PMC

Overexpression of mLEEK does not globally induce the UPR. (a) Schematic of the reporter system used to monitor XBP1 splicing. (b) HT1080 cells stably expressing the XBP1-luciferase reporter were transiently transfected with either pcDNA/HA, pcDNA/mLEEK-HA or pcDNA/YFP-HA as indicated. Cells were either treated with Tg or vehicle control. Luciferase activity in vehicle control transfected with vector was set at 1. Averages from three independent experiments are presented with s.d. values (bars). (c) HT1080 cells were transfected with the same plasmids used above and phosphorylation of eIF2α was monitored by western blotting of lysates. Cells were treated with Tg or vehicle control for 6 h.

Expression of mLEEK diminishes induction of the UPR. (a) HT1080/XBP1-luciferase cells were transiently transfected with pcDNA/HA, pcDNA/mLEEK-HA or pcDNA/YFP-HA. Cells were treated with Tg or vehicle control for the time points indicated before determination of luciferase activity. Averages from triplicate experiments were calculated and data from treated cells are normalized to untreated cells. (b) Same as (a), except that cells were treated with dithiothreitol or vehicle control. (c) Same as (a), except cells were treated with tunicamycin (Tm) or vehicle control. (d) HT1080 cells were transiently transfected with pcDNA/mLEEK-HA or pcDNA/HA. Cells were treated with Tg or vehicle control for 7 h before extraction of RNA. CHOP mRNA levels were measured by qRT–PCR and are reported as the ratio of plus Tg/− Tg for each data point.

mLEEK is localized to the nucleus. (a) Localization of mLEEK in U87MG cells was visualized by indirect immunofluorescence with mLEEK antibody. Peptide competition experiments with immunizing peptide corresponding to the exon 1–23 junction or nonspecific peptide were included as controls. As an additional control, cells were stained with preimmune sera. (b) U87MG cells were fractionated into cytoplasmic (C) and nuclear (N) fractions and lysates from equivalent amounts of cellular starting material were analyzed by western blotting with mLEEK antibody. Purity of the fractions was determined by blotting against lamin and GAPDH to distinguish the nuclear and cytoplasmic fractions, respectively. (c) Immunohistochemistry was performed on paraffin-embedded human tissue with mLEEK antibody. Testes (top left); liver (top right); urinary bladder (bottom left); and cerebrum (bottom right). Scale bar is 100 µm.

mLEEK is essential for cell viability and mLEEK knockdown leads to caspase-mediated cell death and sensitization to ER stress. (a) Sequence of mLEEK-10 and mLEEK-12 siRNA. (b) Western blot of HeLa cells transiently transfected with mLEEK-10, mLEEK-12 or non-targeting control (NC) siRNA. Cells were harvested at 36 h and lysates blotted with indicated antibodies. Westerns were performed using fluorescently labeled secondary antibodies and detected with the Odyssey infrared imager. The signals from mLEEK and GAPDH were quantified and the ratio of mLEEK/GAPDH was determined. These ratios were normalized relative to the NC siRNA control (designated 1.0) to obtain the fold decrease. (c) Membranes from blots shown above were reprobed with anti-GRP78 antibody and quantified as above. (d) Decrease in HeLa cell viability as a result of mLEEK siRNA transfection. Cell viability was determined 72 h post-transfection using the CellTiter-Blue assay. Averages from three experiments are presented with s.d. values (bars). (e) Induction of apoptosis in cells transfected with mLEEK siRNA. Caspase-3/7 activity was measured 3 days after transfection using the Caspase-Glo 3/7 Assay. Values represent the average percentage of caspase 3/7 activity relative to that of NC siRNA-transfected cells. Results are averages with s.d. of four independent experiments. (f) HeLa cells were transfected with mLEEK-10, mLEEK-12 or non-targeting controls siRNA (NC). The cells were treated with tunicamycin or vehicle control for 24 h. Cell viability was determined as above. *P<0.001; **P<0.005, significantly different from the viability in Tm-treated non-targeting siRNA-transfected cells.

mLEEK upregulates the expression of GRP78 by interacting with the transcriptional machinery. (a) A subset of mRNAs identified in microarray experiments that were consistently >2 fold upregulated by overexpression of mLEEK relative to empty vector. (b) Induction of promoter activity by mLEEK was assayed using a panel of luciferase reporter constructs, which were co-transfected into HT1080 cells along with pcDNA/mLEEK-HA or pcDNA/HA. Averages from three independent experiments are presented with s.d. values (bars). (c) Endogenous GRP78 protein levels were assayed by western blotting of lysates from HT1080 cells transiently transfected with pcDNA/mLEEK-HA or pcDNA/HA. As a positive control, cells were treated with Tg for 6 h. (d) HT1080 cells were transiently transfected with pcDNA/mLEEK-HA or vector control. As a positive control, cells were treated with Tg for 6 h. Normalized GRP78 mRNA levels were measured by quantitative RT–PCR and shown relative to levels in vector control-transfected cells. Error bars represent s.d. from triplicate experiments. (e) Left, schematic diagram of GRP78-luciferase reporter constructs. Numbers indicate nucleotide position from transcription start site. Boxes represent ERSE motifs in the promoter. ERSEs with mutated sequences are indicated by crosses. Mutations have been described previously (). Right, Relative luciferase activity upon transfection into cells. As a reference for induction of transcription, subsets of cells were treated with Tg for 6 h before determination of luciferase activity. Averages from three experiments are presented with s.d. values (bars). (f) Top, schematic of the GRP78 gene, showing regions amplified by PCR in ChIP assays corresponding to the ERSE region and a region approximately + 5 kb from the transcriptional start site. Bottom, crosslinked chromatin from U87 cells stably expressing mLEEK-HA or an expression vector containing the HA tag alone (vector) was subjected to ChIP using anti-HA or immunoglobulin G and analyzed by quantitative PCR using the primers sets described above. Data are represented as the IP fold enrichment of the assay site relative to sample specific background (immunoglobulin G).

mLEEK: a novel variant of the EGFR. (a) Primer sets used in RT–PCR and nested PCRs. A, sense primer; B, antisense primer. (b) RT–PCR of coding region of human EGFR using human breast tumors (1–3) and 1A/1B primers, followed by nested PCR using 2A/2B primers. Predicted sizes for full-length EGFR and EGFRvIII are 2782 and 1981 bp, respectively. *The presence of an alternative EGFR amplification product. (c) RT–PCR using 1A/3B primers and RNA from U87MG cells as template. (d) Sequencing of the alternative product identified above reveals that mLEEK is the result of the joining of exons 1–23. Numbering refers to human EGFR cDNA sequence (Accession no. X00588). (e) RNase protection detects the exon 1–23 fusion. A 196 nt. labeled antisense probe spanning the junction was used where 72 nt. are from exon 1 and 96 nucleotides come from exon 23 with an additional 28 nt. of non-complementarity to permit discrimination between full-length probe and protected fragments. The protected fragment corresponding to the junction of exons 1 and 23 is 168 nt. RNA was from A431 cells (lane 1). Undigested probe is included as a reference (lane 2). RNase protection with yeast RNA served as a negative control (lane 3). (f) Schematic of mLEEK protein structure compared to EGFR. SP, signal peptide; TM, transmembrane domain; TK, tyrosine kinase domain; PRR, proline-rich region. The known phosphorylation sites (residues numbered) are indicated. (g) Western blot of NIH3T3 cells stably expressing empty vector or mLEEK with an HA tag, probed with anti-HA, anti-mLEEK or anti-actin. (h) Top, Western blots of colon tumor lysates (T) and paired normal tissue (N), probed with anti-mLEEK antibody. Bottom, Western blot of glioblastoma tumor tissue (1–6) and normal brain tissue lysate (7–9) probed with anti-mLEEK or anti-EGFR.