Investigating hnRNP E1 Knockdown in Cell Lines

Human lung epithelial cell line A549 (ATCC), its hnRNP E1 knockdown (A549/E1KD or E1KD), normal mouse mammary epithelial (NMuMG) cell line, and its hnRNP E1 knockdown (NMuMG/E1KD or E1KD) derivative cells (Hussey et al, 2011 (link), 2012 (link)) were cultured in DMEM supplemented with 5% FBS and 5% FCS at 37°C in a 5% CO₂ humidified chamber. Antibodies used in the work were against the following proteins: hnRNP E1 (mouse; Abnova; mouse, MBL; and rabbit, raised against purified hnRNP E1), TRF2 (rabbit, NB10056506; Novus), Hsp90 (mouse; SCBT), FLAG (rabbit; Sigma-Aldrich), 6XHis (rabbit; Cell signaling), phospho-ATM (MAB 2401; Abnova), ATM (2873S; Cell signaling), phospho-ATR, ATR (13934S; Cell signaling), phospho-p53 (9284S; Cell signaling), p53 (2524; Cell signaling), γ-H2A.X (rabbit, 9718S; Cell signaling), Histone H2A.X (7631T; Cell signaling), PCNA (mouse, Cat no. 2586S; SCBT), Usp1 (rabbit, 8033S; Cell signaling), Usp7 (mouse, HAUSP; SCBT), RPA32 (rat, 2208S; Cell signaling), Rad6 (R6A/R6B, rabbit, 4944S; Cell signaling), Rad18 (rabbit, 9040S; Cell signaling), and Fen1 (rabbit; SCBT). Plasmids containing hnRNP E1 and its three KH domains namely KH1, KH2, and KH3 have been described previously (Chaudhury et al, 2010b (link); Brown et al, 2016 (link)); each of the four ORFs contained an N-terminal GST tag.

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Mohanty B.K., Karam J.A., Howley B.V., Dalton A.C., Grelet S., Dincman T., Streitfeld W.S., Yoon J.H., Balakrishnan L., Chazin W.J., Long D.T, & Howe P.H. (2021). Heterogeneous nuclear ribonucleoprotein E1 binds polycytosine DNA and monitors genome integrity. Life Science Alliance, 4(9), e202000995.

Publication 2021

NB10056506 Hsp90 FLAG 6XHis phospho-p53 γ-H2A.X Histone H2A.X PCNA RPA32 Rad18

Antibodies Cell line Cells Epithelial cell Histone h2a x Hnrnp e1 Hsp90 Human Lung Mammary Mouse Novus Orfs Pcna Plasmids Proteins Rabbit Rad18 Usp1 Usp7

Corresponding Organization : MUSC Hollings Cancer Center

Other organizations : University of Indianapolis, Indiana University – Purdue University Indianapolis, Vanderbilt University

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Variable analysis

independent variables

HnRNP E1 knockdown in A549 cells (A549/E1KD or E1KD)
HnRNP E1 knockdown in NMuMG cells (NMuMG/E1KD or E1KD)

dependent variables

Protein expression levels of hnRNP E1, TRF2, Hsp90, phospho-ATM, ATM, phospho-ATR, ATR, phospho-p53, p53, γ-H2A.X, Histone H2A.X, PCNA, Usp1, Usp7, RPA32, Rad6, Rad18, and Fen1

control variables

A549 cell line (human lung epithelial cell line)
NMuMG cell line (normal mouse mammary epithelial cell line)
DMEM media supplemented with 5% FBS and 5% FCS
Incubation at 37°C in a 5% CO2 humidified chamber

controls

Positive control: A549 cell line (human lung epithelial cell line)
Negative control: Not explicitly mentioned

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