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Figure 1: Overexpression of Rheb promotes protein synthesis and
increases ER stress markers. (A) HEK293 cells were transfected with
indicated vectors (EV = empty vector) and grown for 48 h prior to
harvest. Lysates were harvested for SDS-PAGE western blot analysis with
the indicated antibodies. SuNSET assays were then performed; the growth
medium was changed for EBSS 90 min prior to addition of 30 µM puromycin
for 30 min. Samples indicated ‘CHX’ were pre-treated with cycloheximide,
to inhibit ongoing protein synthesis. (B) Quantification of
data from (A). (C) Lysates of cells that had been transfected
with the indicated vectors were analysed by SDS-PAGE and immunoblot
using the indicated antibodies. (D-G) RT-qPCR analysis for
known ER stress markers was performed on cDNA extracted from HEK293
cells expressing the indicated Rheb mutants or an empty vector (EV).
Figures represent mean ± s.d for 3 independent experiments. All graphs
represent mean ± s.d for 3 independent experiments. The data were
analysed via Student’s t-test compared to EV. *: P < 0.05; **
P < 0.01; *** P < 0.001.
Figure 2: Rheb-T23M and E40K drive increased ER volume.(A) Immunofluorescence of HEK293 cells transiently expressing
the indicated Rheb mutants or EV. Cells were transfected with indicated
vectors 24 h prior to fixation and addition of the indicated primary
antibodies. (B) Quantification of (A) for the ratio of area
containing Calnexin : area containing actin. Graph represent mean ± s.d
for 3 independent experiments. The data were analysed via Student’s
t-test. *: P < 0.05; ** P < 0.01.
Figure 3: Rheb mutants drive constitutive mTORC1 signalling in
GLuc-CHO cells. (A) SDS-PAGE and western blot analysis of
lysates harvested from GLuc-CHO cells transiently expressing the
indicated Rheb mutants or transfected with an empty vector (EV); some
were co-transfected with FLAG-TSC1/2, as indicated. The growth medium
was replaced with medium lacking FBS for 16 h followed by D-PBS for 60
min immediately prior to harvesting. (B) As in (A) for FLuc-CHO
cells. (C) GLuc-CHO cells expressing the indicated Rheb mutants
or an empty vector (EV) were grown in fully supplemented media with cell
number recorded every 24 h for 7 days. (D) As in (C) with cells
grown in media lacking FBS. Figures are representative of mean ± S.D for
3 independent experiments. (E) BrdU incorporation assay of
FLuc-CHO cells expressing the indicated Rheb mutants or EV. Cells were
allowed to grow in fully supplemented media for 24 h before addition of
BrdU for 1 h. (F) As in (E) for GLuc-CHO cells. All graphs
represent mean ± s.d for 3 independent experiments. The data were
analysed via Student’s t-test compared to EV. *: P < 0.05; ***
P < 0.001.
Figure 4: Rheb mutants promote increased secretion of Gaussia
Luciferase. (A) CHO cells stably expressing Firefly luciferase
(FLuc) were transfected with the indicated Rheb mutants or an empty
vector (EV); FLuc assays were then performed every 48 h for 7 days.
Results are normalized to cell number. (B) Quantification of
data in (A) for day 7. (C) CHO cells stably expressing
Gaussia Luciferase (GLuc) were transfected with the indicated Rheb
mutants or an empty vector (EV) and treated with either DMSO (solid
line) or 1 µM AZD8055 (dashed line) for the duration of the experiment.
GLuc assays performed on growth media every 48 h for 7 days. Results are
normalized to cell number. (D) Quantification of data in(C) for day 7. (E) As in (C), but cells were treated
with a cocktail of 1 µM iPERK and 1 µM ISRIB (dashed line). Experiments
shown (C) and (E) were performed simultaneously but are presented
separately for clarity. (F) SDS-PAGE western blot analysis of
cell lysates harvested from GLuc-CHO cells transiently transfected with
the indicated Rheb mutants or an empty vector (EV). All graphs represent
mean ± s.d for 3 independent experiments. The data were analysed via
Student’s t-test. *: P < 0.05; ** P < 0.01; *** P
< 0.001.
Figure 5: Rheb T23M and E40K drive increased secretion of mAb
from CHO-S cells. (A) SDS-PAGE western blot analysis of
lysates harvested from Expi-CHO cells stably over-expressing the
indicated Rheb mutant or transfected with empty vector (EV).(B) SDS-PAGE western blot analysis of lysates harvested from
Expi-CHO cells stably over-expressing the indicated Rheb mutant or
transfected with empty vector (EV). (C) EXPI-CHO cells stably
expressing the indicated Rheb mutant or endogenous Rheb (Endo) were
grown for 48 h prior treatment with 30 µMt puromycin for 30 m. Lysates
were then harvested for SuNSET assays. (D) Expi-CHO cells
stably expressing the indicated Rheb mutants or endogenous Rheb (Endo)
were allowed to proliferate with cell number recorded every 24 h for 7
days. (E) BrdU incorporation assay of Expi-CHO cells stably
expressing the indicated Rheb mutants or endogenous Rheb (Endo). Cells
were allowed to proliferate for 24 h before addition of BrdU for 1 h.(F) IgG yield from Expi-CHO cells stably expressing the
indicated Rheb mutant or endogenous Rheb after 10 days of expression.(G) Time course of IgG secretion from Expi-CHO cells stably
expressing the indicated Rheb mutant. Figure represents n=1. All other
figures are representative of 3 independent experiments. All graphs
represent mean ± s.d for 3 independent experiments. The data were
analysed via Student’s t-test compared to Endo. *: P < 0.05;
** P < 0.01; *** P < 0.001.
Supplementary Figure 1: Mutant Rheb drives an increase in levels
of proteins of the ER stress response . Related to Figure 1Quantification of SDS-PAGE western blot data for Fig. 1a. All graphs
represent mean ± s.d for 3 independent experiments. The data were
analysed via Student’s t-test compared to Endo. *: P < 0.05.
Supplementary Figure 2: Rheb-T23M displays increased
proliferation on media containing reduced FBS. Related to Figure 3. (A)GLuc-CHO cells expressing the indicated Rheb mutants or an empty vector
(EV) were grown in media supplemented with 1% FBS with cell number
recorded every 24 h for 7 days. (B) f-CHO cells expressing the
indicated Rheb mutants or EV were grown in media supplemented with 0.5%
FBS with cell number recorded every 24 h for 7 days. All graphs
represent mean ± s.d for 3 independent experiments. The data were
analysed via Student’s t-test compared to Endo. *** P < 0.001.
Supplementary Figure 3: Characterization of Expi-CHO cells
stably over expressing Rheb mutants. Related to Figure 5. Sanger
sequencing results of Expi-CHO monoclonal cell lines transfected with
vector encoding both the indicated Rheb mutant and NeoR to grant
resistance to G-418. Cells were grown for 6 weeks in the presence of 1
µM G-418 before DNA was extracted and sequenced using primers specific
to the inserted plasmid.