EMBO/soda-nel huggingface dataset:
The yeast mitochondrial pyruvate carrier is a hetero-dimer in its functional state (2019)
Figure 1

Figure 1 - Purification and stability analysis of Mpc proteins.

A Strategy for purification of the Mpc1/Mpc3 hetero-complex by nickel-affinity chromatography.

B Expression of Mpc proteins in mitochondria assessed by SDS-PAGE and immunoblot analysis of crude mitochondrial preparations. The individual untagged Mpc1 (Mpc1), histidine-tagged Mpc1 (Mpc1his), histidine-tagged Mpc3 (Mpc3his) or the Mpc1/Mpc3 hetero-complex (Mpc1/Mpc3his) were detected with antibodies raised against Mpc1 (left panel) or Mpc3 (right panel) and are shown with dashed arrows.

C Five micrograms of each affinity-purified Mpc protein were analysed by SDS-PAGE and the bands were visualised by Coomassie Blue stain. Peptide mass finger printing was used to identify the major protein bands (Table EV1).

D The stability of the purified proteins was assessed via thermal denaturation by fluorescent CPM-adduct formation. The thermal denaturation profiles (left) were used to calculate the first derivative (right), which provides the apparent melting temperature, indicated with the same colour coding.

E The stability of the same samples, as in panel (D), was assessed by NanoDSF. The changes in the 330 nm/350 nm ratio with temperature (left) were used to calculate the first derivative (right). Color coding is as in panel (D).
The yeast mitochondrial pyruvate carrier is a hetero-dimer in its functional state (2019)
Figure 2

Figure 2 - The Mpc proteins form dimeric complexes.

A Nickel-affinity-purified Mpc1/Mpc3 hetero-complex used for SEC-MALLS analysis, showing a 1:1 stoichiometry of the protomers.

B SEC-MALLS analysis of the hetero-complex. The light scattering trace for Mpc1/Mpc3 is shown as a black line. The masses of the protein-detergent-lipid complex (PDL, green), the detergent-lipid micelle (DL, blue) and the protein (P, red) are indicated. Protein fractions across the peak were assessed by SDS-PAGE and visualised by Coomassie Blue staining (B, inset).

C Nickel-affinity-purified Mpc3 protein used for SEC-MALLS analysis.

D SEC-MALLS analysis of Mpc3, colour designation as in (B). The protein fractions across the peak (D, inset) were assessed as in (B).

Data information: Data in (B and D) represent a characteristic experiment repeated independently five times for Mpc1/Mpc3 and three times for Mpc3. All biological repeats are summarised in Table EV2.
The yeast mitochondrial pyruvate carrier is a hetero-dimer in its functional state (2019)
Figure 3

Figure 3 - The Mpc1/Mpc3 hetero-complex transports pyruvate.

A Time course of pyruvate homo-exchange by the Mpc1/Mpc3 hetero-complex in liposomes (n=8) in comparison to empty liposomes (n=6) at a ΔpH of 1.6.

B Kinetic analysis of pyruvate homo-exchange at ΔpH of 1.6 (n=3). The hetero-complex was assayed for initial rates of uptake in the concentration range of 25-600 μM. The calculated KΜ was 299 μΜ in this experiment and 318 and 409 μΜ in two additional biological repeats.

C Time course of pyruvate homo-exchange by the Mpc1/Mpc3 hetero-complex in physiological pH (n=4) compared to empty liposomes (n=4).

D In the absence of a ΔpH, the time course of pyruvate homo-exchange was similar for the Mpc1/Mpc3 proteoliposomes and the empty liposomes (n=4).

E Inhibition of [14C]-pyruvate homo-exchange by UK5099 (1-100 μM), Zaprinast (1-1000 μM), lonidamine (10-10000 μM) and 7ACC2 (5-500 μM). Data points represent the mean of three technical replicates of a typical experiment. The IC50 measurements have also been independently replicated, three times for UK5099 Zaprinast and 7ACC2 (average IC50 9 ± 7 μM, 18 ± 8 μM and 27 ± 13 μM, respectively) and two times for lonidamine (average IC50, 118 ± 24 μM).

F [14C]-pyruvate homo-exchange inhibition by the TZDs, pioglitazone and rosiglitazone (n=6).

Data information: Data have been independently replicated: (A) four biological repeats, (B) three biological repeats, (C, D) 2 biological repeats (E, F) three biological repeats for UK5099, Zaprinast, 7ACC2 and two biological repeats for lonidamine and the TZDs. The error bars represent the standard error of the mean in (A) and the standard deviation in (C, D and F).
The yeast mitochondrial pyruvate carrier is a hetero-dimer in its functional state (2019)
Figure 4

Figure 4 - Mpc1 or Mpc3 alone are not functional.

A Time course of pyruvate homo-exchange in proteoliposomes at a ΔpH of 1.6 was compared for Mpc3 (n=4), the Mpc1/Mpc3 hetero-complex (n=6) and empty liposomes (n=4).

B Time course of pyruvate homo-exchange at a ΔpH of 1.6 was compared for Mpc1 (n=4), Mpc1/Mpc3 hetero-complex (n=4) and empty liposomes (n=4).

C Thermostability analysis via cpm. In detergent solution, Zaprinast (250 μM) increased the thermostability of the Mpc1/Mpc3 hetero-complex (black lines) (n=3) but not of Mpc3 (blue lines) (n=3).

D Thermostability analysis via nanoDSF. In detergent solution, Zaprinast (250 μM) increased the thermostability of the Mpc1/Mpc3 hetero-complex (black lines) (n=3) but not of Mpc1 (orange lines) (n=3).

Data information: Data have been independently replicated in two biological repeats. The error bars represent standard deviations.

Expanded View Figure Legends
LncRNA SOX1-OT V1 Acts as a Decoy of HDAC10 to Promote SOX1-dependent hESC Neuronal Differentiation (2021)
Figure 1

Fig.1 SOX1-OT V1 is required for hESC neuronal differentiation

(A) QRT-PCR assay of PAX6, SOX1, NKX2-1, NKX2-2, TBR1 and TBR2 during dorsal (up) and ventral (down) neural differentiation.

(B) QRT-PCR assay of SOX1-OT V1 during dorsal (up) and ventral (down) neural differentiation

(C) QRT-PCR assay of SOX1-OT V1 in control and V1-PAKI cells on day 16 of dorsal neural differentiation, p=0.0059.

(D) Immunostaining assay of PAX6 (green) in control and V1-PAKI cells on day 10 of neural differentiation. Scale bar, 100 μm.

(E) QRT-PCR assay of PAX6 and SOX1 in control and V1-PAKI cells on day 16 of dorsal neural differentiation, p=0.0146.

(F and G) Immunostaining assay of PAX6 (green) and SOX1 (red) in control and V1-PAKI cells on day 16 of dorsal neural differentiation (F), and quantiﬁcation of SOX1+ cells (G), p=0.0001. Scale bar, 100 μm.

(H and I) Immunostaining assay of MAP2 (green) in control and V1-PAKI cells on day 35 of dorsal neural differentiation (H), and quantiﬁcation of MAP2+ cells (I), p=0.0001. Scale bar, 100 μm.

(J and K) Immunostaining assay of PAX6 (green) and SOX1 (red) in control and V1-PAKI cells on day 16 of ventral neural differentiation (J), and quantiﬁcation of SOX1+ cells (K), p=0.0003. Scale bar, 100 μm.

(L and M) Immunostaining assay of GABA (green) in control and V1-PAKI cells on day 35 of ventral neural differentiation (L), and quantiﬁcation of GABA+ cells (M), p=0.0001. Scale bar, 100 μm.

Data information: Data are presented as mean ±SEM from three independent experiments (n=3). *p<0.05, **p<0.01, ***p<0.001 (unpaired two-tailed Student's t-test). See also Appendix Figure S1 and S2.
LncRNA SOX1-OT V1 Acts as a Decoy of HDAC10 to Promote SOX1-dependent hESC Neuronal Differentiation (2021)
Figure 2

Fig.2 SOX1 is the functional downstream gene of SOX1-OT V1 in neural differentiation

(A) Western blot assay of SOX1 in control and SOX1KO cells on day 16 of dorsal neural differentiation.

(B) Immunostaining assay of TBR2 (red) in control and SOX1KO cells on day 25 of dorsal neural differentiation. Scale bar, 100 μm.

(C and D) Immunostaining assay of MAP2 (green) in control and SOX1KO cells on day 35 of dorsal neural differentiation (C), and quantiﬁcation of MAP2+ cells (D), p=0.0001. Scale bar, 100 μm.

(E) Immunostaining assay of NKX2-1 (red) in control and SOX1KO cells on day 25 of ventral neural differentiation. Scale bar, 100 μm.

(F and G) Immunostaining assay of GABA (green) in control and SOX1KO cells on day 35 of ventral neural differentiation (F), and quantiﬁcation of GABA+ cells (G), p=0.0001. Scale bar, 100 μm.

(H and I) Immunostaining assay of MAP2 (green) after SOX1 overexpression in V1-PAKI cells on day 35 of dorsal neural differentiation (H), and quantiﬁcation of MAP2+ cells (I), p=0.0001 (V1-PAKI), p=0.0001 (V1-PAKI+ptSOX1+dox). Scale bar, 100 μm.

(J and K) Immunostaining assay of GABA (green) after SOX1 overexpression in V1-PAKI cells on day 35 of ventral neural differentiation (J), and quantiﬁcation of GABA+ cells (K), p=0.0001 (V1-PAKI), p=0.0002 (V1-PAKI+ptSOX1+dox). Scale bar, 100 μm.

Data information: Data are presented as mean ±SEM from three independent experiments (n=3). ***p<0.001 versus Ctrl group (D and G); (unpaired two-tailed Student's t-test). ***p<0.001 versus Ctrl group (I and K); ###p<0.001 versus -dox group (I and K); (one-way ANOVA). See also Appendix Figure S2 and S3.
LncRNA SOX1-OT V1 Acts as a Decoy of HDAC10 to Promote SOX1-dependent hESC Neuronal Differentiation (2021)
Figure 3

Fig.3 SOX1-OT V1 is crucial for proper histone acetylation at SOX1 promoter

(A and B) Enrichment of H3K14Ac (left) and H3K27Ac (right) on SOX1 promoter region in control and V1-PAKI cells on day 16 of dorsal neural differentiation (A), p=0.0046 (H3K14Ac), p=0.0223 (H3K27Ac) and ventral neural differentiation (B), p=0.0499 (H3K14Ac), p=0.0022 (H3K27Ac).

(C and D) Immunostaining assay of SOX1 (red) after 4 HDAC inhibitors (4i), including RG2833 (2833, 2μM), CAY10683 (CAY, 2μM), PCI-34051 (34051, 5μM) and Bufexamac (Buf, 12.5μM), treatment or remove one inhibitor from these 4 HDAC inhibitors (4i) in V1-PAKI cells on day 16 of dorsal neural differentiation (C), and quantiﬁcation of SOX1+ cells (D), p=0.0001 (V1-PAKI), p=0.0001 (4i), p=0.0001 (4i-2833), p=0.0001 (4i-34051), p=0.0001 (4i-CAY). Scale bar, 100 μm.

(E) QRT-PCR assay of SOX1 after 4 HDAC inhibitors (4i) treatment or remove one inhibitor from these 4 HDAC inhibitors (4i) in V1-PAKI cells on day 16 of dorsal neural differentiation, p=0.0053 (V1-PAKI), p=0.0240 (4i), p=0.0255 (4i-2833), p=0.0097 (4i-34051), p=0.0272 (4i-CAY).

(F) QRT-PCR assay of SOX1 after Bufexamac treatment in V1-PAKI cells on day 16 of dorsal neural differentiation, p=0.0283 (V1-PAKI), p=0.0288 (+Buf).

(G and H) Immunostaining assay of SOX1 (red) after Bufexamac treatment in V1-PAKI cells on day 16 of dorsal neural differentiation (G), and quantiﬁcation of SOX1+ cells (H), p=0.0001 (V1-PAKI), p=0.0001 (+Buf). Scale bar, 100 μm.

(I) Enrichment of H3K14Ac (up, p=0.0082 (V1-PAKI), p=0.0117 (+Buf)) and H3K27Ac (down, p=0.0106 (V1-PAKI), p=0.0014 (+Buf)) on SOX1 promoter region after Bufexamac treatment in V1-PAKI cells on day 16 of dorsal neural differentiation.

(J and K) Immunostaining assay of MAP2 (green) after Bufexamac treatment in V1-PAKI cells on day 35 of dorsal neural differentiation (J), and quantiﬁcation of MAP2+ cells (K), p=0.0001 (V1-PAKI), p=0.0001 (+Buf). Scale bar, 100 μm.

(L) Enrichment of RNA polymerase Ⅱ on SOX1 promoter region after Bufexamac treatment in V1-PAKI cells on day 16 of dorsal (left, p=0.0007 (V1-PAKI), p=0.0002 (+Buf)) and ventral (right, p=0.0083 (V1-PAKI), p=0.0276 (+Buf)) neural differentiation.

Data information: Data are presented as mean ±SEM from three independent experiments (n=3). *p<0.05, **p<0.01 versus Ctrl group (A and B); (unpaired two-tailed Student's t-test). *p<0.05, **p<0.01, ***p<0.001 versus Ctrl group (D, E, F, H, I, K and L); #p<0.05, ##p<0.01, ###p<0.001 versus V1-PAKI group (D, E, F, H, I, K and L); (one-way ANOVA). See also Appendix Figure S4.
LncRNA SOX1-OT V1 Acts as a Decoy of HDAC10 to Promote SOX1-dependent hESC Neuronal Differentiation (2021)
Figure 4

Fig.4 SOX1-OT V1 serves as decoy of HDAC10 to block HDAC10 binding to SOX1 promoter

(A) MS2bp-YFP RNA pull-down analysis for the interaction between SOX1-OT V1 and HDAC10 (up) or HDAC6 (down) in HEK293FT extracts.

(B and C) RIP analysis for endogenous interaction between HDAC10 and SOX1-OT V1 on day 16 of dorsal neural differentiation (B), p=0.0085 and ventral neural differentiation (C), p=0.0152.

(D and E) Enrichment of HDAC10 on SOX1 promoter region in control and V1-PAKI cells on day 16 of dorsal neural differentiation (D), p=0.0052 and ventral neural differentiation (E), p=0.0001.

(F) MS2bp-YFP RNA pull-down analysis for the interaction between HDAC10 and SOX1-OT V1-5P (up) or SOX1-OT V1-3P (down) in HEK293FT extracts.

(G and H) Immunostaining assay of SOX1 (red) after SOX1-OT V1, SOX1-OT V1-5P or SOX1-OT V1-3P overexpression in V1-PAKI cells on day 16 of dorsal neural differentiation (G), and quantiﬁcation of SOX1+ cells (H), p=0.0001 (V1-PAKI), p=0.0001 (ptV1+dox), p=0.0001 (pt5P+dox). Scale bar, 100 μm.

(I and J) Immunostaining assay of SOX1 (red) after SOX1-OT V1, SOX1-OT V1-5P or SOX1-OT V1-3P overexpression in V1-PAKI cells on day 16 of ventral neural differentiation (I), and quantiﬁcation of SOX1+ cells (J), p=0.0001 (V1-PAKI), p=0.0001 (ptV1+dox), p=0.0001 (pt5P+dox). Scale bar, 100 μm.

(K and L) Enrichment of HDAC10 on SOX1 promoter region after SOX1-OT V1, SOX1-OT V1-5P or SOX1-OT V1-3P overexpression in V1-PAKI cells on day 16 of dorsal neural differentiation (K), p=0.0219 (V1-PAKI), p=0.0181 (ptV1+dox), p=0.0183 (pt5P+dox) and ventral neural differentiation (L), p=0.0194 (V1-PAKI), p=0.0082 (ptV1+dox), p=0.0026 (pt5P+dox).

Data information: Data are presented as mean ±SEM from three independent experiments (n=3). *p<0.05, **p<0.01, ***p<0.001 versus IgG group (B and C) or versus Ctrl group (D and E); (unpaired two-tailed Student's t-test). *p<0.05, ***p<0.001 versus Ctrl group (H, J, K and L); #p<0.05, ##p<0.01, ###p<0.001 versus -dox group (H, J, K and L); (one-way ANOVA). See also Appendix Figure S5 and S6.
LncRNA SOX1-OT V1 Acts as a Decoy of HDAC10 to Promote SOX1-dependent hESC Neuronal Differentiation (2021)
Figure 5

Fig.5 ASCL1 is the effector molecule of SOX1 to promote hESC neuronal differentiation

(A and B) Immunostaining assay of ASCL1 (green) in control, V1-PAKI and SOX1KO cells on day 25 of dorsal neural differentiation (A), and quantiﬁcation of ASCL1+ cells (B), p=0.0021 (V1-PAKI), p=0.0024 (SOX1KO). Scale bar, 100 μm.

(C and D) Immunostaining assay of ASCL1 (green) in control, V1-PAKI and SOX1KO cells on day 25 of ventral neural differentiation (C), and quantiﬁcation of ASCL1+ cells (D), p=0.0003 (V1-PAKI), p=0.0004 (SOX1KO). Scale bar, 100 μm.

(E and F) Immunostaining assay of ASCL1 (green) after SOX1 overexpression in V1-PAKI cells on day 25 of dorsal neural differentiation (E), and quantiﬁcation of ASCL1+ cells (F), p=0.0001 (V1-PAKI), p=0.0001 (ptSOX1+dox). Scale bar, 100 μm.

(G and H) Immunostaining assay of ASCL1 (green) after SOX1 overexpression in V1-PAKI cells on day 25 of ventral neural differentiation (G), and quantiﬁcation of ASCL1+ cells (H), p=0.0001 (V1-PAKI), p=0.0001 (ptSOX1+dox). Scale bar, 100 μm.

(I and J) Immunostaining assay of MAP2 (green) after ASCL1 overexpression in V1-PAKI cells on day 35 of dorsal neural differentiation (I), and quantiﬁcation of MAP2+ cells (J), p=0.0001 (V1-PAKI), p=0.0001 (ptASCL1+dox). Scale bar, 100 μm.

(K and L) Immunostaining assay of GABA (green) after ASCL1 overexpression in V1-PAKI cells on day 35 of ventral neural differentiation (K), and quantiﬁcation of GABA+ cells (L), p=0.0001 (V1-PAKI), p=0.0001 (ptASCL1+dox). Scale bar, 100 μm.

Data information: Data are presented as mean ±SEM from three independent experiments (n=3). **p<0.01, ***p<0.001 versus Ctrl group (B and D); (unpaired two-tailed Student's t-test). ***p<0.001 versus Ctrl group (F, H, J and L); ###p<0.001 versus -dox group (F, H, J and L); (one-way ANOVA). See also Appendix Figure S7.
LncRNA SOX1-OT V1 Acts as a Decoy of HDAC10 to Promote SOX1-dependent hESC Neuronal Differentiation (2021)
Figure 6

Fig.6 Deletion of SOX1-OT V1 impairs neuronal differentiation in organoid culture

(A and B) Immunostaining assay of SOX1 (red) in control, V1-PAKI and SOX1KO cells on day 40 of dorsal (A) and ventral (B) organoids differentiation. Scale bar, 100 μm.

(C) Immunostaining assay of MAP2 (green) in control, V1-PAKI and SOX1KO cells on day 40 of dorsal organoids differentiation. Scale bar, 100 μm.

(D) Immunostaining assay of GABA (green) in control, V1-PAKI and SOX1KO cells on day 40 of ventral organoids differentiation. Scale bar, 100 μm.

(E and F) Immunostaining assay of SOX1 (red) after SOX1-OT V1 overexpression in V1-PAKI cells on day 40 of dorsal (E) and ventral (F) organoids differentiation. Scale bar, 100 μm.

(G) Immunostaining assay of MAP2 (green) after SOX1-OT V1 overexpression in V1-PAKI cells on day 40 of dorsal organoids differentiation. Scale bar, 100 μm.

(H) Immunostaining assay of GABA (green) after SOX1-OT V1 overexpression in V1-PAKI cells on day 40 of ventral organoids differentiation. Scale bar, 100 μm.

(I) Summary of the HDAC10/SOX1-OT V1-SOX1-ASCL1 regulatory axis in hESC neuronal differentiation.

Data information: See also Appendix Figure S8.
STK38 kinase acts as XPO1 gatekeeper regulating the nuclear export of autophagy proteins and other cargoes (2019)
Figure 2

Figure 2. XPO1 activity is required for STK38 cytoplasmic accumulation and autophagy upon nutrient starvation.

(A) STK38 interacts with XPO1. HekRasV12 cells were transiently transfected with myc-STK38(wt) together with either Flag-XPO1(wt) plasmid, Flag-control (ctrl = Sirt3) plasmid or without DNA. 24h later, cells were incubated with Okadaic Acid (OA), (final concentration = 1µM) for 1 hour or with DMSO. Flag fusions were pulled-down and co-immunoprecipited proteins were analyzed by western blotting (WB). Upper panel displays whole cell lysates (WCL) and lower panel represents immunoprecipitated proteins.

(B-C) STK38 accumulates in the cytoplasm upon nutrient starvation in a XPO1-dependant manner. (B) HeLa cells were transfected with myc-STK38(wt) plasmid. The next day, cells were incubated with DMEM or EBSS in the presence of XPO1 inhibitors KPT-185 or KPT-330 as indicated (final concentration = 1 µM) or DMSO for 4 hours. Cells were then fixed and stained for myc-tag. Representative images are shown and scale bars are 40 µm. (C) Quantification of myc-STK38(wt) nuclear/cytoplasmic staining (n > 30 cells from 3 independent experiments, mean ± SEM; *p<0.05, **p<0.01, ***p<0.001, Mann-Whitney test).

(D-E) XPO1 activity is required for nutrient starvation-induced autophagy. (D) Immunoblotting with indicated antibodies of whole cell lysates of cells in (B) and (C) and its graphical representation (n = 3 independent experiments, mean ± SEM; ***p<0.001, ns, not significant, Student's t test). As expected, p62 degradation in starvation conditions was inhibited in presence of XPO1 inhibitors. (E) HeLa cells stably expressing the GFP-LC3-RFP-LC3ΔG reporter autophagic probe [26] were incubated with DMEM or EBSS in the presence of XPO1 inhibitors KPT-185 or KPT-330 (final concentration = 1 µM) or DMSO for 4 hours. The GFP-LC3 (degraded upon autophagy) and RFP-LC3ΔG (non-degraded upon autophagy) signals were recorded by FACS analysis and then shown as a ratio recapitulating overall LC3 level (n=3 independent experiments, mean ± SEM; *p<0.05, **p<0.01, ns, not significant, Mann-Whitney test). Here again, incubation with XPO1 inhibitors significantly impaired the autophagy process (see Appendix Figure S2 C&D for stable cell line validation).
STK38 kinase acts as XPO1 gatekeeper regulating the nuclear export of autophagy proteins and other cargoes (2019)
Figure 3

Figure 3. STK38 kinase activity is necessary and sufficient to induce its cytoplasmic relocalization and autophagy.

(A-B) STK38 kinase activity is required and sufficient for its cytoplasmic accumulation upon nutrient starvation. (A) HeLa cells silenced for endogenous STK38 were transfected with myc-STK38(wt) expressing plasmid, myc-STK38(K118R) (STK38 kinase-dead version) plasmid or with HA-STK38(PIF) (STK38 constitutively active version) plasmid. 24 hours later, cells were incubated with DMEM or EBSS for 4 hours, fixed and stained for myc-tag or HA-tag. Representative images are shown and scale bars are 40 µm.

(B) Graphical representation of tag-STK38 variants nuclear staining/cytoplasmic staining (n > 30 cells from 3 independent experiments, mean ± SEM; **p<0.01, ***p<0.001, ns, not significant, Mann-Whitney test). (C-D) STK38 kinase activity is required and sufficient for nutrient-starvation induced autophagy. (C) HeLa cells stably expressing the GFP-LC3-RFP-LC3ΔG autophagic probe [26] were transiently transfected with siRNA targeting the 3'UTR region of endogenous STK38 (or with non-targeting siRNA (siNT)). The next day, cells were transiently transfected with the indicated STK38 mutants expressing plasmids (identical to (A-B)). 24 hours after plasmid transfection, cells were incubated with DMEM or EBSS for 4 hours. The GFP-LC3 (degraded upon autophagy) and RFP-LC3ΔG (non-degraded upon autophagy) signals were recorded by FACS analysis and then shown as a ratio recapitulating overall LC3 level (n=4 independent experiments, mean ± SEM; *p<0.05, ***p<0.001, ns, not significant, Mann-Whitney test). As published [8], depleting STK38 prevents autophagy to take place. This effect was partially reversed by expressing the wt ORF as well as constitutively active STK38 whereas kinase-dead version failed to reproduce endogenous STK38 effect. Expression of constitutively active STK38 is sufficient to induce a substantial change in the autophagic flux upon nutrient rich conditions (see Appendix Figure S3A for STK38 replacement). (D) HeLa cells were transiently transfected with siRNA targeting the 3'UTR region of endogenous STK38 (or with non-targeting siRNA (siNT)) and then transiently transfected with the indicated STK38 mutants expressing plasmids 48h after. The next day, the cells were incubated with DMEM or EBSS for 4 hours and overall p62 level was quantified for autophagy estimation (see Appendix Figure S3B for blots) (n = 3 independent experiments, mean ± SEM; ***p<0.001, ns, not significant, Student's t test).
STK38 kinase acts as XPO1 gatekeeper regulating the nuclear export of autophagy proteins and other cargoes (2019)
Figure 4

Figure 4. STK38 is required for phosphorylation of XPO1 on Ser1055.

(A-B) STK38 is required for XPO1_S1055 phosphorylation. (A) HeLa cells were transiently transfected with the indicated siRNA and subjected to Flag-XPO1(wt) transient transfection the following day. 48 hours later, cells were incubated with Okadaic Acid (OA, final concentration = 1 µM) for 1 hour or with DMSO. Immunoblotting was performed on whole cell lysates with indicated antibodies. (B) Graphical representation of the phospho S1055-XPO1 signal on total XPO1 (n = 3 independent experiments, mean ± SEM; *p<0.05, ns, not significant, Mann-Whitney test). As expected, XPO1 is phosphorylated on its Ser1055 upon OA treatment but not when STK38 is silenced (see Fig. S5 for antibody validation and STK38 silencing quantification).(C) STK38 kinase activity is required for XPO1_S1055 phosphorylation. HekRasV12 cells silenced (or not) for endogenous STK38 were transfected with Flag-XPO1(wt) expression plasmid in addition to myc-STK38(wt) or myc-STK38(K118R) (STK38 kinase-dead version) plasmids. The next day, cells were treated with 1 µM OA or with vehicle (DMSO) for 1 hour. Flag fusions were immunoprecipited and pulled-down proteins were analyzed by western blotting. Upper panel displays immunoprecipited proteins and lower panel represents whole cell lysates.