Early megakaryocyte lineage-committed progenitors in adult mouse bone marrow

Hematopoietic stem cells (HSCs) have been considered to progressively lose their self-renewal and differentiation potentials prior to the commitment to each blood lineage. However, recent studies have suggested that megakaryocyte progenitors are generated at the level of HSCs. In this study, we newly identified early megakaryocyte lineage-committed progenitors (MgPs) in CD201-CD48- cells and CD48+ cells separated from the CD150+CD34-Kit+Sca-1+Lin- HSC population of the bone marrow in C57BL/6 mice. Single-cell transplantation and single-cell colony assay showed that MgPs, unlike platelet-biased HSCs, had little repopulating potential in vivo, but formed larger megakaryocyte colonies in vitro (on average eight megakaryocytes per colony) than did previously reported megakaryocyte progenitors (MkPs). Single-cell RNA-sequencing supported that these MgPs lie between HSCs and MkPs along the megakaryocyte differentiation pathway. Single-cell colony assay and single-cell RT-PCR analysis suggested the coexpression of CD41 and Pf4 is associated with megakaryocyte colony-forming activity. Single-cell colony assay of a small number of cells generated from single HSCs in culture suggested that MgPs are not direct progeny of HSCs. In this study, we propose a differentiation model in which HSCs give rise to MkPs through MgPs.

RNA polymerase II subunit 3 regulates vesicular, overexpressed in cancer, prosurvival protein 1 expression to promote hepatocellular carcinoma

Objective To explore the relationships between hepatocellular carcinoma (HCC) and the expression of RNA polymerase II subunit 3 (RPB3) and vesicular, overexpressed in cancer, prosurvival protein 1 (VOPP1), and to determine whether RPB3 regulates VOPP1 expression to promote HCC cell proliferation, tumor growth, and tumorigenesis. Methods HCC and adjacent liver samples were collected from 51 patients with HCC who underwent surgical excision between September 20, 2010 and June 22, 2017. Immunohistochemical staining, western blot, quantitative PCR, plate colony assay, and RNA microarray were used to detect relevant indexes for further analyses. Results VOPP1 was shown to function as a target gene of RPB3 in facilitating HCC proliferation, and was downregulated after RBP3 silencing. Additionally, hepatic tumor tissues demonstrated high VOPP1 expression. Furthermore, VOPP1 silencing suppressed tumor growth and cell proliferation and elicited apoptosis. Conclusion RPB3 regulates VOPP1 expression to promote HCC cell proliferation, tumor growth, and tumorigenesis.

Screening Antibody Libraries with Colony Assay Using scFv-Alkaline Phosphatase Fusion Proteins

Screening antibody libraries is an important step in establishing recombinant monoclonal antibodies. The colony assay can identify positive clones without almost any false-positives; however, its antibody library is smaller than those used in other recombinant screening methods such as phage display. Thus, to improve the efficiency of colony assays, it is necessary to increase library size per screening. Here, we report developing a colony assay with single-chain variable fragment (scFv) fused to the N-terminus of bacterial alkaline phosphatase (scFv-PhoA). The scFv-PhoA library was constructed in an expression vector specifically designed for this study. Use of this library allowed the successful and direct detection of positive clones exhibiting PhoA activity, without the need for a secondary antibody. Colony assay screening with scFv-PhoA is simple, rapid, offers a higher success rate than previous methods based on scFv libraries, and—most importantly—it enables high-throughput procedures.

Combined Effect of Iodine Contrast Media, Cisplatin and External Beam Radiotherapy on Anaplastic Thyroid Cancer Cells

Introduction: The current study investigated the combination of high Z atoms (iodine-, platinium-based drugs) with using low energy irradiation (120kvp) in Anaplastic Thyroid cancer cells.Materials and Methods: For this purpose, eight groups were designed: control (CNT), different concentrations of Iodine contrast media (ICM), irradiation with various doses, Cis-platin (CDDP) with different concentrations, (ICM + CDDP), (ICM + RAD), (CDDP + RAD) and (ICM + CDDP + RAD). The viability was measured by MTT and Colony assay. In MTT assay, the viability of 8305c cells RAD (2 Gy)+ICM (10mg/mL) group was significantly lower than those treated with RAD or ICM alone. CDDP +ICM+RAD group significantly decreased the viability. In colony assay, cells in ICM + RAD (2 Gy) group reduced the number of colonies more significant than RAD group. The difference of colony forming ability between CDDP and CDDP + RAD (2 Gy) was significant. The difference of ICM + CDDP + RAD (2 Gy) and CDDP +RAD (2Gy) group was significant. All data were statistically analysed using one-way analysis of variance (ANOVA) followed by Chafe’s multi-comparisons tests. All data were presented as mean ± standard deviation (SD) and analysed using statistical package for social sciences (SPSS 16). Significance was considered to be p<0.05.Results: In MTT assay, the viability of 8305c cells RAD (2 Gy) + ICM (10mg/mL) group was significantly lower than those treated with RAD or ICM alone. CDDP + ICM + RAD group significantly decreased the viability. In colony assay, cells in ICM + RAD (2 Gy) group reduced the number of colonies more significantly than RAD group. The difference of colony forming ability between CDDP and CDDP + RAD (2 Gy) was significant. The difference of ICM + CDDP + RAD (2 Gy) and CDDP + RAD (2 Gy) group was significant.Conclusion: Exposure of ATC to ICM in the presence of CDDP increases tissue X-rays absorbance by Auger electrons and photo electrons leading to more fatal effects against the tumour