Translation visualization in HEK293T cells. (A) HEK293T cells from left to right: vehicle, puromycin, and HHT at first image cycle (top images) and tenth image cycle (bottom images) after treatment (see Materials and Methods for definition of image cycle). Red arrows point to puncta. (B–E) Graphs of number of puncta in V4-expressing cells (B,C) or V4-Arc expressing cells (D,E) normalized to first cycle after treatment (V4; puro n = 5 cells, vehicle n = 8 cells, HHT n = 11 cells. V4-Arc: puro n = 3 cells, vehicle n = 9 cells, HHT n = 10 cells). Experiments are from a minimum of three independent cultures. Puncta were counted with an automated program (Materials and Methods). Vehicle groups from HHT (water) and puromycin (0.0675% Ethanol) were not significantly different from each other and were pooled. The same pooled vehicle groups are used in B,C and D,E. An interaction between time and group was observed for V4 (two-way ANOVA F(18,189) = 2.4, P = 0.002) and V4-Arc (two-way ANOVA F(18,162) = 4.9, P = <0.0001). For V4, control vs. HHT and control vs. Puro, Tukeys post-hoc tests P = 0.0002 and <0.0001, respectively, and for V4-Arc control vs. HHT and control vs. Puro, Tukey's post-hoc tests P < 0.0001 and <0.0001, respectively. (F) Total number of puncta in V4 and V4-Arc cells at first image cycle, P > 0.05 n = 24 (V4 cells), 22 (V4-Arc cells). (G) Number of puncta at tenth image cycle (∼12 min after first image cycle) normalized to first image cycle immediately after treatment (n is same as B–E) with puncta counted manually. Number of puncta was significantly different between controls and cells treated with HHT or puromycin for both V4 (one-way ANOVA F(23,2) P < 0.01), Tukey's post-hoc test P < 0.01 and V4-Arc (one-way ANOVA F(21,2) P < 0.01), Tukey's post-hoc test P < 0.01. All errors are shown as standard errors of the mean. Asterisks are applied as follows: (**) P ≤ 0.01, (****) P ≤ 0.0001.
