 Good morning. I'm Shreya Nandilajan, and I'm working with Dr. Karan and his lab. My topic is a spontaneous evaluation for breast tumor cell line, featuring aberrant ATT crosstalk. Let me introduce the X-ray cell line. It's a mammary epithelial cell line isolated from the balance at mid-pregnancy. Normal mammary cells are arranged like that. They have an outer layer of basal cells and an inner layer of luminal cells. The basal cells express the cytokine K5 and the luminal cells K8. These are differentiated cells. Why do we use a cell line? It's because primary cells are very difficult to culture. And why the X-ray cell line is because they have a lot of properties very similar to the normal mammary gland. They're also P53 nutrient. Now I'm explaining the significance of the X-ray cell line. It was found that under conditional stress, the X-ray cell line underwent a transformation. And when sorted by field cytometry, they gave two variant populations. The ACAM positive population, which we're going to call the P-variant cells, when injected into mice did not initiate any tumors. However, there was another population of cells, the ACAM negative cells, which were found to initiate tumors when injected into mice. This makes the study of these T-ray cells very important. These were pictures taken under the microscope. You can see that there is a significant difference between the P and T cells. The T cells are more spread out. This may be due to the loss of the cell agression molecule at the AM. These are pictures taken under a microscope. The green cells are basal cells, but the red cells are luminal cells. Like you can see, the T cells are not sure either of these cytoketin markers. This shows that the T cells are undifferentiated cells. What do we know so far? The T cells are tumorogenic, and they initiate tumors when injected into mice. They are an adaptive cell type, and they are able to transform under environmental stresses. Therefore, it becomes very important to study the differences between these two cell types. We now treat the T and P cells with different growth factors. EGF is found to introduce cell differentiation and insulin for cell proliferation. When we treated both T and P cells, we found that under all conditions, the T cells do express the cytoketin markers. However, T cells do not show these markers under any conditions. We counted those cells in the P-fractional, P-pore, basal and luminal fractions, and we found that when marginally it increases basal cell population. However, this is not the focus of my study. I will now attempt to characterize the T cells. These are introduced in the P-I-GK and MEK pathways, which are pathways frequently indicated in breast cancer. They are found to be upregulated, and the effect of these pathways is an inhibition of apoptosis and increased cell proliferation. Insulin specifically activates the A and B part, and EGF activates this MEK pathway. Now, why do we study these pathways? Because it was found that when we treated the VMT cells with phosphoresics, this phosphoresics is an upstream protein of the AKT pathway. It was found to be expressed only in the T cells. This suggests a dysregulation of the AKT pathway in T cells. So the question arises, are the signaling responses of the P-I-GK and the AKT pathway different in T cells? We use two methods, one is gene sequencing, and another is we test the responses of these cells to inhibitors of these pathways. This is a protocol I use for gene sequencing. We use genome, DNA, RNA and CDNA. We use CDNA because we could cover the largest sections of the genome. Then we run it on a gel and sequenced if it ran. But more often than not, it would not amplify and we were having problems with sequencing. This is the strategy we use for sequencing H-RAS. We concentrated on two genes, one is H-RAS and one is PIC3CA. Why these genes is because they're constitutively activated in breast cancers, and this leads to permanent activation of the pathways. For rounds 12, 13 and 61 are frequently mutated in H-RAS, and we used these two primers and covered almost the entire gene. Like you can see, we have covered these codons and we did not find any mutation. For PIC3CA, it's a much larger gene, and we are found to show hotspots of mutation in the axons one, two, nine and 20. So in order to cover as much of the gene as possible, we designed three sets of primers to cover this section, this section, that. What did we find? We found that H-RAS does not have any mutation. Exam nine of PIC3CA does not have any mutation in both P and T cells. These sequences, when I say they don't have any mutation compared to the normal valve sequence. And we could not complete the sequencing. Exam 20, we found multiple bands were forming on our gel. We don't really know why. We probably have to redesign the primers or use different conditions. I'm going to introduce the cell proliferation assay, also called the mist one assay, which I'm going to use in my future experiments. The principle is the reduction of tetrasol to formazones by viable cells. And we measure the absorbance at 440 nanometers. The protocol I use as I see cells, I change the medium. I add mist one on a third day and I measure absorbance for hours after treatment. This is a very interesting result we got. We found that wind significantly increases the proliferation of T cells alone as compared to the P cells. Wind on a normal conditions does not induce proliferation of any type of cell, mammary cell, ethyl cell, but it does affect T cells. This correlates with data by Soyang, where she found that phosphor AKT was significantly increased in T cells as compared to the P cells under the influence of wind. And since wind is an anti-radial AKT pathway, this again suggests a dysregulation of the pathway. So what did we do? We treated the pathway with two different inhibitors, the Met pathway at this point with the U01 to 6, and the PLTK pathway with L1 to 9402. And we found that both cells seem to be equally susceptible to inhibition of the PLTK pathway. However, we found that when treated with an inhibitor of MEK, the T cells were less susceptible to inhibition of MEK. We now, this is a previous study, which was found that when the HCLL cell line was treated as a whole, inhibition of the MEK pathway seemed to upregulate the PLTK pathway. And like you can see there's an increase in phosphor AKT when treated with MEK. So this would be our future study. To summarize, the T variant cells, they suppress differentiated cell molecules when it's K5 and K8. They divide it approximately the same rate as the B2B cells. They express phosphor S6, which is a marker for AKT. They show AKT stimulation in response to wind library. They are resistant to MEK inhibitors and they do not fill in any mutations in the F-cells unit. Well, thank you. I would like to thank the Khurana program, DVD and Adi Lassice here. Dr. Khairana, let's have a lecture on you. Syam and Rod Clark. All my love, it's Dr. Ansari and all of you guys. Thank you so much. In your sequenced, H, we asked, do you know how, did you have a program that could recognize heterogeneous mutations or did you examine the traces? Did you compare it with the Lassice sequence? Directly, the traces directly?