Journal: bioRxiv

Article Title: Glucocorticoid involvement in reproductive biology

doi: 10.1101/2022.11.03.515013

Figure Lengend Snippet: BrdU distribution following pulse and six-week chase in (a, c) WT ovary; (b, d,) Cyp11b1-/- ovary; wild type uterus (f) Cyp11b1 KO uterus. (g,h) morphology of WT and Cyp11b1 KO uterus. Label-retaining cells in ovary are; granulocytes (yellow arrow); theca cells (red arrow); stroma (black arrows). CL - corpus luteum; LPF – large preantral follicle; SF – secondary follicle; AF – atretic follicle; St – stroma.

Article Snippet: Taqman gene-specific RT-PCR primer pairs were used with the ABI Prism 7000 (as follows: Cyp11b1 – Mm01204952_m1; Cyp21a – Mm00487230_g1; Hsd11b2 – Mm01251104_m1) and results were analysed using the ABI Prism 7000 software.

Techniques:

Journal: bioRxiv

Article Title: Glucocorticoid involvement in reproductive biology

doi: 10.1101/2022.11.03.515013

Figure Lengend Snippet: (a) corpus lutea in Cyp11b1-/- ovary following superovulation, revealed by OPT; (b) Cyp11b1-/- ovary section immunostained with anti-pdcd4; (c, d) ovary from WT female or (e, f) superovulated Cyp11b1 KO female, stained with anti Col4a2; (e, f) higher magnification of Col4a2 stained follicle (F) and corpus luteum (CL)F

Article Snippet: Taqman gene-specific RT-PCR primer pairs were used with the ABI Prism 7000 (as follows: Cyp11b1 – Mm01204952_m1; Cyp21a – Mm00487230_g1; Hsd11b2 – Mm01251104_m1) and results were analysed using the ABI Prism 7000 software.

Techniques: Staining

Journal: bioRxiv

Article Title: Glucocorticoid involvement in reproductive biology

doi: 10.1101/2022.11.03.515013

Figure Lengend Snippet: OPT scan of WT e6.5 implantation sites (sagittal section) using (a) brightfield and (b) autofluorescence. Brightfield OPT scans of (c) e6.5 and (d) e7.5 Cyp11b1 KO implantation sites

Article Snippet: Taqman gene-specific RT-PCR primer pairs were used with the ABI Prism 7000 (as follows: Cyp11b1 – Mm01204952_m1; Cyp21a – Mm00487230_g1; Hsd11b2 – Mm01251104_m1) and results were analysed using the ABI Prism 7000 software.

Techniques:

Journal: bioRxiv

Article Title: Glucocorticoid involvement in reproductive biology

doi: 10.1101/2022.11.03.515013

Figure Lengend Snippet: (a, c) Embryo location (e7.5 WT) visualized with H&E stain and marked with dotted line. (b) in situ of implantation site showing Cyp11b1-speciific staining in decidual tissue flanking the embryo-derived tissue.

Article Snippet: Taqman gene-specific RT-PCR primer pairs were used with the ABI Prism 7000 (as follows: Cyp11b1 – Mm01204952_m1; Cyp21a – Mm00487230_g1; Hsd11b2 – Mm01251104_m1) and results were analysed using the ABI Prism 7000 software.

Techniques: Staining, In Situ, Derivative Assay

Journal: bioRxiv

Article Title: Glucocorticoid involvement in reproductive biology

doi: 10.1101/2022.11.03.515013

Figure Lengend Snippet: (a-c) Graphs showing RT-PCR analysis of implantation sites from e5.5 to e8.5 for Cyp11b1, 21-OH and Hsd11b2. In each case, the red line indicates the level of expression seen in inter-implantation site tissue at e5.5

Article Snippet: Taqman gene-specific RT-PCR primer pairs were used with the ABI Prism 7000 (as follows: Cyp11b1 – Mm01204952_m1; Cyp21a – Mm00487230_g1; Hsd11b2 – Mm01251104_m1) and results were analysed using the ABI Prism 7000 software.

Techniques: Reverse Transcription Polymerase Chain Reaction, Expressing

Journal: Clinical Cancer Research

Article Title: Negative Regulation of the Osteoblast Function in Multiple Myeloma through the Repressor Gene E4BP4 Activated by Malignant Plasma Cells

doi: 10.1158/1078-0432.ccr-08-0219

Figure Lengend Snippet: Fig. 1. Microarray gene screening of myeloma-conditioned osteoblasts (OBs) and induction of the E4BP4 transcriptional repressor. A, osteoblasts from a healthy donor were incubated for 48 h with U-266 and the relative cDNA was screened on a microarray chip containing probes relative to 20,000 gene sequences. Spot intensities of gene expression in U-266-conditioned osteoblasts (Cy5) were related to their basal levels in unconditioned osteoblasts (Cy3) and the Cy5/Cy3 ratio provided the relative variation value (deregulated genes in red).To identify only highly deregulated genes, the exclusion range was increased to comprise 0.33 to 3.0 Cy5/Cy3 ratio values.The test thus selected the 38 genes (7 up-regulated and 31down-regulated) listed inTable1in relation to their role in bone metabolism and identified E4BP4 as the highest up-regulated gene.Values were calculated by dedicated software (MWG Biotech), which defined the values in the shaded area as not significant. B, changes in E4BP4 RNA transcription as 2-DDCt absolute value induced by different myeloma cell lines compared with control B cell lines, including CEM and Raji. In all instances, the myeloma cell lines produced a significant increase (2-DDCt > 1.5) of the basal transcription value, whereas no variation was induced by the control lines.

Article Snippet: Osteoblast RNA (1 Ag) was transcribed to cDNA (Applied Biosystems) and E4BP4 was measured by real-time PCR (ABI Prism 7000 Sequence Detector by Assay-On-Demand product Hs00356605_g1) in parallel with glyceraldehyde 3-phosphate dehydrogenase (TaqMan assay).

Techniques: Microarray, Incubation, Gene Expression, Software, Control, Produced

Journal: Clinical Cancer Research

Article Title: Negative Regulation of the Osteoblast Function in Multiple Myeloma through the Repressor Gene E4BP4 Activated by Malignant Plasma Cells

doi: 10.1158/1078-0432.ccr-08-0219

Figure Lengend Snippet: Fig. 2. PTHrP production by myeloma cells and in vitro stimulation of osteoblasts. A, left, PCR analysis of PTHrP expression by myeloma cell lines compared with control breast cancer cells (MCF7).The 249-bp PCR product was variably amplified by the cDNA of all myelomas (left) that released different amounts of soluble PTHrP molecules. Measurement of PTHrP in pmol/L was completed on 96 h cultures by dedicated ELISA and confirmed the release of PTHrP by all myelomas with lower release in both GRA-2 and MCC-2 cell cultures and by MCF7. Parallel stimulation with transforming growth factor-h at 10 ng/mL increased the secretion of PTHrP in all instances. Right, PTHrP was removed by immunoprecipitation in U-266, BUT-1, and PAS-2 supernatants whose residual E4BP4 activation in normal osteoblasts was measured by real-time PCR.These PTHrP-deprived supernatants induced lower E4BP4 RNA transcription (P < 0.1) inline with the specificity of the protein on E4BP4 gene expression. B, stimulation of osteoblasts by PTHrP, IL-3, and dexamethasone and measurement of E4BP4 (left) and BMP-2, Runx2, and Osterix (right). Significant real-time PCR values (2-DDCt > 1.5) of E4BP4 transcription were detected in PTHrP-stimulated osteoblasts, whereas no effect was observed by IL-3. By contrast, dexamethasone significantly inhibited (2-DDCt< 0.5) E4BP4 RNA transcription.The PCR expression of bone morphogenetic factors calculated with the Quantity One software revealed a substantial absence of any effect of PTHrP, IL-3 and dexamethasone on the expression of BMP-2, whereas Runx2 and particularly Osterix were significantly inhibited by PTHrP (P < 0.05 in both instances). By contrast, both morphogenetic factors were definitely up-regulated in dexamethasone-treated osteoblasts (P < 0.05), whereas no effect was induced by IL-3.

Article Snippet: Osteoblast RNA (1 Ag) was transcribed to cDNA (Applied Biosystems) and E4BP4 was measured by real-time PCR (ABI Prism 7000 Sequence Detector by Assay-On-Demand product Hs00356605_g1) in parallel with glyceraldehyde 3-phosphate dehydrogenase (TaqMan assay).

Techniques: In Vitro, Expressing, Control, Amplification, Enzyme-linked Immunosorbent Assay, Immunoprecipitation, Activation Assay, Real-time Polymerase Chain Reaction, Gene Expression, Software

Journal: Clinical Cancer Research

Article Title: Negative Regulation of the Osteoblast Function in Multiple Myeloma through the Repressor Gene E4BP4 Activated by Malignant Plasma Cells

doi: 10.1158/1078-0432.ccr-08-0219

Figure Lengend Snippet: Fig. 3. Effect of E4BP4 silencing in normal osteoblasts and COX-2 pathway activity. A, left, measurement of E4BP4 by real-time PCR in E4BP4-silenced osteoblasts showed a retarded and minimal RNA transcription in both unstimulated (2-DDCt = 0.0006) and PTHrP-stimulated (2-DDCt = 0.004) cells, whereas control nonsilenced osteoblasts produced efficient transcription of the repressor gene. Right, real-time PCR measurement of both Runx2 and Osterix transcription in E4BP4-silenced compared with control nonsilenced osteoblasts. Basal levels of both Runx2 and Osterix RNAs were virtually mostly unchanged after PTHrP treatment in E4BP4-silenced osteoblasts with respect to control cells. B, real-time PCR measurement of Runx2 and Osterix transcription through the COX-2 pathway. Levels of both Runx2 and Osterix, as major bone morphogenetic factors, were measured by real-time PCR in both PTHrP-treated and E4BP4-silenced osteoblasts with respect to control osteoblasts. Parallel cultures in each group of osteoblasts explored the effect of NS-398 as COX-2 inhibitor, PGE2, or both. In PTHrP-treated osteoblasts, the 2-DDCt values of both factors were constitutively down-regulated compared with control osteoblasts and supplementation with NS-398 failed to produce evident variation of relative RNAs in relation to the functional expression of E4BP4. However, by adding PGE2 at10-7 mol/L, a dramatic increase of both Runx2 and Osterix was detected compared with basal levels (P < 0.05 in both instances), whereas the concurrent presence of NS-398 induced a slight down-regulation of the PGE2-restored transcription. By contrast, E4BP4-silenced osteoblasts showed high constitutive activity of both Runx2 and Osterix, whose enhanced levels were inhibited by NS-398 and slightly increased by PGE2 in response to the inhibitor or activator of the COX-2 pathway, respectively.These effects were associated to the overexpression and vice versa, with the absence of E4BP4 in PTHrP-treated or E4BP4-silenced osteoblasts, respectively, and were confirmed in control osteoblasts sensitive to the inhibition of COX-2 by NS-398 or its activation by PGE2.These results supported the hypothesis that the expression of both Runx2 and Osterix in osteoblasts occurred through the integrity of COX-2 and that the E4BP4-mediated suppression of both morphogenetic factors occurred through the COX-2 inhibition. C, differential expression of granular cytoplasmic alkaline phosphatase in E4BP4-up-regulated osteoblasts by PTHrP compared with E4BP4-silenced and control unstimulated osteoblasts. As shown, the overexpression of the transcriptional repressor induced a maturation arrest revealed by the defective expression of the enzyme (magnification, 100).

Article Snippet: Osteoblast RNA (1 Ag) was transcribed to cDNA (Applied Biosystems) and E4BP4 was measured by real-time PCR (ABI Prism 7000 Sequence Detector by Assay-On-Demand product Hs00356605_g1) in parallel with glyceraldehyde 3-phosphate dehydrogenase (TaqMan assay).

Techniques: Activity Assay, Real-time Polymerase Chain Reaction, Control, Produced, Functional Assay, Expressing, Over Expression, Inhibition, Activation Assay, Quantitative Proteomics

Journal: Clinical Cancer Research

Article Title: Negative Regulation of the Osteoblast Function in Multiple Myeloma through the Repressor Gene E4BP4 Activated by Malignant Plasma Cells

doi: 10.1158/1078-0432.ccr-08-0219

Figure Lengend Snippet: Fig. 4. Detection of E4BP4 protein product in osteoblasts and measurement of osteogenic factors. A, unstimulated (control) osteoblasts, U-266-conditioned and CEM-conditioned osteoblasts, PTHrP-treated, and E4BP4-silenced osteoblasts were screened by immunoblotting in their expression of E4BP4 protein using a polyclonal goat antiserum.The 81-kDa E4BP4 protein was expressed, although at different intensity, by osteoblasts cocultured with the myeloma cell line U-266 and by osteoblasts stimulated by PTHrP. By contrast, no evidence of E4BP4 was shown in osteoblasts stimulated by the CEM cell line.The E4BP4-silenced osteoblasts provided the negative control.The secondary antibody was revealed by ECL Plus reagent (Amersham). h-Actin was detected as loading control for each nuclear extract. B, comparison by real-time PCR quantification of E4BP4 and Osterix transcription in cultured osteoblasts. A discrepant expression of E4BP4 was detected in osteoblasts from bone biopsies of patients with multiple myeloma compared with those from control patients with NHL or MGUS. In contrast with significantly higher transcription of E4BP4 (2-DDCt> 1.5) in osteoblasts from multiple myeloma patients with respect to those from NHL and MGUS patients, all five osteoblast preparations from myeloma bone biopsies showed dramatic Osterix deficiency (2-DDCt < 0.5) compared with the normal reference range (2-DDCt = 1.0 F 0.5) in the controls.This analysis supported the inversely correlated expression of E4BP4 and Osterix existing in vivo in osteoblasts from multiple myeloma bone biopsies. C, scatter plot correlating serum PTHrP with osteocalcinlevels. All multiple myeloma patients displayed variable increases of PTHrP, three with a concurrent reduction of osteocalcin. By contrast, patients with NHL and MGUS expressed normal serum levels of osteocalcin and differential (sometimes increased) PTHrP concentrations. However, the mean PTHrP value in multiple myeloma patients was 4.57 higher than in the controls (P < 0.05). Numbers refer to patients in each group; shaded areas include the corresponding normal ranges.

Article Snippet: Osteoblast RNA (1 Ag) was transcribed to cDNA (Applied Biosystems) and E4BP4 was measured by real-time PCR (ABI Prism 7000 Sequence Detector by Assay-On-Demand product Hs00356605_g1) in parallel with glyceraldehyde 3-phosphate dehydrogenase (TaqMan assay).

Techniques: Control, Western Blot, Expressing, Negative Control, Comparison, Real-time Polymerase Chain Reaction, Cell Culture, In Vivo

Journal: Clinical Cancer Research

Article Title: Negative Regulation of the Osteoblast Function in Multiple Myeloma through the Repressor Gene E4BP4 Activated by Malignant Plasma Cells

doi: 10.1158/1078-0432.ccr-08-0219

Figure Lengend Snippet: Fig. 5. Expression of E4BP4 protein in myeloma bone biopsies. A, immunohistochemistry assays revealed the protein in osteoblasts (top) located within the spongy bone (left) and facing the cortical bone (right), which were positive to both osteopontin (left) as osteoblast marker and to the presence of myeloma cells (right) identified by the detection of intracytoplasmic lambda chain (bottom). B, sequential events driving the osteoblast functional exhaustion through E4BP4. In bone marrow, malignant plasma cells secrete PTHrP that induces E4BP4 in osteoblasts through the PTH-1-R.The transcriptional repressor inhibits COX-2, which in turn leads to the suppression of both Runx2 and Osterix.The final effect is the arrest of osteoblast maturation.

Article Snippet: Osteoblast RNA (1 Ag) was transcribed to cDNA (Applied Biosystems) and E4BP4 was measured by real-time PCR (ABI Prism 7000 Sequence Detector by Assay-On-Demand product Hs00356605_g1) in parallel with glyceraldehyde 3-phosphate dehydrogenase (TaqMan assay).

Techniques: Expressing, Immunohistochemistry, Marker, Functional Assay, Clinical Proteomics