing 10% FCS was added as invasion stimulus. Then, cells were incubated for 24 h either at normoxic or at hypoxic conditions and at the end of the experiment, invaded cells were fixed and stained with H&E. Five pictures from every membrane at 10x magnification were taken and cells were counted. Invaded cells/mm² are displayed in the graph, whereas the white column represents cells under normoxic and the black column represents cells under hypoxic conditions. n = 3. (B) For the MTT Proliferation assay, cells were cultured either at regular or at low oxygen levels for 24 h. Then the proliferation rate was determined using the MTT proliferation kit (ATCC). Relative proliferation rates for hypoxic conditions (white column) are plotted compared to the normoxic control (set as 1). n = 3. (C) BeWo cells were plated and serum starved for 16 h. Then they were either incubated at regular oxygen (- hyx) or at low oxygen (+ hyx) levels for further 24 h. Cell lysates were analysed for phospho-ERK1/2 (pERK), total ERK1/2 (ERK), phospho-p38 (pp38), total p38 (p38), CNN3 and HPRT protein levels on a Western Blot. (D) A densitometric analysis was performed to determine pp38/p38 (white columns) and pERK/ERK (black columns) ratios and plotted in a graph. n = 3. (E) An invasion assay under hypoxic conditions was carried out with either DMSO treated cells as control or with 10 µM U0126 treated cells. The number of invaded cells/mm² is plotted in the graph. n = 3. (F) Shown is the analysis of n = 3 invasion assays under hypoxic conditions with either control cells (DMSO, white columns) or cells treated with 21 nM SB203580 (black columns). (G) BeWo cells were cultured in the presence of either DMSO as control, 10 µM U0126 or 21 nM SB203580 for 24 h. Then the proliferation rate was determined using a MTT proliferation kit (ATCC). Relative proliferation rates for U0126 (white column) and SB203580 (black column) are plotted in the graph compared to the DMSO control (set as 1). n = 3.</p

CNN3 does not promote cell invasion or MAPK activation under hypoxic conditions.

<p>(A) An invasion assay was performed with either siRNA-control transfected cells or with siRNA-CNN3 transfected cells at low oxygen levels. After fixation and staining of the invaded cells, pictures of the membrane were taken and the cell number/mm² was determined and plotted in the graph. n = 3. (B) In a MTT proliferation assay, the proliferation rate of siRNA-CNN3 transfected BeWo cells compared to siRNA-control transfected cells under low oxygen levels was measured. Relative proliferation rates for CNN3 knockdown cells are plotted in the graph compared to the control (set as 1). n = 3. (C) siRNA-control or siRNA-CNN3 transfected BeWo cells were serum starved for 16 h and then incubated under hypoxic conditions. Whole cell lysates were analyzed on a Western Blot for phospho-ERK1/2 (pERK), total ERK1/2 (ERK), phospho-p38 (pp38), total p38 (p38), CNN3 and HPRT protein levels. (D) A densitometric analysis of n = 3 Western Blot membranes was performed and relative ratios of pp38/p38 (white columns) and pERK/ERK (black columns) levels are plotted in the graph.</p

CNN3 does not regulate MMP-2 and MMP-14 levels in BeWo cells.

<p>CNN3 expression was knocked down in BeWo cells by transfection with specific siRNA. 48(A) and (B) A quantitative Real-Time-PCR assay was performed to determine MMP-2 (a) and MMP-14 (b) mRNA expression levels. Expression was normalized to the expression of the housekeeping genes GAPDH (white column), HPRT (grey column) and b2MG (black column). Expression levels of control cells were set as 1 and only levels for CNN3 knockdown cells are plotted in the graph. n = 3. (C) To measure MMP-2 and MMP-14 protein levels, protein lysates from either control or CNN3 knockdown cells were analyzed via Western Blotting. For normalization, HPRT was detected on the same membrane. (D) The intensity of the protein bands was densitometrically measured and MMP-2/HPRT and MMP-14/HPRT levels of CNN3 knockdown cells are plotted in the graph. The MMP-2/HPRT and MMP-14/HPRT levels in control cells were set as 1. n = 5. (E) We also detected pro-MMP-2 (upper band) and MMP-2 (lower band) levels in the supernatant of control or siRNA-CNN3 transfected cells by using a gelatin Zymogel. (F) Densitometric analysis of the bands was performed. Pro-MMP-2 (white column) and MMP-2 (grey column) levels in the control cells were set as 1 and only the levels in CNN3 knockdown cells are displayed in the graph. n = 10.</p

Xenografting of cryopreserved human ovarian tissue.

<p>(A) Ovarian pieces after thawing, (B, C, D) Transplantation of these pieces, (E, F) Ovarian pieces after 45 d xenografting. Scale bar: 2 mm.</p

Translocation of phosphatidylserine in ovarian tissue pre-cooled to 5°C for 24 h before the freezing and then xenografted in SCID mice: representative example of one experiment.

<p>(a, b, c, d) Group 1: pieces were frozen immediately after operation, thawed and just after thawing their quality was FACS analyzed, (e, f, g, h) Group 2: pieces after operation were cooled to 5°C for 24 h, then frozen, thawed and just after thawing their quality was FACS analyzed, (i, j, k, l), pieces were frozen immediately after operation, without pre-cooling, thawed, transplanted to SCID mice and then, after 45 d of culture their quality was FACS analyzed. (m, n, o, p) pieces were frozen after 24 h pre-cooling to 5°C, thawed, transplanted to SCID mice and then, after 45 d their quality was FACS analyzed, (a, e, i, m) forward and scatter dot plot used to select the interest population, (c, g, k, o) histograms displaying fluorescence of PE channel, used to measure fluorescence intensity of Propidium Iodide (PI), (d, h, i, p) dot plot analysis of FITC-Annexin V and PE channels, (Q1) cells negative to Annexin V (FITC A) and positive to PI (could indicate necrotic cells), (Q2) cells positive to both Annexin V and PI (could indicate late apoptotic stage), (Q3) cells negative for both Annexin V and PI (could indicate viable cells), (Q4) cells positive to FITC-Annexin V and negative to PI (could indicate early apoptotic state).</p

Effect of cooling on the quality of follicles (expressed as quantity and normality of follicles).

<p>Group 1: freezing of tissue without pre-cooling, then thawing and evaluation of their quality. Group 2: freezing of tissue after 24h pre-cooling to 5°C, then thawing and evaluation of their quality. Group 3: freezing of tissue without pre-cooling, then thawing, xenografting and evaluation of their quality. Group 4: freezing of tissue without pre-cooling, then thawing, xenografting and evaluation of their quality. No statistical differences between respective groups (Group 1 <i>vs</i> Group 2 and Group 3 <i>vs</i> Group 4 (P>0.1).</p