PMC

Schematic showing the proposed position of CHAC1 within the UPR cascade.

CHAC1 overexpression induces apoptosis. HEK cells untransfected (UNT co) or transfected with CHAC1-V5 (CHAC1), GFP (GFP co), or CHAC1-V5eGFP (CHAC1-GFP). a, Fluorescent staining of nuclei with DAPI (blue), apoptoic nuclei with TUNEL-rhodamine (red), and GFP (green). Examples of apoptotic nuclei are highlighted with white arrows. b, Quantification of TUNEL-positive nuclei. *, p < 0.01.

CHAC1 siRNA treatment activates and overexpression reduces TNFRSF6B. Determination of normalized mRNA levels by real-time RT-PCR of (a) CHAC1 and (b) TNFRSF6B following transfection with CHAC1-specific siRNA vs scrambled control. c and d, Measurement of TNFRSF6B levels by Western blot, normalized to GAPDH in cells transfected with CHAC1-V5eGFP overexpressing plasmid, vs eGFP control. *, p < 0.01.

CHAC1 siRNA treatment does not affect the expression of CHOP, ATF3, or BiP. Determination of normalized mRNA levels by real-time RT-PCR of (a) CHAC1, (b) CHOP, (c) ATF3, and (d) BiP following transfection with CHAC1-specific siRNA vs scrambled control in HAEC that were untreated (media) vs treated with ox-PAPC (ox) or Tunicamycin (Tun). *, p < 0.01.

CHAC1 is induced specifically by chemical UPR inducers. Determination of normalized CHAC1 mRNA measured by real-time RT-PCR in untreated cells (Media) and in cells treated with ox-PAPC (Ox), tunicamycin (Tun), DTT, thapsigargin (Thaps), or the general cell stressors UV and heat shock (HS). Shown are data from primary (a) HAEC, (b) HEK 293 cells, and (c) HeLa cells. Induction of known UPR genes (d) ATF4 and (e) ATF3 in HAEC, by real-time RT-PCR, in untreated cells (media) and in cells treated with chemical UPR inducers. *, p < 0.01.

ATF3 or CHOP siRNA treatment inhibits CHAC1 mRNA expression in the context of UPR induction. Determination of normalized CHAC1 mRNA levels measured by real-time RT-PCR following treatment with media or tunicamycin (Tun) in (a and c) HAEC or (b and d) HEK 293 cells treated with (a and b) ATF3 siRNA or (c and d) CHOP siRNA vs scrambled control. Determination of normalized levels of the target mRNA is also shown in each panel. *, p < 0.01.

XBP1 or ATF6 arms of the UPR do not affect CHAC1 mRNA expression. Determination of normalized CHAC1 mRNA levels measured by real-time RT-PCR following treatment with media, ox-PAPC (Ox), and/or tunicamycin (Tun) in HAEC cells transfected with (a) XBP1 siRNA, or scrambled control, (b) spliced XBP1 (sXBP1) overexpression construct or eGFP control plasmid, or (c) ATF6 siRNA, vs scrambled control. d–f, Determination of normalized CHAC1 mRNA levels as in a–c in HEK 293 cells. Measurement of normalized target gene modulation is also shown in the bottom panels. *, p < 0.01.

CHAC1 overexpression induces, while siRNA treatment reduces, apoptosis. Measurement of cleaved PARP (cPARP) levels by Western blot in (a) cells transfected with CHAC1-V5eGFP overexpression plasmid vs eGFP control normalized to GAPDH, (c) in cells treated with CHAC1 siRNA vs scrambled control, and 0, 10, or 20 μM cadmium chloride, normalized to actin. e, Measurement of cytoplasmic and nuclear AIF levels by Western blot, normalized to GAPDH (Cytosol) or histone H3 (Nucleus) in cells transfected with CHAC1-V5eGFP overexpressing plasmid vs eGFP. b, d, and f, Quantification of band intensities in a, c, and e. *, p < 0.05.

CHAC1 protein is expressed in the cytosol, and protein localization is not changed following induction of the UPR. a, Western blot detecting the V5 epitope, cytosolic control GAPDH, and nuclear histone H3 in cells transfected with C (CHAC1-V5eGFP (55 kDa)) or G (control eGFP). Whole indicates whole-cell extract, Cyto, cytosolic extract; Nuc, nuclear extract; Pellet, pelleted membranes. b, Fluorescence images of eGFP (green) and DAPI (blue) in cells transfected with CHAC1-V5eGFP-expressing plasmid or eGFP control. c, eGFP and DAPI fluorescence of HEK cells transfected with CHAC1-V5eGFP and treated with ox-PAPC or the chemical UPR inducers DTT, thapsigargin, or tunicamycin.