PMC

Western blots of extracts from HepG2 cells incubated for 6 hr in media having either 0 mM or 5 mM glucose, and without (-) or with (+) 10 µM Sal003 as shown above lanes. Antibodies against phospho-eIF2α (eIF2α∼P), total eIF2α and GAPDH as a loading control, were used as indicated to the right of each blot.

Oligonucleotides spanning F7 AARE without mutation (WT) or with mutation in the ATF-motif (ΔA) or the C/EBPβ-motif (ΔC), were incubated with 8 µg extracts of COS-1 cells that were untransfected, or transfected with expression plasmid for human ATF4. Extract used in each lane, and the lanes in which anti-ATF4 antibody were included, are indicated. The arrow indicates supershifted complex (ATF4 ss) detected in presence of antibody. B. A similar experiment, but using extract of COS-1 cells transfected with expression plasmid for human C/EBPβ in the absence or presence of anti-C/EBPβ antibody. The arrow indicates position of the supershifted complex (C/EBPβ ss).

WT oligonucleotide probe spanning the F7 AARE was titrated with 2 to 8 µg of extract from COS-1 cells overexpressing recombinant human ATF4, in the absence (-) or presence (+) of 2 µg of extract from COS-1 cells overexpressing recombinant human LIP. Blocking/supershift of binding complexes with anti-ATF4 and C/EBPβ antibody are shown without LIP in lanes 5 and 6 and with LIP in lanes 11 and 12. The bracket (ss) indicates the region of the gel with supershifted complexes. 2 µg of LIP extract did not produce a detectable complex when tested alone (lane 7).

Replicate cultures of HepG2 cells were transfected with ATF4 siRNA #2 (grey bars), or negative control siRNA (black bars), then cultured with 25 mM, 5 mM, or 0 mM glucose for 24 hr. qRT-PCR was done for expression of ATF4, F7, and C/EBPβ amplicons. For each amplicon, average expression +/− SD at 25 mM glucose with negative control siRNA was considered 100% and other values are shown relative to that. N = 3 per group, p<0.001 at all glucose concentrations for ATF4, F7 and ASNS amplicons, and for the C/EBPβ amplicon at 0 mM glucose. B. Experiment as in panel A, except that C/EBPβ siRNA (white bars) was used with negative control siRNA (grey bars). C/EBPβ siRNA had no effect on ATF4 amplicon expression at all glucose concentrations, but significantly blocked expression and induction of C/EBPβ at all glucose concentrations and induction of F7 at 0 mM glucose (p<0.001), N = 3 per group.

In each lane, 10 µg of nuclear extracts from cells cultured with 25 mM, 5 mM, or 0 M glucose interacted with AARE-WT probe, in the absence and presence of anti-ATF4 or anti-C/EBPβ antibodies. The darker exposure is shown at left; an ATF4 supershifted complex in lane 7 is indicated by arrow (ATF4 ss). Two C/EBPβ supershifted complexes are seen in lanes 9 and 11, one of which has identical mobility to ATF4 ss and the other which is indicated by arrow (C/EBPβ ss). In the lighter exposure of lanes 4 through 7, shown at right, a binding complex with slightly increased mobility in the 0 mM glucose condition (lanes 6 and 7) is more easily distinguished from the complexes of lanes 2–5 and indicted by arrow (new complex).

HepG2 cells were cultured for 24 hr in media supplemented with 10% or 1% fetal bovine serum and either high (25 mM), standard (5 mM), or low/no (1 mM or 0 mM) glucose. The concentration of secreted FVII antigen, expressed as ng per ml, was determined by ELISA. For each experimental set, the average amount secreted by cells in 25 mM glucose was considered 100%, and amounts secreted at the lower glucose concentrations were expressed as percentages +/− SD relative to that. The number of replicates assayed at each condition is shown below the bars. Reducing the concentration of glucose significantly increased the amount of FVII secreted for each experimental set (p<0.001).

(A-C) HepG2 cells were treated in parallel for 24 hr in medium with 25 mM, 5 mM or 0 mM glucose (white bars, with the direction of arrows indicating decreasing concentration) or for 6 hr at 5 mM glucose without (-) or with (+) 500 nM TG (grey bars), and qRT-PCR was performed as described. Expression at 25 mM glucose and without thapsigargin were considered 100% expression for each amplicon; other groups were expressed as a percentage +/− SD relative to this. N = 3/group. Glucose deprivation significantly increased expression of ATF4, ATF6, ASNS, C/EBPβ, XBP1(S), GRP78, CHOP, GADD34, and F7 (all p<0.001). TG did likewise (all p<0.001), except for ATF6 and F7 amplicons. F8 was downregulated by glucose deprivation but unaffected by TG. (D) Western blot of whole-cell extracts of cells treated with glucose and/or TG as shown, above the lanes, for detection of the proteins shown at the right.

siRNA directed to either the 5' UTR (ATF4 si#1, white bars) or the 3' UTR (ATF4 si#2, striped bars) of the human ATF4 gene were introduced into HepG2 cells cultured in media with 5 mM glucose. Parallel cultures were transfected with the same amount of negative control siRNA (control si, light and dark grey bars, respectively). Relative expression of ATF4 and F7 are graphed as a percentage +/− SD of the expression seen with negative control siRNA, which was considered 100%. For ATF4 si#1, N = 5 for ATF4 amplicon and N = 10 for F7 amplicon; for ATF4 si #2, N = 6 per group. All p<0.001 for both ATF4 siRNAs. B. Whole-cell extracts prepared from HepG2 cells transfected with negative control siRNA (control si), 5' UTR-ATF4 siRNA (ATF4 si#1) or without siRNA (none) and cultured 48 hr in media with 5 mM glucose. Replicate aliquots of extracts, 4 µg/lane, were separated on SDS-PAGE and Western-blotted in parallel with antibody to ATF4 (upper panel) or to GAPDH (lower panel) as loading control. C. Conditioned media collected from cells following transfection with negative control siRNA (dark grey bars) or ATF4 si#2 (striped bars) for 48 hr in media with 5 mM glucose, analyzed for FVII antigen by ELISA. N = 18/group, p<0.001.

Replicate cultures of HepG2 cells were transfected with negative control siRNA (grey bars) or ATF4 siRNA #1 (white bars), then cultured in media with 25 mM, 5 mM, or 0 mM glucose for 24 hr. RNA was analyzed by qRT-PCR for expression of amplicons as shown. For each amplicon, average expression at 25 mM glucose with the negative control siRNA was considered 100% and other values are shown as % expression +/− SD relative to that. N = 3 per group. Panel A, coagulation factor genes; panels B and C, stress responsive genes. At 0 mM glucose, expression was significantly increased for F7, ATF4, ATF6, XBP1(S), GADD34, GRP-78, ASNS, C/EBPβ, ATF3 and CHOP (all p<0.001). Please note difference in scale between panel A and panels B and C. ATF4 siRNA blocked all increases (all p<0.005) except for GRP-78, GADD34 and CHOP. Expression of FX, F2 and PROS1 were significantly decreased at 0 mM glucose (all p<0.001) and were not significantly affected by ATF4 siRNA.

HepG2 cells were transfected with promoterless or reporter vectors OGH (N = 6/group), AARE-WT or ΔAARE (N = 12/group), along with either negative control siRNA or ATF4 si#1, then cultured at 5 mM glucose for the final 24 hr before harvest. Reporter expression is shown relative to that of AARE-WT vector in the presence of control siRNA, which was considered 100%. Effect of ATF4 siRNA on expression from AARE-WT vector only, p<0.001. B. HepG2 cells were transfected with reporter vectors and incubated in medium containing 25 mM glucose (dark grey bars), 5 mM glucose (light grey bars) or 0 mM glucose (white bars), with diminishing glucose concentration depicted by wedges. Glucose deprivation increased expression from WT reporter (N = 12; p<0.001), but not from ΔAARE (N = 12/group) or OGH (N = 9/group) reporters. C. HepG2 cells were transfected with reporter vectors OGH, AARE-WT, ΔAARE, ΔA or ΔC, along with 0 ng (dark grey bars), 125 ng (light grey bars) or 250 ng (white bars) of ATF4 expression plasmid, with increasing amounts depicted by the wedges. The number of replicates per group is shown below each bar. Expression from AARE-WT reporter without recombinant ATF4 was considered 100%; expression from the other groups are shown in comparison to this. Note the break in the Y-axis at 800%; expression from WT vector with ATF4 is graphed on the upper portion, all other groups on the lower portion. All effects of ATF4 coexpression were significant (p<0.001).