Download Supplementary Figure Legend

Supplementary information – figure and table legends
Figure S1 (Relates to Figure 2) – Kinase-dead SIK2 and SIK2 knock-down reduce
proliferation of LNCaP cells.
(A) LNCaP cells were stably transfected with a wild-type SIK2-Myc construct (WT), a
kinase-dead SIK2-Myc construct (KI) and the corresponding empty-vector construct (EV).
SIK2 over-expression was assessed by qRT-PCR. mRNA expression levels were normalised
against β-Actin and SDH. Data are represented as fold change compared with EV. (n=3; error
bars represent mean ± SEM; *p<0.05, **p<0.01, ***p<0.001)
(B) SIK2 protein expression was assessed by Western blot. Cell lysates were resolved by
SDS-PAGE, transferred to nitrocellulose membrane and immunoblotted for SIK2 (SigmaAldrich). β-Actin (Abcam) was used as loading control. There were too few cells overexpressing kinase-dead SIK2 to prepare protein lysates
(C) LNCaP cells over-expressing wild-type SIK2 (WT) or kinase-dead SIK2 (KI) after 7
days of culture.
(D) Cell proliferation was assessed using the CellTiter 96® AQueous One Solution Cell
Proliferation Assay (MTS, Promega). Formazan absorption at day 0, day 2, day 4, day 6 and
day 8 is shown. (n=4; error bars represent mean ± SD; *p<0.05, **p<0.01, ***p<0.001)
(E) Cell proliferation was assessed using the IncuCyte™ system. The percentage of cell
confluence every four hours for 10 days is shown. (n=4; error bars represent mean ± SD)
(F) Cell proliferation of LNCaP cells transiently knocked-down for SIK2 was assessed using
the CellTiter 96® AQueous One Solution Cell Proliferation Assay (MTS, Promega).
Formazan absorption every day for 7 days is shown. The control non-targeting siRNA (NT)
is shown in black, the SIK2 knocked-down cell lines (A, B, C) are shown in dashed. (n=3;
error bars represent mean ± SD; *p<0.05, **p<0.01, ***p<0.001)
(G) Cell proliferation was assessed using the IncuCyte™ system. Cell confluence was
measured every four hours for 10 days. (n=4; error bars represent mean ± SD)
(H) Anchorage-independent growth was assessed using a soft agar assay. LNCaP cells were
transfected with the control non-targeting siRNA (NT) or SIK2-siRNA A, B and C (A, B, C),
trypinised 24 h later and re-seeded in soft agar. After 7 days, the number of colonies was
determined using the CyQuant GR dye. The control non-targeting siRNA (NT) is shown in
black, the SIK2 knocked-down cell lines (A, B, C) are shown in white. (n=6; error bars
represent mean ± SD; *p<0.05, **p<0.01, ***p<0.001)
Figure S2 (Relates to Figure 2) – SIK2 knock-down reduces proliferation and viability of
LNCaP-Bic, C4-2 and DuCaP cells.
(A) SIK2 knock-down was confirmed by Western blot at day 4 and day 7 after transfection
with the control non-targeting siRNA (NT) or SIK2-siRNAs (A, B, C). Cell lysates were
resolved by SDS-PAGE, transferred to nitrocellulose membrane and immunoblotted for SIK2
(Sigma-Aldrich). β-Actin (Abcam) was used as loading control.
(B) Cell proliferation of LNCaP-Bic, C4-2 and DuCaP cells transiently knocked-down for
SIK2 was assessed by counting the cells using a Beckman Coulter™ Vi-Cell and Trypan
Blue staining. The number of viable cells at day 0, day 4 and day 7 is shown. The control
non-targeting siRNA (NT) is shown in black, the SIK2 knocked-down cell lines (A, B, C) are
shown in white. (n=3; error bars represent mean ± SEM; *p<0.05, **p<0.01, ***p<0.001)
(C) Cell viability was assessed with a Beckman Coulter™ Vi-Cell and Trypan Blue staining.
The percentage of viable cells at day 0, day 4 and day 7 is shown. (n=3; error bars represent
mean ± SEM; *p<0.05, **p<0.01, ***p<0.001)
Figure S3 (Relates to Figure 4) – SIK2 knock-down induces a cell cycle arrest in G1
phase and delays mitotic progression of LNCaP cells.
p27 and cyclin D1 protein expression was assessed by Western blot 48 h after transfection of
LNCaP cells with the control non-targeting siRNA (NT) and SIK2-siRNAs (A, B, C). Cell
lysates were resolved by SDS-PAGE, transferred to nitrocellulose membrane and
immunoblotted for p27 and cyclin D1 (Cell Signaling). SIK2 levels after knock-down are
also shown (Sigma-Aldrich). β-Actin (Abcam) was used as loading control.
Figure S4 (Relates to Figure 4) – SIK2 localises at the centrosomes of SKOv3 and LNCaP
cells.
SKOv3 (A) and LNCaP (B) cells were fixed and stained for immunofluorescence using γTubulin (Sigma-Aldrich) and SIK2 (BioLegend) antibodies. Cells are shown in interphase,
metaphase and anaphase. The γ-Tubulin staining is shown in red and SIK2 staining is shown
in green. DAPI was used for nuclear staining (blue). Images were taken using a Leica
Tandem confocal microscope and correspond to z sections of 14 µM. Scale bar, 10 µM.
Figure S5 (Relates to Figures S4 and S6) – Validation of SIK2, TORC1, TORC2 and
TORC3 antibodies by Western-Blot.
(A) The specificity of SIK2 antibody (BioLegend) was assessed by Western blot on parental
C4-2 cells (P) and on C4-2 cells transfected with a wild-type SIK2-Myc construct (WT), a
kinase-dead SIK2-Myc construct (KI), the corresponding empty-vector construct (EV) or
knocked-down for SIK2 (si). Cell lysates were resolved by SDS-PAGE, transferred to
nitrocellulose membrane and immunoblotted for SIK2 (BioLegend,). β-Actin (Abcam) was
used as loading control.
(B) LNCaP cells were transfected with TORC1, TORC2 and TORC3 siRNAs to transiently
knock-down TORC1, 2 and 3 respectively. TORC1, TORC2 and TORC3 protein expression
was assessed by Western blot four days after transfection. Cell lysates were resolved by SDSPAGE, transferred to nitrocellulose membrane and immunoblotted for TORC1, TORC2 and
TORC3 (Cell Signaling). TORC1, TORC2 and TORC3 are indicated with arrows. The
control non-targeting siRNA (NT) is also shown.
Figure S6 (Relates to Figure 5) – SIK2 regulates CREB1 activity via the phosphorylation
of TORC2 and TORC3 and their sequestration in the cytoplasm.
(A) LNCaP cells were treated with a SIK2 inhibitor (ARN3236) (Arrien Pharmaceuticals)
[22] before CREB1 activity was assessed using a CRE(1) reporter construct (Affymetrix) and
a dual-luciferase reporter assay system (Promega) 48 h (i.) or 12 h, 24 h, 36 h and 48 h after
treatment with the drug (ii.). A control Forskolin treatment (Forsk., 10 µM) or vehicle
(DMSO) was also included (n=6; error bars represent mean ± SD; *p<0.05, **p<0.01,
***p<0.001)
(B) Phosphorylation status of TORC2 and TORC3 were checked after treatment of total
protein lysates obtained from LNCaP cells 12 h after transfection with EV, WT, and KI using
an alkaline phosphatase (New England Biolabs) (+ CIP). Cell lysates were resolved by SDSPAGE, transferred to nitrocellulose membrane and immunoblotted for TORC2 and TORC3
(Cell Signaling). SIK2 levels after over-expression are also shown (Sigma-Aldrich). β-Actin
(Abcam) was used as loading control.
(C) Cellular fractionation was performed on LNCaP cells 12 h after transfection with EV,
WT, and KI. TORC2 and TORC3 protein expression in cytoplasmic (cytop.) and nuclear
(nucl.) fractions was assessed by Western blot. Cell lysates were resolved by SDS-PAGE,
transferred to nitrocellulose membrane and immunoblotted for TORC2 and TORC3 (Cell
Signaling). β-Tubulin and Histone H3 (Abcam) were used to confirm the purity of the
fractionation.
Figure S7 (Relates to Figure 6) – Validation of microarray hits.
(A) C1orf112, FOXO3, HOXA5, PTPRF, CCRN4L, DNAJC12 and ERRFl1 gene expression
was assessed by qRT-PCR in LNCaP cells after treatment with Forskolin (Forsk.), overexpression of kinase-dead SIK2 (KI) or SIK2 knock-down (si). mRNA expression levels
were normalized against β-Actin and SDH. Data are represented as fold change compared
with control Vehicle, Empty vector or non-targeting siRNA respectively. (n=4; error bars
represent mean ± SEM; p-values are indicated)
(B) ID1, NR4A1, HOXA5 and SIK1 gene expression was assessed by qRT-PCR in LNCaP
cells after treatment with Forskolin (F), over-expression of wild-type SIK2 (WT), kinasedead SIK2 (KI) or SIK2 knock-down (si). mRNA expression levels were normalized against
β-Actin and SDH. Data are represented as fold change compared with control Vehicle, Empty
vector or non-targeting siRNA respectively. (n=4; error bars represent mean ± SEM; p-values
are indicated)
Figure S8 (Relates to Figure 6) – SIK2 regulates a cell cycle network and a ER stress
network related to cancer.
(A) The gene ontology data base Ingenuity Pathway Analysis (IPA) (Ingenuity Systems Inc.
2011) was interrogated to determine the enriched networks related to the categories “diseases
and disorders” and “molecular and cellular functions” contained in the data set. The top five
diseases and disorders and molecular and cellular functions corresponding to the downregulated genes (green) and down-regulated genes (red) for SIK2 knock-down (SIK2siRNA), wild-type SIK2 (WT-SIK2) and kinase-dead SIK2 (SIK2-KI) are shown.
(B) All down-regulated genes corresponding to the molecular function “cell cycle” (211
genes) and all up-regulated genes corresponding to the molecular function “cell death and
survival” (152 genes) were submitted to GeneCodis. The top 10 enriched biological processes
are shown. The rank, number of genes and p-value are indicated. The DNA motifs enriched
in the top four biological processes were identified. The rank, transcription factor and p-value
are indicated.
Table S1 – qRT-PCR primer sequences. When primer sequences were not from published
studies, they were designed using Integrated DNA Technologies software. [52]
Table S2 – Detailed lists of differentially expressed genes (DEG) up- or down-regulated after
treatment with Forskolin (Forsk.), over-expression of wild-type SIK2 or kinase-dead SIK2
(KI) and SIK2 knock-down (SIK2si). The number of DEG in each list is indicated.
Table S3 – Detailed list of differentially expressed genes (DEG) up- or down-regulated after
both Forskolin treatment and kinase-dead SIK2 over-expression (Forsk. / KI), Forskolin
treatment and SIK2 knock-down (Forsk. / SIK2si), kinase-dead SIK2 over-expression and
SIK2 knock-down (KI / SIK2si) and in all three conditions. The number of DEG in each list
is indicated.
Table S4 - A detailed list of genes involved in the processes of "mitotic cell cycle", "protein
folding" and "response to stress". Genes are ranked according to their levels of expression
after SIK2 knock-down (SIK2si) or over-expression of kinase-dead SIK2 (KI).