ATF6 induces XBP1-independent expansion of the endoplasmic reticulum

JCS045625 Supplementary Material

Files in this Data Supplement:

• Supplemental Figure S1 -

  Fig. S1. Fluorescence microscopy analysis of the ER in CHO cells expressing HA-ATF6α(1-373) and HA-ATF6α(1-373)m1. CHO cells were transfected with the pDsRed2-ER expression vector (Clontech) plus expression vectors encoding either HA-ATF6α(1-373) (top) or HA-ATF6α(1-373)m1 (bottom). The DsRed2-ER protein (the Discosoma sp. red fluorescent protein, DsRed2, modified by addition of the calreticulin ER targeting sequence at its N-terminus and the ER-retention sequence, KDEL, at its C-terminus) localizes to the ER. At 40 hours post-transfection, cells were fixed and stained with anti-HA monoclonal antibody followed by Alexa Fluor 350-labeled goat anti-mouse IgG antibody and analyzed by fluorescence microscopy. (Left) HA-ATF6α(1-373) and HA-ATF6α(1-373)m1 proteins visualized through a DAPI filter. (Middle) DsRed2-ER visualized through a TRITC filter. (Right) Merged DAPI and TRITC images. In ATF6α(1-373)-transfected cells, a mesh-like ER network appeared more highly developed and prominent than that observed in ATF6α(1-373)m1-transfected cells. Scale bars: 10 µm.

• Supplemental Figure S2 -

  Fig. S2. Electron microscopy analysis of the ER in NIH-3T3 cells expressing HA-ATF6α(1-373) or HA-XBP1(S). NIH-3T3 cells were transfected with the indicated expression vectors. At 40 hours post-transfection, EGFP⁺ cells were collected by FACS and then examined by TEM at the indicated magnifications. Arrows point to representative ER. For vector alone, 0/20 cells, for ATF6α(1-373), 11/20 cells, and for HA-XBP1(S), 16/20 cells, examined by EM exhibited ER expansion. Scale bars: 500 nm.

• Supplemental Figure S3 -

  Fig. S3. Electron microscopy analysis of the ER in 293 cells expressing HA-ATF6α(1-373). 293 cells were transfected with the indicated expression vectors. At 40 hours post-transfection, EGFP⁺ cells were collected by FACS and then examined by TEM at the indicated magnifications. Arrows point to representative ER. For vector alone, 0/10 cells, and for ATF6α(1-373), 8/10 cells, examined by EM exhibited ER expansion. Scale bars: 500 nm.

• Supplemental Figure S4 -

  Fig. S4. Electron microscopy analysis of the ER in HeLa cells expressing HA-ATF6α(1-373). HeLa cells were transfected with the indicated expression vectors. At 40 hours post-transfection, EGFP⁺ cells were collected by FACS and then examined by TEM at the indicated magnifications. Arrows point to representative ER. For vector alone, 0/10 cells, and for ATF6α(1-373), 6/10 cells, examined by EM exhibited ER expansion. Scale bars: 500 nm.

• Supplemental Figure S5 -

  Fig. S5. A partial cDNA for Chinese hamster Xbp1. The isolated 531 nt are translated into the one-letter code for amino acids (aa) without the first 2 nt (AG). The underlined sequences correspond to primers used for RT-PCR analysis of hamster Xbp1 mRNA splicing. The bold sequence represents the intron (26 nt) removed by IRE1-dependent splicing. The percentage identities were predicted with the corresponding nt and aa sequences of Xbp1 homologs using the William Pearson's lalign program (http://www.ch.embnet.org/software/LALIGN_form.html). At the nt level, unspliced hamster Xbp1 shares 93% identity with rat Xbp1 (GenBank accession no. BC079450), 92% identity with mouse Xbp1 (AF027963), and 89% identity with human XBP1 (AB076383). At the aa level, hamster XBP1 shares 91% identity with rat XBP1, 87% identity with mouse XBP1, and 83% identity with human XBP1.

• Supplemental Figure S6 -

  Fig. S6. Analysis of NIH-3T3 cells transduced with ATF6α(1-373) retrovirus. (A) Electron microscopy analysis of the ER. NIH-3T3 cells were transduced with either empty vector or ATF6α(1-373) retroviruses. At 40 hours post-transduction, cells were prepared and examined by TEM as described previously (Sriburi et al., 2004) in the Loyola University Imaging Facility. Arrows point to representative ER. Stereological analysis of electron micrographs (32/group) revealed 3.8- and 3.0-fold increases in the volume and area of rough ER, respectively, in ATF6α(1-373)-transduced cells. (B) RT-PCR analysis of Xbp1 mRNA splicing. NIH-3T3 cells were transduced with either empty vector or ATF6α(1-373) retroviruses. At 40 hours post-transduction, cells were harvested and total RNA isolated. As controls, total RNA was isolated from NIH-3T3 cells left untreated (−) or treated with tunicamycin (Tm) at 1 µg/ml for 6 hours. RT-PCR analysis of mouse Xbp1 mRNA was performed as described (Gunn et al., 2004). The primers used to PCR amplify Xbp1 from cDNA flank the 26 nt intron that is excised upon UPR activation and yield products of 237 and 211 bp from unspliced (Un) and spliced (S) Xbp1 mRNA, respectively.

• Supplemental Table S1 - Adobe PDF

• Supplemental Table S2 - Adobe PDF