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Hello again. My apologies for the delay in posting – I’ve been busy with other projects lately. As a follow up to my previous post, we wanted to further confirm our EZH1 antibody and test it in a knockout line. To do this, we generated a CRISPR knockout of EZH1 in an AML cell line Read More …
Hello again! My previous post discussed how I finally had a specific antibody to EZH1 in a cell line (HEK293). As a follow up, I wanted to determine if we could detect EZH1 expression in patient cells. I’m happy to report I was able to detect the EZH1 protein in patient cells (Figure 1). Experimental Read More …
Hello again! As a follow up to my previous post,I’ve continued the search for an antibody to detect EZH1. I’m happy to report that I finally found one. I was able to detect a band in HEK293 cells using the Cell Signaling Antibody. Using knockdowns targeting EZH1, I was able to determine that the band Read More …
Following up on my previous post, I tested two antibodies I was planning on trying from Abcam and Novus that were used in previous publications. This time I tested directly in patient sample lysates and either saw no band, or many nonspecific bands (Figure 1). I have since reached out to the Orlando lab who Read More …
Hello again! Here is my first attempt to find an EZH1 antibody. I have mainly focused on EZH2 in the PRC2 complex as the methyltransferase responsible for the H3K27me3 mark. However, EZH1 can functionally substitute for EZH2 in PRC2. We have a good antibody to detect EZH2 levels in patient AML samples. However, for some Read More …
Hello again! In previous posts I talked about how we were using a co-culture system to grow our leukemic cells. Although this is a great way to maintain cells, it can complicate some analyses where we cannot distinguish mouse (stroma) from human (AML) cells. We wanted to determine H3K27me3 levels (the mark that EZH2 is Read More …
Previously, we established that OCI-AML-20 requires stroma to maintain CD34 expression (https://zenodo.org/record/1187083#.Wr5-1C7wbIV). In this experiment, we wanted to see whether those cells grown in suspension are able to regain CD34 expression when cells are reintroduced to stroma. We find that OCI-AML-20 regains CD34 expression when put back on to stroma (either MS5 or OP9). Full Read More …
We are currently working on a manuscript to characterize one of the cell lines that we established from growing patient samples. I previously discussed how we established OCI-AML-20 and I’m currently working on some experiments to complete the manuscript. One of those experiments is determining whether the CD34 expression seen in OCI-AML-20 relies on the Read More …
Hello again! I have written a summary report of how we are culturing patient leukemic cells using two patients as an example, 150333 and OCI-AML-20. The report can be found at Zenodo. We are monitoring the growth of cultured cells by looking at the expression of CD34 and CD38 markers over a number of weeks. These Read More …
Our experiments use cells from patients with leukemia. These primary cells are notoriously difficult to culture, especially long term. To overcome this, we are using a co-culture system where we grow the leukemia cells with mouse stromal cells (OP9) to try to mimic some aspects of the bone marrow environment present in leukemia. Although essential Read More …
