EMBO/soda-nel huggingface dataset:
Intercepting IRE1 Kinase-FMRP Signaling Prevents Atherosclerosis Progression (2022)
Figure 2

Figure 2. FMRP deficiency enhances RCT while reducing foam cell formation in vivo.

Fmr1+/+ and Fmr1-/- mice were injected with AAV_PCSK9 and fed with 16 weeks of WD. Residential PM were stained with Oil Red O (ORO) and imaged (n=7 mice per group; Scale bar = 50 µm).

Apoe-/- mice were fed with WD (12 weeks) and injected with vehicle (DMSO) or AMG-18 (30 mg/kg/day) in the last 4 weeks of WD. Residential PM were stained with ORO and imaged (n=5 mice per group; Scale bar = 50 µm).

C-E Flow cytometry analysis of BMDMs after dil-ac-LDL (25 µg/ml) loading for 24 hours; (C) control- or Fmr1-siRNA transfected BMDM (n=4 biological replicates), (D) Fmr1+/+ and Fmr1-/- BMDM (n=4 biological replicates), (E) BMDM pre-treated with either vehicle (DMSO) or AMG-18 (5 µM; 1 hour) (n=6 biological replicates).

F-G Macrophages were pre-loaded with fluorescently labeled cholesterol (16 hours) followed by incubation in efflux medium including APOA1 (25 µg/ml) or HDL (50 µg/ml) as acceptors for 6 hours. % Efflux was calculated as cholesterol signal in medium/cholesterol signal in medium and cell: Cholesterol efflux in (F) Fmr1+/+ and Fmr1-/- BMDM (n=4 biological replicates) and in (G) BMDM that were pre-treated either with DMSO or AMG-18 (5 µM; 1 hour) (n=4 biological replicates).

H-K RCT experiment: (H) Schematic representation of C57BL6 mice were injected with [3H]-cholesterol-loaded foamy Fmr1+/+ and Fmr1-/- BMDM, (I) plasma cholesterol levels after 24 and 48 hours, (J) liver cholesterol levels after 48 hours, and (K) feces cholesterol levels after 48 hours (n=12 mice per group).

Data information: Data are mean ± SEM. Unpaired t-test with Welch's correction.
Intercepting IRE1 Kinase-FMRP Signaling Prevents Atherosclerosis Progression (2022)
Figure 3

Figure 3. FMRP-deficiency increases efferocytosis in vivo.

A-E In vitro and in vivo efferocytosis experiments, where percentage of macrophages F4/80+ (red) that ingested apoptotic cells (AC) labeled with carboxyfluorescein succinimidyl ester (CFSE)+ (green) were reported as % efferocytosis. (A) BMDMs were transfected with Fmr1- or control-siRNA and incubated CFSE-labeled AC for the indicated hours (n=4 biological replicates). (B) Fmr1+/+ and Fmr1-/- BMDMs were treated with PA (500 µM) for 6 hours and then incubated with CFSE-labeled ACs for 4 hours (n=4 biological replicates). (C) Fmr1+/+ and Fmr1-/- mice were fed WD (16 weeks) and injected intraperitoneally with CFSE-labeled AC (1.5 hours), followed by PM elicitation (n=4-5 mice per group). (D) BMDM were pre-treated either with vehicle (DMSO) or AMG-18 (5 µM) for 1 hour then incubated with CFSE-labeled ACs for 4 hours (n=3 biological replicates). (E) C57BL/6 mice were injected with AMG-18 (30 mg/kg) or vehicle (DMSO) for 8 hours, followed by intraperitoneal injection with CFSE-labeled ACs for 1.5 hours and PM elicitation (n=4 mice per group).

F-G In vitro continuous efferocytosis experiments, where macrophages were stained for F4/80+ (red), AC were labeled with CFSE (AC-1; green) or Violet (AC2; violet). % continuous efferocytosis was determined by the ratio of F4/80+, CFSE+ and Violet+ (triple positive) cells to total F4/80+ and CFSE+ (double positive) cells. (F) Fmr1+/+ and Fmr1-/- BMDM were incubated with AC-1 for 2 hours, and after 2 hours interval, incubated with AC-2 for 2 more hours (n=4-3 biological replicates). (G) BMDM were pre-treated either with vehicle (DMSO) or AMG-18 (5 µM) for 1 hour, incubated with CFSE-labeled AC-1 for 2 hours, followed by incubation with Violet-labeled AC-2 for 2 hours and PM collection (n=4 biological replicates).

Data information: For all images scale bar = 50 µm; Red: Macrophages, Green: AC/AC-1, Violet: AC-2. Data are mean ± SEM. Unpaired t-test with Welch's correction.
Intercepting IRE1 Kinase-FMRP Signaling Prevents Atherosclerosis Progression (2022)
Figure 4

Figure 4. FMRP targets in macrophages.

A-B RNA lysates from Fmr1+/+ and Fmr1-/- BMDM that were treated with PA (500 µM; 6 hours) were fractionated using a 10%-50% sucrose gradient and separated to polysome, monosome/NTR fractions. The absorbance (260 nm) of RNA was measured and plotted as a function of time (n=3 biological replicates). (A) Representative profile for RNA distribution from genotypes based on UV absorbance readings after sucrose gradient fractionation. (B) The ratio of the Abca1, Abcg1, Mertk, Lrp1, Cd36, Cd47 and Rac1 mRNA in polysome to NTR fraction (n=3 biological replicates).

C BMDM were isolated from Fmr1+/+ and Fmr1-/-, and protein lysates were analyzed by western blotting using specific antibodies for ABCA1, ABCG1, MerTK, LRP1, FMRP and β-Actin antibodies and fold inductions relative to β-Actin are depicted above the blots (n=6 biological replicates).

D Fmr1-/- MEF cells were transfected with EV, WT-FMRP or STSA-FMRP plasmids followed by PA treatment (500 µM; 6 hours). Protein lysates were analyzed by western blotting using specific antibodies for ABCA1, MerTK, LRP1, pFMRP, FMRP and β-Actin and fold inductions relative to β-Actin are depicted above the blots (n=5 biological replicates).

Data information: A representative blot is shown. In C and D data are cumulative results of 2 and 3 independent experiments respectively. In western blots, the fold protein expression change was calculated relative to β-Actin and depicted above the blots and a representative blot was shown. Data are mean ± SEM. Unpaired t-test with Welch's correction.
Functional Platelet-Derived Mitochondria Induce the Release of Human Neutrophil Microvesicles (2022)
Figure 1

Figure 1. Modulatory effects of freeMitos on human polymorphonuclear leucocytes.

A, B Functional freeMitos association with PMN. Overlay dot plot population, representative of 4 biological replicates of the flow cytometry gating strategy used to quantify the interaction between PMN (anti-CD11b-PE) and freeMitos. Freshly isolated platelets were labeled with MitoTrackerTM Deep Red and freeMitos isolation was subsequently performed. CD11b-labeled PMN were incubated in absence (A) or presence (B) of freeMitos at a 1:5 ratio (PMN:mitochondria).

C Confocal microscopy imaging of PMN cell membrane (red) labeled with CellMask Orange in absence (upper panel) or presence (lower panel) of MitoTrackerTM Deep Red-labeled freeMitos (magenta). PMN nuclei were stained with DAPI (cyan). The white scale bars shown in the lower right of the images represent 10 μm in size.

D, E FreeMitos associate with PMN and modulate gene expression. Volcano plot, of 4 biological replicates, representing the gene expression modulation induced by mitochondrial DAMPs preparation (D) and freshly isolated functional freeMitos (E) in PMN.

F-H FreeMitos effect on the respiratory state of PMN. O2 consumption rates of resting or activated (incubated with TNF-a and GM-CSF) PMN were measured using high-resolution O2 respirometry (Oroboros Oxygraph) and revealed no significant differences between the basal state (left panel), leak state (middle panel) and the maximum electron transport system capacity (ETS, right panel) of cellular respiration (F). Immunoblot of mitochondrial cytochrome c oxidase (COX IV) and histone H3 as a loading control of PMN after co-incubations with freeMitos (G) with subsequent densitometry analysis (H).

Data information: For panels F and H, data are shown as means ± SEM, of 3 biological replicates. Two-way ANOVA tests followed by Tukey's multiple comparisons tests for each sample was performed for panels F and H. Values without at least one common superscript letter are different (P < 0.05).
Loss of C9ORF72 impairs autophagy and synergizes with polyQ ATXN2 to induce motor neuron dysfunction and cell death (2016)
Figure 1

Figure 1. C9ORF72 in complex with SMCR8 and WDR41 is a GEF for RAB8 and RAB39.

(A) Silver staining of proteins extracted from N2A mouse neuronal cells expressing Flag-HA-tagged C9ORF72 and captured through consecutive anti-Flag and anti-HA affinity purification steps. (B) Immunoblot analysis of HA-immunoprecipitated proteins and lysate of HEK293 cells co-expressing HA-tagged C9ORF72 and/ or Flag-tagged SMCR8 and/or Flag-tagged WDR41. (C) Coomassie blue staining of Nickel-NTA affinity purification of HIS-C9ORF72, SMCR8 and WDR41 co-expressed in baculovirus infected insect cells. (D) Immunoblot analysis of HA-immunoprecipitated proteins and lysate of HEK293 cells co-expressing HA-tagged C9ORF72 and HA-tagged SMCR8 with various Flag-tagged Rab GTPases. (E) Immunoblot against endogenous C9orf72, Wdr41, Rab8a, Rab39b, Rab5 and Rab7 of control (IgG alone) or endogenous Smcr8 immunoprecipitated from adult mouse brain. (F) αP32-radiolabelled GDP release from GST-tagged purified RAB8a as a function of increased concentration of either recombinant purified C9ORF72 alone or in complex with SMCR8 and WDR41. (G) Identical GDP release assay as in (F) but using recombinant purified GST-tagged RAB39b instead of RAB8a. Error bars indicate s.e.m. Experiments were repeated 3 times (n=3). (H) Schematic representation of the C9ORF72 complex acting as a Rab-guanine nucleotide exchange factor.
Loss of C9ORF72 impairs autophagy and synergizes with polyQ ATXN2 to induce motor neuron dysfunction and cell death (2016)
Figure 2

Figure 2. Reduced expression of C9ORF72 partly impairs autophagy.

(A) Left panel, representative images of organotypic cultures of E18 mouse cortical neurons transfected with GFP-RFP-LC3B and transduced with lentivirus expressing either control shRNA or shRNA targeting C9orf72 mRNA and treated or not with Torin. Right panel, quantification of LC3B puncta. (B) Upper panel, immunoblot analysis of endogenous LC3B (Map1lc3b), C9orf72 and control Actin of E18 mouse cortical neurons transduced with lentivirus expressing either control shRNA or shRNA targeting C9orf72 mRNA and treated or not with Torin. Lower panel, real time RT-qPCR quantification of endogenous C9orf72 mRNA expression relative to Rplp0 mRNA. (C) Left panel, representative images of immunofluorescence labeling of endogenous P62 (Sqstm1) on organotypic cultures of E18 mouse cortical neurons transduced with lentiviral particles expressing either control shRNA or shRNA targeting C9orf72. Right panel, quantification of P62 aggregates. (D) Left panel, representative images of immunofluorescence labeling of transfected constructs (Green) and endogenous P62 (Sqstm1, red) on GT1-7 neuronal cells transfected with siRNA targeting C9orf72 and plasmids expressing control GFP, HA tagged wild type or mutant RAB39b (CA, Q68L or CN, S22N), RAB8a or RAB7. Right panel, quantification of P62 aggregates. Scale bars, 10 µm. Nuclei were counterstained with DAPI. Error bars indicate s.e.m. Student T-test, * indicates p<0.05, *** indicates p<0.001. n=3.
Loss of C9ORF72 impairs autophagy and synergizes with polyQ ATXN2 to induce motor neuron dysfunction and cell death (2016)
Figure 3

Figure 3. SMCR8 is phosphorylated by TBK1.

(A) Immunoblot analysis of HA-immunoprecipitated proteins and lysate of HEK293 cells co-expressing HA-tagged C9ORF72 and HA-tagged SMCR8 with Flag-tagged TBK1, NAP1, TANK or SINTBAD. (B) Immunoprecipitated HA-tagged C9ORF72, HA-tagged SMCR8 and HA-tagged WDR41 expressed in HEK293 were subjected to in vitro TBK1 kinase assay in the presence of γP32-radiolabelled ATP. Proteins were separated by SDS page migration and phosphorylation was detected by autoradiography (upper panel), while expression was detected by western blotting (lower panel). (C) Mass spectrometry identification of SMCR8 phosphorylation sites by in vitro TBK1 kinase assay of HIS-tagged C9ORF72:SMCR8:WDR41 complex purified from baculovirus infected insect cells. (D) Left panel, representative images of immunofluorescence labeling of transfected constructs (Green) and endogenous P62 (Sqstm1, red) on GT1-7 neuronal cells transfected with siRNA targeting the 3’UTR of Smcr8 mRNA and plasmids expressing control GFP, HA tagged wild type or mutant SMCR8 (TA, S402A and T796A; TD, S402D and T796D; UD, S400D, S492D, S562D and T666D). Right panel, quantification of P62 aggregates. (E) Left panel, representative images of immunofluorescence labeling of transfected constructs (Green) and endogenous P62 (Sqstm1, red) on GT1-7 neuronal cells transfected with siRNA targeting Tbk1 and plasmids expressing control GFP, HA tagged wild type or mutant SMCR8 or RAB39b. Right panel, quantification of P62 aggregates. Scale bars, 10 µm. Nuclei were counterstained with DAPI. Error bars indicate s.e.m. Student T-test, * indicates p<0.05, *** indicates p<0.001. n=3.
Loss of C9ORF72 impairs autophagy and synergizes with polyQ ATXN2 to induce motor neuron dysfunction and cell death (2016)
Figure 4

Figure 4. Reduced expression of C9ORF72 promotes aggregation of TDP-43.

(A) Immunoblot analysis of endogenous Tdp-43, C9orf72 and control Gapdh of E18 mouse cortical neurons transduced with lentivirus expressing either control shRNA or shRNA targeting C9orf72 mRNA. (B) Left panel, representative images of immunofluorescence labeling of endogenous phosphorylated Ser409/410-Tdp-43 on primary cultures of E18 mouse cortical neurons transduced with lentiviral particles expressing either control shRNA or shRNA targeting C9orf72. Right panel, quantification of cytoplasmic Tdp-43 aggregates. (C) Left panel, representative images of immunofluorescence labeling of D169G mutant GFP-tagged TDP-43 on primary cultures of E18 mouse cortical neurons transfected with either HA-tagged control or HA-C9ORF72 plasmid. Right panel, quantification of cells with cytoplasmic aggregates of TDP-43. Scale bars, 10 µm. Nuclei were counterstained with DAPI. Error bars indicate s.e.m. Student T-test, *** indicates p<0.001. n=3.
Loss of C9ORF72 impairs autophagy and synergizes with polyQ ATXN2 to induce motor neuron dysfunction and cell death (2016)
Figure 5

Figure 5. Reduced expression of C9ORF72 synergizes ATXN2 toxicity.

(A) Left panel, representative images of organotypic cultures of E18 mouse cortical neurons co-transfected with HA-tagged ATXN2 with control (Q22x) or intermediate (Q30x) polyQ size and transduced with lentivirus expressing either control shRNA or shRNA targeting C9orf72 mRNA. Right panel, quantification of ATXN2 aggregates. Scale bars, 10 µm. Nuclei were counterstained with DAPI. (B) Cell viability (tetrazolium assay) of GT1-7 neuronal cells co-transfected with HA-tagged ATXN2 with control (Q22x) or intermediate (Q30x) polyQ size and control siRNA or siRNA targeting C9orf72 mRNA. Error bars indicate s.e.m. Student T-test, *** indicates p<0.001. n=3. (C) Tracing of the swimming trajectories of 48 hours post-fertilization zebrafish larvae following light touch. (D-F) Quantification of the touch-evoked swimming distance (D), average velocity (E), and maximum velocity attained (F) shows significant functional impairment of the zebrafish injected with HA-tagged ATXN2 with intermediate length of polyQ (Q30x) and antisense morpholino oligonucleotides (AMOs) against C9orf72 compared to control HA-tagged ATXN2 (Q22x) or to the sole injection of AMO against C9orf72. (G) Representative images of motor neurons visualized with anti-Sv2 immunohistochemistry show severe axonopathy in fish injected with both ATXN2 Q30x and AMO against C9orf72 compared to zebrafish injected with control ATXN2 Q22x or with AMO against C9orf72 alone. (H) Quantification of the motor neuron axonal length demonstrates a significant decrease of axonal length in 48 hours post-fertilization zebrafish larvae injected with both ATXN2 Q30x and AMO against C9orf72 compared to controls. Error bars indicate s.e.m. One-way ANOVA, ** indicates p<0.01. n=3.
SCF (Fbxl17) ubiquitylation of Sufu regulates Hedgehog signaling and medulloblastoma development (2016)
Figure 1

Figure 1. Fbxl17 interacts with Sufu and regulates Sufu levels

A. Detection of Sufu and Skp1 after immunoprecipitation of the indicated Flag-tagged F-Box Proteins (FBPs). An Empty Vector (EV) was used as a negative control. HEK293T cells were treated with MLN4924 (2 M) for 5 hours prior collection.

B, C. Immunoprecipitation of endogenous Sufu from PC3 (B) and DAOY cells (C) transfected with either an empty vector (EV) or Myc-tagged Fbxl17. Non-specific rabbit immunoglobulin G (IgG) was used as a negative control. Detection of light chains of immunoglobulin (IgG) was used to assess the amount of IgG used for each immunoprecipitation reaction. Treatment with MLN4925 (2 M) was started 5 hours before cell collection.

D. Quantification of Fbxl17 mRNA levels in PC3 and DAOY transfected with a non-targeting siRNA (Control) or two siRNAs to Fbxl17 (1) and (2). Cells were serum starved for 24 hours in serum-reduced medium and treated with SAG (100 nM) for 24 hours prior collection.

E. Sufu protein levels in PC3 and DAOY cells treated as in D. GAPDH was used as a loading control. Representative image of three independent experiments is shown.

F. Sufu protein levels in PC3 and DAOY cells infected with an empty backbone retrovirus (EV) or a retrovirus expressing Myc-tagged Fbxl17. Representative image from three independent experiments is shown.

G. Detection of Sufu protein levels following cycloheximide (CHX) treatment for the indicated hours, and Fbxl17 depletion using two different siRNAs. (Hrs=hours). Representative image of two independent experiments is shown
SCF (Fbxl17) ubiquitylation of Sufu regulates Hedgehog signaling and medulloblastoma development (2016)
Figure 2

Figure 2. SCFFbxl17 mediates Sufu polyubiquitylation

A. Detection of polyubiquitylated species of endogenous Sufu co-immunopurified from DAOY cells transfected with Myc-tagged ubiquitin (Ub) either in the presence of non-targeting siRNA (Control) or two siRNAs against Fbxl17 (1) and (2). MG132 (10µM) was added in all samples.

B. Detection of polyubiquitylated species of Sufu upon co-transfection of HEK293T cells with HA-tagged Sufu and Myc-tagged ubiquitin (Ub) either in the presence of non-targeting siRNA (Control) or two siRNAs against Fbxl17 (1) and (2). MG132 (10µM) was added in all samples.

C. Fbxl17 mRNA relative levels in DAOY and HEK293T cells upon Fbxl17 depletion with either a non-targeting siRNA (Control) or two siRNAs to Fbxl17 (1) and (2).

D. Detection of ubiquitylated Sufu co-immunoprecipitated from HEK293T cells co-transfected with HA-tagged Sufu, Myc-ubiquitin (Ub) along with either Flag-tagged Fbxl17 wild type (WT) or a mutant lacking the F-box domain (Fbxl17ΔF). MG132 (10µM) was added in all samples.

E. Detection of ubiquitylated Sufu co-immunoprecipitated from HEK293T cells co-transfected with Myc-tagged ubiquitin (Ub), Flag-tagged Fbxl17 and HA-tagged Sufu WT or Sufu mutant K257R. MG132 (10µM) was added in all samples.
SCF (Fbxl17) ubiquitylation of Sufu regulates Hedgehog signaling and medulloblastoma development (2016)
Figure 3

Figure 3. Fbxl17-Sufu interaction is impaired by Sufu phosphorylation and favored by the presence of Gli

A. Detection of Myc-tagged Fbxl17 after immunoprecipitation of HA-tagged Sufu WT or Sufu S342/6A and S342/6D, as indicated. HEK293T cells were treated with MLN4924 (2 M) for 5 hours prior collection.

B. Detection of Flag-tagged Gli1 and Myc-tagged Fbxl17 binding to immunoprecipitated HA-tagged Sufu Wild-Type (WT) or to Sufu S352F. An Empty Vector (EV) was used as a negative control. HEK293T cells were treated with MLN4924 (2 M) for 5 hours prior collection.

C. Detection of phosphorylated Sufu on S352/T353 after immunoprecipitation of HA-tagged Sufu WT and Sufu 351-353 AAA, as indicated.

D. Detection of Myc-tagged Fbxl17 binding to Sufu full length (FL) or to the following Sufu peptides: Sufu 340-360(APSRKDSLESDSSTAIIPHEL); Sufu 340-360 [S352P/T353P] (phosphorylated on the residues S352 and T353); Sufu 351-372 (SSTAIIPHELIRTRQLESVHLK).

E. Detection of HA-tagged Sufu binding to a Myc-tagged Fbxl17 construct encompassing the residues 318 to 701 (corresponding to the F-box motif and C-terminus region) assessed by in vitro binding assay. Both proteins were synthetized in vitro using a T7-coupled reticulocyte lysate system.

F. Detection of ubiquitylated Sufu immunoprecipitated from HEK293T cells co-transfected with HA-tagged ubiquitin (Ub), Myc-tagged Fbxl17 and Flag-tagged Gli1. MG132 (10µM) was added in all samples.

G. Detection of Flag-tagged Gli1 binding to Myc-tagged Fbxl17 co-immunoprecipitated from HEK293T cells transfected with a non-targeting siRNA (Control) or two siRNA to Sufu (1) and (2), as indicated.
SCF (Fbxl17) ubiquitylation of Sufu regulates Hedgehog signaling and medulloblastoma development (2016)
Figure 4

Figure 4. Sufu degradation by Fbxl17 allows Gli dissociation for Hedgehog signaling activation

A. Sufu protein levels in mouse embryonic fibroblasts (MEFs) Ptch1+/+, Ptch1+/- and Ptch1-/- transfected with a non-targeting siRNA (Control) or two siRNAs targeting mouse Fbxl17 (1) and (2).

B. Quantification of Fbxl17 mRNA levels in MEFs Ptch1+/+, Ptch1+/- and Ptch1-/- transfected as in A.

C. Analysis of Gli1 mRNA levels in MEFs Ptch1+/+ and Ptch1-/- upon Fbxl17 depletion using two different siRNAs. (mean ± SEM from 3 independent experiments, ***p<0.0005).

D. Analysis of Bcl2 mRNA levels in MEFs Ptch1+/+ and Ptch1-/- upon Fbxl17 depletion using two different siRNAs. (mean ± SEM from 3 independent experiments, **p<0.005; ***p<0.0005).

E. Detection of Flag-tagged Gli1 binding to HA-tagged Sufu in MEFs Ptch1-/- cells upon expression of Myc-tagged Fbxl17, or Fbxl17 depletion by two siRNAs. For each condition, 200 µg of whole cell lysate was immunoprecipitated.

F. Immunostaining of Fbxl17 using anti-Myc antibody in Ptch1-/- MEFs stably transduced with a pBABE vector expressing Fbxl17 tagged with Myc either at the N-terminus [Myc(N)-Fbxl17] or at the C-terminus [Myc(C)-Fbxl17]. Scale bars: 20 m.

G. Detection of Fbxl17 using anti-Myc antibody in Ptch1-/- MEFs stably expressing Myc(N)-Fbxl17 (Fbxl17 tagged at the N-terminus) and Myc(C)-Fbxl17 (Fbxl17 tagged at the C-terminus).

H. Detection of HA-tagged Fbxl17 binding to endogenous Sufu immunoprecipitated from cytoplasmic and nuclear extracts from Ptch1-/- MEFs. Asterisk (*) indicates a non specific band. Identification of cytoplasmic and nuclear fractions was performed by Lamin A/C and GAPDH detection.
SCF (Fbxl17) ubiquitylation of Sufu regulates Hedgehog signaling and medulloblastoma development (2016)
Figure 5

Figure 5. Fbxl17 mediated degradation of Sufu promotes medulloblastoma cell proliferation

A. Cell proliferation of DAOY cells upon Fbxl17 depletion using a non-targeting siRNA (Control), two siRNAs against Fbxl17 (1) and (2) or upon reintroduction of a Myc-tagged Fbxl17 construct in Fbxl17-depleted DAOY cells using siRNA Fbxl17 (2) (mean ± SEM from 3 independent experiments, **p<0.005; ***p<0.0005).

B. Quantification of Fbxl17 mRNA levels in DAOY cells transfected with a non-targeting siRNA (Control) or two siRNAs against Fbxl17 (1) and (2) (mean ± SEM from 3 independent experiments, **p<0.005).

C. Sufu protein levels in DAOY cells treated as in A. Representative image of three independent experiments is shown.

D. Cell proliferation of DAOY cells transfected with non-targeting siRNA (Control), siRNA against Fbxl17 or a combination of siRNA targeting Fbxl17 and Sufu. (mean ± SEM from 3 independent experiments, **p<0.005; ***p<0.0005).

E. Quantification of Fbxl17 mRNA levels in DAOY cells treated as in D mean ± SEM from 3 independent experiments, ***p<0.0005).

F. Sufu protein levels in DAOY cells treated as in D. Representative image of three independent experiments is shown.