EMBO/soda-nel huggingface dataset:
Complex inhibitory effects of nitric oxide on autophagy. (2011)
Figure 1

NO Inhibits Autophagosome Synthesis

(A) Immunoblot analysis with anti-LC3 antibody shows that NO donors reduced LC3-II levels in rat primary cortical neurons and decreased autophagosome synthesis in bafilomycin A1-treated rat primary cortical neurons and HeLa cells.

(B–E) Confocal microscopy images and analysis of autophagic flux by automated Cellomics microscopy show that NO donors reduced autolysosome numbers in mRFP-GFP-LC3 HeLa cells grown in full medium (FM) (B and D) or starved with HBSS (C and E). Cells in HBSS had more autolysosomes compared to FM (C). Autolysosome number in untreated cells in FM is set at 100%.

(F) NO donors increased EGFP-HDQ74 aggregates in a dose-dependent manner in EGFP-HDQ74–transfected Atg5+/+, but not in Atg5−/−, MEFs. Atg5−/− MEFs had more EGFP-HDQ74 aggregates than Atg5+/+ MEFs.

(G and H) Confocal microscopy images (H) and immunofluorescence analysis with anti-Atg16 antibody (G) show that NO donors reduced the percentage of HeLa cells with Atg16-positive structures (arrows) under starvation. Graphical data denote mean ± SEM.
Complex inhibitory effects of nitric oxide on autophagy. (2011)
Figure 2

NO Reduced Phosphorylation of JNK1 and Bcl-2, Leading to Increased Bcl-2–Beclin 1 and Decreased Beclin 1–hVps34 Interactions

(A) NO donors reduced JNK1 phosphorylation in HeLa cells, as analyzed by immunoprecipitation with anti-JNK1 agarose-conjugated beads and immunoblotting with anti-phospho-JNK antibody.

(B) NO donors reduced Bcl-2 phosphorylation in HeLa cells, as analyzed by immunoblotting with anti-phospho-Bcl-2 antibody.

(C) Immunoblot analysis with anti-phospho-Bcl-2 antibody shows that DETA NONOate could not reduce phospho-Bcl-2 in Flag-CA JNK1-transfected HeLa cells, where phospho-Bcl-2 was higher compared to mock-transfected cells.

(D and E) Immunoprecipitation with anti-Flag M2 affinity agarose gel and immunoblotting with anti-Myc antibody shows that NO donors increased the interaction of Flag-Beclin 1 with WT Myc-Bcl-2 (D), but not with AAA Myc-Bcl-2 (E), in HeLa cells transfected with WT Myc-Bcl-2 (D) or AAA Myc-Bcl-2 (E) along with either empty Flag or Flag-Beclin 1. Asterisk denotes IgG band (E).

(F) Immunoprecipitation with anti-Flag M2 affinity agarose gel and immunoblotting with anti-Vps34 antibody shows that NO donors decreased Flag-Beclin 1–hVps34 interaction in HeLa cells transfected with hVps34 along with either empty Flag or Flag-Beclin 1.

(G) Immunoprecipitation with anti-Flag M2 affinity agarose gel and immunoblotting with anti-Vps34 antibody shows that AAA Myc-Bcl-2 decreased Flag-Beclin 1–hVps34 interaction in the presence (right panel) or absence (left panel) of DETA NONOate in HeLa cells transfected with hVps34 along with either empty Flag or Flag-Beclin 1, and with pcDNA3.1 or AAA Myc-Bcl-2. Graphical data denote mean ± SEM.
Complex inhibitory effects of nitric oxide on autophagy. (2011)
Figure 3

NO Impairs Autophagy in AAA Bcl-2 and Jnk1−/− MEFs by Activating mTORC1

(A) Immunoblot analysis with anti-LC3 antibody shows that DETA NONOate reduced autophagosome synthesis in bafilomycin A1-treated WT Bcl-2 and AAA Bcl-2 MEFs.

(B and C) NO donors decreased EGFP-LC3 vesicles in EGFP-LC3–transfected WT Bcl-2 and AAA Bcl-2 MEFs (C). Images were acquired by a confocal microscope (B). WT Bcl-2 and AAA Bcl-2 MEFs were analyzed separately.

(D) NO donors increased EGFP-HDQ74 aggregates in EGFP-HDQ74–transfected WT Bcl-2 and AAA Bcl-2 MEFs. WT Bcl-2 and AAA Bcl-2 MEFs were analyzed separately.

(E) Immunoblot analysis with anti-LC3 antibody shows that DETA NONOate reduced autophagosome synthesis in bafilomycin A1-treated WT Jnk, Jnk1−/−, and Jnk2−/− MEFs.

(F) Immunoblot analyses with anti-phospho-S6K and anti-phospho-S6 antibodies show that NO donors activated mTORC1 in HeLa cells.

(G) Confocal microscope images of immunofluorescence with anti-phospho-S6 antibody show that DETA NONOate increased S6 phosphorylation; arrowhead shows a cell where this effect was not observed.

(H) Immunoblot analyses with anti-phospho-S6K and anti-phospho-S6 antibodies show that NO donors activated mTORC1 in WT Bcl-2 and AAA Bcl-2 MEFs.

(I) Confocal microscope images of immunofluorescence with anti-phospho-mTOR and anti-LAMP1 antibodies in HeLa cells show that DETA NONOate increased phospho-mTOR but did not alter its distribution with lysosomes. Graphical data denote mean ± SEM.
Complex inhibitory effects of nitric oxide on autophagy. (2011)
Figure 4

Activation of mTORC1 by NO Is Dependent on TSC2 and IKKβ

(A) Immunoblot analyses with anti-phospho-S6K and anti-phospho-S6 antibodies show that NO donors activated mTORC1 in Tsc2+/+ MEFs, but not in Tsc2−/−, MEFs.

(B) NO donors reduced phosphorylation of IKKα/β and AMPKα in MEFs, as analyzed by immunoblotting with anti-phospho-IKKα/β and anti-phospho-AMPKα antibodies, respectively.

(C) Immunoblot analyses with anti-phospho-S6K and anti-phospho-S6 antibodies show that NO donors activated mTORC1 in Ikkβ+/+ MEFs, but not in Ikkβ−/− MEFs.

(D) Immunoblot analysis with anti-LC3 antibody shows that DETA NONOate reduced autophagosome synthesis in bafilomycin A1-treated Tsc2+/+ and Tsc2−/− MEFs.

(E) DETA NONOate decreased EGFP-LC3 vesicles in Tsc2+/+ and Tsc2−/− MEFs transfected with EGFP-LC3, along with either pcDNA3.1 or AAA Myc-Bcl-2. Although it further reduced EGFP-LC3 vesicles in Tsc2+/+ MEFs expressing AAA Myc-Bcl-2 compared to mock-transfected Tsc2+/+ MEFs, this effect was not seen in Tsc2−/− MEFs. Tsc2+/+ and Tsc2−/− MEFs were analyzed separately. Graphical data denote mean ± SEM.
Complex inhibitory effects of nitric oxide on autophagy. (2011)
Figure 5

Overexpression of Mammalian NOS Isoforms Inhibits Autophagosome Synthesis

(A–D) Immunoblot analyses with anti-LC3, along with anti-nNOS, anti-iNOS, and anti-eNOS antibodies, show decreased LC3-II levels in stable HEK293 cell lines overexpressing nNOS (A and D), iNOS-GFP (B and D), or eNOS (C and D) compared to HEK293 control cells in the presence or absence of bafilomycin A1.

(E and F) mCherry-LC3 vesicles were reduced in mCherry-LC3–transfected stable HEK293 NOS cell lines compared to HEK293 control cells cultured in HBSS, an effect that was restored in the NOS cells by L-NAME (F). Images were acquired by a confocal microscope (E).

(G and H) Confocal microscopy images (G) and immunofluorescence analysis with anti-Atg16 antibody show that stable HEK293 NOS cell lines had fewer Atg16-positive structures under starvation, compared to HEK293 control cells (H). Graphical data denote mean ± SEM.
Complex inhibitory effects of nitric oxide on autophagy. (2011)
Figure 6

NOS Overexpression Primarily Inhibits the JNK1–Bcl-2 Pathway to Impair Autophagy

(A–F) Immunoblot analyses with anti-nNOS, anti-iNOS, anti-eNOS, anti-phospho-JNK1, anti-phospho-Bcl-2, anti-phospho-S6K, and anti-phospho-S6 antibodies show that stable HEK293 cell lines overexpressing nNOS (A and D), iNOS-GFP (B and E), or eNOS (C and F) inhibited both the JNK1–Bcl-2 and mTORC1 pathways compared to the HEK293 control cells under basal (FM) (A–C) or starvation (HBSS) (D–F) conditions. Graphical data denote mean ± SEM.
Complex inhibitory effects of nitric oxide on autophagy. (2011)
Figure 7

L-NAME Induces Autophagy and Reduces Mutant Huntingtin Aggregation/Neurodegeneration in Models of Huntington's Disease

(A) Immunoblot analyses with anti-LC3 antibody show that rapamycin (8 hr) and L-NAME (24 hr) increased autophagosome synthesis in bafilomycin A1-treated mouse primary cortical neurons and HeLa cells.

(B and C) Confocal microscopy images (B) and analysis of autophagic flux by automated Cellomics microscope (C) show that L-NAME increased autolysosomes in mRFP-GFP-LC3 HeLa cells.

(D) L-NAME reduced EGFP-HDQ74 aggregates in EGFP-HDQ74–transfected Atg5+/+, but not in Atg5−/−, MEFs.

(E) Drosophila expressing mutant huntingtin exon 1 (Q120) shows a significant decrease in neurodegeneration (p < 0.001, paired t test) upon L-NAME treatment compared to DMSO.

(F) Expression of an endogenous negative regulator of NOS activity (NOS4) significantly attenuates neurodegeneration (p < 0.05, paired t test) in Drosophila expressing mutant huntingtin exon 1 (Q120).

(G–I) Images from the retina of transgenic HD zebrafish show mutant huntingtin aggregates (arrows) (G). Treatment with rapamycin or L-NAME reduced the number of aggregates (H). L-NAME did not reduce aggregates in the presence of NH4Cl, which increased aggregate count (I).

(J) NO inhibits autophagy by S-nitrosylating and inhibiting JNK1 phosphorylation, thereby reducing phospho-Bcl-2 and increasing Bcl-2–Beclin 1 interaction, which disrupts hVps34–Beclin 1 association. NO also S-nitrosylates and inhibits IKKβ phosphorylation, leading to reduced phospho-AMPK and TSC2 activity, which alleviates the inhibitory effect of TSC1/2 on Rheb (denoted by “×”), thereby allowing Rheb to activate mTORC1 and inhibit autophagy. Overexpression of NOS isoforms impairs autophagy by inhibiting the JNK1 pathway, whereas NOS inhibition by L-NAME induces autophagy by mechanism distinct from the NO pathways. Graphical data denote mean ± SEM, except in (E) and (F).
The tRNA-like small noncoding RNA mascRNA promotes global protein translation (2020)
Figure 1

Figure 1. mascRNA forms a stable complex with cytosolic components

Gel shift assay of biotin-labeled mascRNA and 5S rRNA with the cytosol (Cyto) and the postnuclear membrane lysate (Mem).

Northern blotting of mascRNA (top panel) and western blotting of β-Tubulin and Mortalin (bottom two panels) in the cytosolic fractions and the postnuclear membrane fractions of HEK293 and HepG2 cells.
The tRNA-like small noncoding RNA mascRNA promotes global protein translation (2020)
Figure 2

Figure 2. mascRNA interacts with the multi-tRNA synthetase complex (MSC) component QARS

SDS-PAGE analysis and coomassie staining of the proteins in the RNA pulldown samples. Biotin-labeled mascRNA (S) and the antisense RNA (AS) were used to pull down the RNA-protein complexes.

Representive data of mass spectrometry analysis of the RNA pulldown samples. Proteins most enriched in mascRNA (S) RNA pulldown sample are listed.

Western blots of the proteins in mascRNA S and AS pulldown samples (top 4 panels), and mascRNA and menRNA pulldown samples (Bottom 2 panels). β-tubulin was used as a loading control.

qRT-PCR analysis of RNAs extracted from ARS immunoprecipitation (RIP) samples (erro bars: mean ± SD; biological replicates n = 3). Eight complex-forming ARSs (DARS, EPRS, IARS, KARS, LARS, MARS, QARS and RARS) and GARS tagged with FLAG were overexpressed in HEK cells. ARS-RNA complexes were imminoprecipitated with anti-FLAG Magnetic Beads. The cell line constructed with PQCXIP vector (PQ) was used as a negative control. GAPDH was used as an internal control for qRT-PCR.

Northern blot validation of mascRNA in the RIP samples. 5S rRNA from the RIP samples was used as an internal control.

Northern blot (top 2 panels) and western blot (bottom 3 panels) analysis of ARS immunoprecipitation samples (RIP). RIP was performed using EPRS, QARS and GARS antibodies.

Gel shift assay of biotin-labeled mascRNA with the cytosol in the presence or the absence of a QARS antibody.
The tRNA-like small noncoding RNA mascRNA promotes global protein translation (2020)
Figure 3

Figure 3. The anticodon stem-loop is required for mascRNA-QARS interaction

Biotin-labeled mascRNA (sense (S) and antisense (AS)) and the acceptor stem mutants (Mut1-8) were used to pulldown the RNA-protein complexes. Top panel: western blotting of QARS in the pulldown samples. β-Tubulin was used as an cytosolic input control for the pulldown. Bottom panel: Biotin detection of the biotin-labeled mascRNA and the mutants.

Biotin-labeled mascRNA (sense (S) and antisense (AS)) and the anticodon stem-loop mutants (Anti-Mut1-9) were used to pulldown the RNA-protein complexes. Top panel: western blotting of QARS in the pulldown samples. β-Tubulin was used as an cytosolic input control for the pulldown. Bottom panel: Biotin detection of the biotin-labeled mascRNA and the mutants.

The secondary structure of mascRNA with the acceptor stem and the anticodon stem-loop in red boxes

The acceptor stems of mascRNA and the mutants with the mutated nucleotides in red.

The anticodon stem-loops of mascRNA and the mutants with the mutated nucleotides in red.

Gel shift assay of biotin-labeled mascRNA or the mutants (Anti-Mut1 and Mut1) with the cytosol.

Comparison of human mascRNA and tRNAGlnUUG secondary structures. The identical nucleotides were marked in red.
The tRNA-like small noncoding RNA mascRNA promotes global protein translation (2020)
Figure 4

Figure 4. mascRNA promotes global protein translation

Northern blot analysis of mascRNA in mascRNA overexpressing HEK cells (mascRNA) and the cells expressing the scrambled RNA (Scramble). 5S rRNA was used as an internal control.

qRT-PCR analysis of MALAT1 (total RNAs used as templates) in mascRNA overexpressing (mascRNA) and the scrambled RNA expressing (Scramble) cells.

Detection of the newly synthesized proteins in HEK cells stably expressing mascRNA (mascRNA) or the scrambled RNA (Scramble). Left: biotin detection of the labeled nascent proteins; right: coomassie staining of total proteins.

Quantification of the levels of the labeled nascent proteins in Panel C .

Northern blot analysis of mascRNA in HEK cells transiently transfected with in vitro synthesized eGFP RNA fragment or mascRNA. 5S rRNA was used as an internal control.

Detection of the newly synthesized proteins in HEK cells transiently transfected with in vitro synthesized eGFP RNA fragment or mascRNA. Left: biotin detection of the labeled nascent proteins; right: coomassie staining of total proteins.

Quantification of the levels of the labeled nascent proteins in Panel F.

Northern blot analysis of mascRNA in mascRNA overexpressing (mascRNA), and the mutants (Anti-Mut1 and Mut1) or the scrambled RNA (Scramble) expressing HEK cells. 5S rRNA was used as an internal control.

Detection of the newly synthesized proteins in HEK cells stably expressing mascRNA (mascRNA), the mutants (Anti-Mut1 and Mut1) or the scrambled RNA (Scramble). Left: biotin detection of the labeled nascent proteins; right: coomassie staining of total proteins.

Quantification of the levels of the labeled nascent proteins in Panel I.

Decay of the pulse Biotin-labeled proteins in HEK cells stably expressing mascRNA (mascRNA) or the scrambed RNA (Scramble)

Data information: Statistical comparisons are performed using unpaired t-tests (biological replicates n = 3); **P<0.01, ***P<0.001. Data are presented as mean ± standard error of the mean (s.e.m.).
The tRNA-like small noncoding RNA mascRNA promotes global protein translation (2020)
Figure 5

Figure 5. mascRNA increases QARS protein stability

Western blot detection of ARS proteins in mascRNA overexpressing (mascRNA) or the scrambled RNA expressing (Scramble) cells. β-Tubulin was used as a loading control.

Quantification of the protein levels in Panel A.

Western blot detection of ARS proteins in HEK cells stably expressing mascRNA (mascRNA), the mutants (Anti-Mut1 and Mut1) or the scrambled RNA (Scramble). β-Tubulin was used as a loading control.

Quantification of the protein levels in Panel C.

qRT-PCR analysis of MALAT1 RNA levels in MALAT1 knockdown and the control cells.

Northern blot analysis of mascRNA in MALAT1 knockdown and the control cells. 5S rRNA was used as an internal control.

Western blot detection of ARS proteins in MALAT1 knockdown and the control cells.

Quantification of the protein levels in Panel G.

qRT-PCR analysis of GARS and QARS mRNAs in mascRNA overexpressing (mascRNA) or the scrambled RNA expressing (Scramble) cells.

Western blot detection of ARS proteins in mascRNA overexpressing (mascRNA) or the scrambled RNA expressing (Scramble) cells, before and after emetine and cycloheximide treatment. β-Tubulin was used as a loading control.

Quantification of the relative QARS protein levels in Panel J.

Quantification of the relative GARS protein levels in Panel J.

Data information: Statistical comparisons are performed using unpaired t-tests (biological replicates n = 3); **P<0.01, ***P<0.001. Data are presented as mean ± standard error of the mean (s.e.m.).
The tRNA-like small noncoding RNA mascRNA promotes global protein translation (2020)
Figure 6

Figure 6. mascRNA promotes global protein translation by increaseing QARS protein levels

Western blot detection of endogenous and FLAG-tagged ARS proteins in HEK cells transiently transfected with PQCXIP vector (PQ), QARS expressing (QARS) or GARS expressing (GARS) plasmid. β-Tubulin was used as a loading control.

Detection of the newly synthesized proteins in HEK cells transiently transfected with PQCXIP vector (PQ), QARS expressing (QARS) or GARS expressing (GARS) plasmid. Left, biotin detection of the labeled nascent proteins; right, coomassie staining of total proteins.

Quantification of the labeled nascent protein levels in Panel B.

Western blot detection of FLAG-tagged ARS proteins in mascRNA overexpressing cells transiently transfected with PQCXIP vector (PQ), QARS expressing (QARS) or GARS expressing (GARS) plasmid. β-Tubulin was used as a loading control.

Detection of the newly synthesized proteins in mascRNA overexpressing cells transiently transfected with PQCXIP vector (PQ), QARS expressing (QARS) or GARS expressing (GARS) plasmid. Left, biotin detection of the labeled nascent proteins; right, coomassie staining of total proteins.

Quantification of the labeled nascent protein levels in Panel E.

Western blot detection of FLAG-tagged ARS proteins in mascRNA anticodon stem-loop mutant Anti-Mut1 expressing cells transiently transfected with PQCXIP vector (PQ), QARS expressing (QARS) or GARS expressing (GARS) plasmid. β-Tubulin was used as a loading control.

Detection of the newly synthesized proteins in mascRNA Anti-Mut1 expressing cells transiently transfected with PQCXIP vector (PQ), QARS expressing (QARS) or GARS expressing (GARS) plasmid. Left, biotin detection of the labeled nascent proteins; right, coomassie staining of total proteins.

Quantification of the labeled nascent protein levels in Panel H.

Western blot detection of FLAG-tagged ARS proteins in MALAT1 knockdown cells with stable QARS (QARS) or GARS (GARS) overexpression or without (PQ). β-Tubulin was used as a loading control.

Detection of the newly synthesized proteins in wild-type HEK cells (WT) and MALAT1 knockdown cells (sh-MALAT1), or MALAT1 knockdown cells with stable QARS (QARS) or GARS (GARS) overexpression or without (PQ). Left, biotin detection of the labeled nascent proteins; right, coomassie staining of total proteins.

Quantification of the labeled nascent protein levels in Panel K.

Detection of the newly synthesized proteins in QARS (shQARS) or GARS (shGARS) knockdown cells and the control cells stably overexpressing mascRNA (mascRNA) or expressing the scrambled RNA (Scramble). Left, biotin detection of the labeled nascent proteins; right, coomassie staining of total proteins.

Quantification of the labeled nascent protein levels in Panel M.

Data information: Statistical comparisons are performed using unpaired t-tests (biological replicates n = 3); *P<0.05, **P<0.01, ***P<0.001 Data are presented as mean ± standard error of the mean (s.e.m.).
The tRNA-like small noncoding RNA mascRNA promotes global protein translation (2020)
Figure 7

Figure 7. mascRNA promotes cell proliferation

Northern blotting of mascRNA and 5S rRNA in the lysates of HEK cells before and after FBS starvation.

Northern blotting of mascRNA and 5S rRNA in the lysates of human embryonic stem cells on different days of differentiation.

Soft agar colony formation assay of HEK, HeLa or MCF-7 cells overexpressing mascRNA or expressing the the scrambled RNA.

Quantification of stained colones in Panel C.

Soft agar colony formation assay of HEK cells overexpressing mascRNA, or expressing the mutants (Anti-Mut1 and Mut1) or the the scrambled RNA (Scramble).

Soft agar colony formation assay of QARS (shQARS) or GARS (shGARS) knockdown cells and the control cells stably overexpressing mascRNA (mascRNA) or expressing the the scrambled RNA (Scramble).

Soft agar colony formation assay of MALAT1 knockdown cells (shMALAT1) and the control cells (Control) with stable QARS (QARS) or GARS (GARS) overexpression or without (PQ).

Data information: Statistical comparisons are performed using unpaired t-tests (biological replicates n = 3); **P<0.01, ***P<0.001. Data are presented as mean ± standard error of the mean (s.e.m.).

EXPANDABLE VIEW FIGURE LEGENDS
RNF43 truncations trap CK1 to drive niche-independent self-renewal in cancer (2020)
Figure 1

Fig 1. Cancer truncations endow RNF43 with oncogenic properties

A. Schematic representation of the RNF43 protein. SP; signal peptide, PA; protease-associated domain, TM; transmembrane domain, RING; E3 ligase catalytic domain, DIR; Dishevelled-interaction region, SRR; Serine-rich region, HRR; Histidine-rich region, PRR; Proline-rich region. The region in which oncogenic truncations occur (aa 504-563) is indicated.

B. β-catenin-mediated reporter activity in HEK293T cells expressing the indicated RNF43 cancer mutants. Cells were treated with control medium (no Wnt3a) or Wnt3a-conditioned medium (Wnt3a) overnight. Average β-catenin-mediated reporter activities ±s.d. in n = 2 independent wells are shown.

C. Western blot analysis showing the effect of RNF43 cancer mutants on V5-FZD5 expression in HEK293T cells. Open and closed arrows indicate mature (post-Golgi) and immature (ER-associated) FZD5, respectively.

D. Confocal microscopy of surface labeled SNAP-FZD5 in HEK293T cells upon expression of RNF43 WT or R519X. Cells were chased for 30 min. Scale bars represent 10 μm.

E. Confocal microscopy of β-catenin localization in HEK293T cells expressing the indicated RNF43 cancer mutants. Scale bars represent 10 μm.

F. Western blot analysis of RNF43 WT and R519X for cytoplasmic β-catenin levels.

G. β-catenin-mediated reporter activity in HEK293T cells expressing Wnt3a and WT RNF43, oncogenic RNF43 (R519X) or a LOF RNF43 variant (I48T) after o/n treatment with DMSO or the PORCN inhibitor C59 (1 μM).

Data information: IB; immunoblot, WT; wild-type.
RNF43 truncations trap CK1 to drive niche-independent self-renewal in cancer (2020)
Figure 2

Fig 2. Oncogenic RNF43 variants activate Wnt signaling downstream of the receptor complex and upstream of β-catenin

A. β-catenin-mediated reporter activity in HEK293T cells expressing WT RNF43 and oncogenic RNF43 (R519X) harboring mutations C290S/H292S in the RING domain (M1). Cells were treated with control medium (no Wnt3a) or Wnt3a-conditioned medium (Wnt3a) overnight. Average β-catenin-mediated reporter activities ±s.d. in n = 2 independent wells are shown.

B. Schematic of RNF43 constructs in which the extracellular and transmembrane domains (ECD and TM) are replaced by those of the unrelated CD16 and CD7, respectively. CCR; CD16-CD7-RNF43.

C. β-catenin-mediated reporter activity in HEK293T cells expressing the indicated RNF43 constructs. Cells were treated with control medium (no Wnt3a) or Wnt3a-conditioned medium (Wnt3a) overnight. Average β-catenin-mediated reporter activities ±s.d. in n = 2 independent wells are shown.

D. β-catenin-mediated reporter activity in HEK293T cells co-expressing oncogenic RNF43 (R519X) and the Dishevelled DEP-C tail. Cells were treated with control medium (no Wnt3a) or Wnt3a-conditioned medium (Wnt3a) overnight. Average β-catenin-mediated reporter activities ±s.d. in n = 2 independent wells are shown.

E. β-catenin-mediated reporter activity in HEK293T cells co-expressing oncogenic RNF43 (R519X) and dominant negative ∆N-TCF4. Cells were treated with control medium (no Wnt3a) or Wnt3a-conditioned medium (Wnt3a) overnight. Average β-catenin-mediated reporter activities ±s.d. in n = 2 independent wells are shown.

Data information: WT; wild-type.