Figure 2. Multiple attempts were made to establish cell lines stably expressing NSM-tuberin.

(a) Tetracycline regulated the expression of each tuberin variant using Tet-on 3G system in HEK293 Tet-On 3G cells. Expression of each tuberin variant was appropriately induced by the presence or absence of doxycycline (Dox). Note that the previously reported band shift of GSM-tuberin compared to WT- or NSM-tuberin band was reproducible¹⁵,²³, suggesting that the system is working properly. The blots have been cropped, focusing on the bands or interest; See Supplementary Fig. S2 for full-length blots. (b) Tetracycline regulated the expression of each tuberin variant and ZsGreen using Tet-on 3G system in HEK293 Tet-On 3G cells. Representative immunofluorescent images are shown. Simultaneous tuberin variant and ZsGreen expression utilising IRES sequence was properly regulated by the presence or absence of Dox. (c) Representative flow cytometry (FCM) data showing the purification of ZsGreen-positive cells. The horizontal and vertical axes show the logarithmic fluorescent intensity and the number of cells, respectively. The percentages of the total live cells that were positive for ZsGreen are shown in each panel. Left panel; FCM before cell sorting (drug selection only). Although most cells are ZsGreen-positive, there are two peaks of cells in fluorescent intensity. To solve this heterogeneity in fluorescent intensity (indicating variable translational efficiency), cells were subjected to cell sorting. Right panel; FCM after cell sorting by gating stronger-ZsGreen-intensity peak. Subgroups of cells with weaker ZsGreen intensity (left peak in the left panel) were eliminated and subgroups with stronger ZsGreen intensity (right peak in the left panel) were purified by cell sorting. Cell lines stably expressing WT- and GSM-tuberin were successfully cultured, but despite numerous improvements in the methodology, cell lines that stably express NSM-tuberin were not obtained.