Fig. 3: Expression of Ceg14 in HEK293T cells caused ATP depletion.

a Expression of Ceg14 inhibits the growth of HEK293T cells. HEK293T cells of ~50% confluence were transfected with the empty vector (EV) or plasmid that expressed Flag-tagged Ceg14, Ceg14_S/A, Ceg14_H/A, or Ceg14_E/A for 24 h prior to image acquisition (left panels). Note the low cell confluency of the sample expressing Ceg14. The expression of Ceg14 and its mutants in an equal number of transfected HEK293T cells was detected by immunoblotting using the Flag antibody. The glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was probed as a loading control (right panels). Scale bar, 20 μm. b, c HEK293T cells transfected to express the indicated proteins for 24 h were assessed for cell viability using the CCK-8 method (b) or for LDH release (c). Results (mean ± s.e.) shown were from three independent experiments each done in triplicate. d Ceg14 depleted cellular ATP. Total ATP was measured in HEK293T cells transfected to express Ceg14 or LnaB. Cells transfected with the empty vector were used as controls. All samples contained an equal number of cells. Results (mean ± s.e.) shown were from three independent experiments (upper). The expression of Flag-Ceg14 and its mutants was detected with Flag antibody and GAPDH was probed as a loading control (lower panels). e HEK293T cells were transfected with either an empty plasmid or a Flag-Ceg14 expression plasmid for 4 h, followed by treatment with different doses of cytochalasin D for an additional 20 h. Equal amounts of cells were then collected to measure ATP levels. Data shown in (a, d) (lower panels) are representative from three independent experiments. Data shown in b-e were mean ± s.e. from three replicates. Unpaired two-tailed Student’s t-tests were performed. Source data are provided as a Source Data file.