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. 2022 Mar 15;11:e63404. doi: 10.7554/eLife.63404

Figure 5. Induction of the memory state requires neither transcription nor prolonged 20E exposure.

(A) E74 mRNAs measured by qPCR and normalized relative to rp49 with varying duration of 20E exposure during first induction. The data represent the mean of three independent experiments and the error bars the standard deviation of the mean. (B). Flavopiridol or Triptolide inhibitors were added 30 min prior 20E first induction at 1 μM and 5 μM, respectively. After 4 hr of induction, both, the hormone and the transcriptional inhibitors were washed-out and cells recovered for 24 hr. 20E re-induced cells were collected and, E74 expression was measured by qPCR from three independent experiments. Fold change values were normalized using rp49 and error bars represent the standard deviation of the mean. (C). Cells were treated with Flavopiridol or Triptolide as described in B and E74 mRNAs were monitored by smFISH. Error bars represent standard deviation of the mean. (D). E74 mRNAs levels were measured by qPCR and normalized against rp49 using two different recovery times (6 hr and 24 hr). The data represent the mean of three independent experiments ± standard deviation. (E). Calibrated Memory Index (CMI) of E74, E23, and E75 genes.

Figure 5.

Figure 5—figure supplement 1. Establishment of the memory state is independent of transcription.

Figure 5—figure supplement 1.

(A) Flavopiridol or Triptolide inhibitors were added 30 min prior to 20E induction and the nascent transcript of housekeeping gene rp49 was used to monitor the efficiency of transcriptional blockage. After 4 hr of induction, the hormone and inhibitor were washed out and cells allowed to recover for 24 hr. qPCR was normalized against spliced rp49 transcript. The data represent the mean of three independent experiments and the error bars the standard deviation of the mean. (B). Representative images of E74 gene in cells treated with Flavopiridol or Triptolide, and analyzed in Figure 5C. (C). Histograms showing the distribution of measured total instantaneous transcriptional activity in normalized units (C.U.) obtained from smFISH of E74 after 2 hr of second induction in untreated, Flavopiridol and Triptolide-treated cells. (D–E). Expression analysis of ecdysone-induced genes: E23 (D) and E75 (E) in cells treated as described in Figure 5B. The data represent the mean of three independent experiments ± standard deviation.