Journal: PLoS ONE

Article Title: Loss of Igfbp7 Causes Precocious Involution in Lactating Mouse Mammary Gland

doi: 10.1371/journal.pone.0087858

Figure Lengend Snippet: (A) The transcriptome profiles of the Wild-Type (WT) and the Igfbp7 −/− glands on the lactation day 3 were analyzed using the RNA-Seq technique. Differentially expressed transcripts were identified by imposing a minimum of 1.5 fold change in the transcript expression level and a P-value of 0.05 or lower and are depicted on a Volcano plot. (B and C) The differentially expressed transcripts in the Igfbp7 −/− mammary epithelial cells (MECs) are categorized into the different KEGG Signaling Pathways using the DAVID Bioinformatics Resources. The fold enrichment scores are calculated based on the number of differentially regulated genes that belong to a particular KEGG signaling pathway out of the total gene set. The Benjamini statistics is used to identify the statistically significant gene enrichment for specific KEGG signaling pathways. Using this analysis the down regulated transcripts were found to be enriched in extracellular matrix receptor interaction, focal adhesion and cell adhesion molecules. The up regulated transcripts in the Igfpb7 −/− MECs showed enrichment for the WNT, TGF beta and adherens junction proteins. (D) To examine if the differentially regulated transcripts in the Igfbp7 −/− MECs show enrichment for involution-related genes, we first obtained a common involution gene signature by comparing 3 publically available transcriptome profile of involuting glands. Set 1 is from , Set 2 is from , Set 3 is from . The Ven diagram depicts a 72 gene-set that is common among all 3 data sets.

Article Snippet: The protein gels were transferred onto PVDF membranes (Millipore, Cat#IPVH00010) and the expression of specific proteins were determined using rabbit anti-Stat5a (1∶1000; Santa Cruz Biotechnology, cat#sc-1081), anti Stat3 (1∶1000; Cell Signaling Technology, cat#9139), mouse anti pStat3 (1∶2000; Cell Signaling Technology, cat#9145), anti Igf-1R (1∶1000; Cell Signaling Technology, cat#3027), and AKT (1∶1000; Cell Signaling Technology, cat#4691), anti pAKT (1∶1000; Cell Signaling Technology, cat#4060), anti Igfbp5 (1∶500; Abcam, cat#ab4255), anti Igfbp7 (1∶1000; Abcam, cat#ab74169), anti beta casein (1∶1000 BETHYL laboratories Inc. cat#IHC-00179) and anti-beta actin (1∶10,000; Sigma, cat#WH0000060M1) antibodies using chemiluminescence as per standard protocols.

Techniques: RNA Sequencing, Expressing, Protein-Protein interactions

Journal: PLoS ONE

Article Title: Loss of Igfbp7 Causes Precocious Involution in Lactating Mouse Mammary Gland

doi: 10.1371/journal.pone.0087858

Figure Lengend Snippet: To determine if Igfbp7 −/− glands exhibit molecular changes that are the hallmark of involution process we prepared protein extracts from the Wild Type (WT) or the Igfbp7 −/− glands on lactation day 3 (WT LD3, KO LD3 respectively) or from WT lactating glands were weaned for 5 days (WT Inv D5) to induce post-lactational involution. The expression of Stat5a, Stat3, phospho Stat3 (pStat3), AKT, pAKT, Igfbp5, and IGF-1R proteins was determined by Western Blots. (A, C–D) Representative Western Blots are shown. The protein expression levels for Stat5a and Stat3, AKT, Igfbp5, and IGF-1R have been normalized to beta actin expression while the expression of pStat3, pAKT have been normalized to total corresponding protein expression in each sample. The bar graphs show the average expression obtained from of 3 independent protein extracts and the statistical significance was calculated based on two-tailed t-test (*P = <0.05, **p<0.005, ***P<0.0005). As shown, lactating KO LD3 glands show elevated Stat3, pSTat3, and Igfbp5, along with decreased Stat5a, IGF-1R, and pAKT; indicating that Igbp7 −/− glands are undergoing involution. (B) Immunohistochemical staining was used to detect the expression of Stat3 in tissue sections obtained from WT or Igbp7 −/− glands on lactation days 1 or 3. As shown, expression of Stat3 can be detected as early as the first day of lactation in the Igbp7 −/− glands while WT glands show detectable expression of Stat3 .

Article Snippet: The protein gels were transferred onto PVDF membranes (Millipore, Cat#IPVH00010) and the expression of specific proteins were determined using rabbit anti-Stat5a (1∶1000; Santa Cruz Biotechnology, cat#sc-1081), anti Stat3 (1∶1000; Cell Signaling Technology, cat#9139), mouse anti pStat3 (1∶2000; Cell Signaling Technology, cat#9145), anti Igf-1R (1∶1000; Cell Signaling Technology, cat#3027), and AKT (1∶1000; Cell Signaling Technology, cat#4691), anti pAKT (1∶1000; Cell Signaling Technology, cat#4060), anti Igfbp5 (1∶500; Abcam, cat#ab4255), anti Igfbp7 (1∶1000; Abcam, cat#ab74169), anti beta casein (1∶1000 BETHYL laboratories Inc. cat#IHC-00179) and anti-beta actin (1∶10,000; Sigma, cat#WH0000060M1) antibodies using chemiluminescence as per standard protocols.

Techniques: Expressing, Western Blot, Two Tailed Test, Immunohistochemical staining, Staining