EMBO/soda-nel huggingface dataset:
FBXL2- and PTPL1-mediated degradation of p110-free p85β regulatory subunit controls the PI(3)K signalling cascade (2013)
figf1

(a) FBXL2 binds p85α and p85β. HEK293T cells were transfected with either an empty vector (EV) or the indicated FLAG-tagged F-box proteins (FBPs). 24 h post-transfection, cells were treated with MG132 for 3 h before collection for immunoprecipitation (IP) and immunoblotting as indicated. WCL, whole-cell lysate. (b) FBXL2 does not bind p110α and p110β. HEK293T and NIH3T3 cells were transfected with either an empty vector (EV) or the indicated FLAG-tagged proteins. The experiment was performed as described in a. (c) The CaaX motif of FBXL2 is required to bind p85α and p85β. HEK293T cells were transfected with either an empty vector (EV) or the indicated FLAG-tagged F-box proteins. The experiment was performed as described in a. (d) Table summarizing the results presented in a–c and Supplementary Fig. S1a. NT, not tested. Uncropped images of blots are shown in Supplementary Fig. S8.
FBXL2- and PTPL1-mediated degradation of p110-free p85β regulatory subunit controls the PI(3)K signalling cascade (2013)
figf2

(a) HEK293T cells were transfected with an empty vector (EV) or the indicated FLAG-tagged constructs. 24 h after transfection, cells were treated with either MG132 or solvent for 3 h before collection for immunoblotting as indicated. (b) HeLa cells stably expressing FLAG–HA-tagged FBXL2 were transfected with either siRNAs targeting FBXL2 (four different siRNAs) or a non-silencing siRNA (NS). Cells were lysed, and proteins were immunoblotted as indicated. NT, non-transfected. (c) During a 72-h serum starvation, NHFs were transfected with either an siRNA targeting FBXL2 (no. 1) or a non-silencing siRNA (NS). Cells were subsequently stimulated with media containing serum and collected at the indicated time points for immunoblotting. The graph shows FBXL2 mRNA levels in the different samples analysed using real-time PCR in triplicate measurements. The values represent the ratios between FBXL2 and GAPDH mRNAs. SR, serum re-addition. (d) HEK293T cells were transfected with HA-tagged p85β, FLAG-tagged FBXL2, FLAG-tagged FBXL2(ΔF-box) or an empty vector (EV) as indicated. After immunopurification with anti-FLAG resin, in vitro ubiquitylation of p85β was performed in the presence of E1, E2s and ubiquitin (Ub). Where indicated, an excess of methylated ubiquitin (MeUb) was also added. Samples were analysed by immunoblotting with the indicated antibodies. The ladder of bands with a relative molecular mass of >85,000 (lane 3) corresponds to ubiquitylated p85β. Immunoblots of whole-cell lysates (WCL) are shown at the bottom. Uncropped images of blots are shown in Supplementary Fig. S8
FBXL2- and PTPL1-mediated degradation of p110-free p85β regulatory subunit controls the PI(3)K signalling cascade (2013)
figf3

(a) Schematic representation of p85β mutants. Binding of p85β to FBXL2 and p110α is indicated with the symbol +; NT, not tested. ‘++’ denotes enhanced binding. (b) HEK293T cells were transfected with GFP-tagged FBXL2 and the indicated FLAG-tagged p85β mutants. Whole-cell lysates (WCL) were immunoprecipitated (IP) with anti-FLAG resin, and immunocomplexes were probed with antibodies against the indicated proteins. (c) p85β(ΔSH2C) is more stable than wild-type p85β. RPE1-hTERT cells were infected with either a retrovirus expressing wild-type p85β or p85β (ΔSH2C). Cells were incubated with cycloheximide (CHX) for the indicated times, collected and analysed by immunoblotting as indicated. In the graph, the amount of p85β (wild-type or mutant) is represented relative to the amount at time 0. (d) p85β (QR/AA) is more stable than wild-type p85β. HEK293T cells were infected with either a retrovirus expressing wild-type p85β or p85β (QR/AA). Cells were incubated with cycloheximide (CHX) for the indicated times, collected and analysed by immunoblotting as indicated. In the graph, the amount of p85β (wild-type or mutant) is represented relative to the amount at time 0. (e) p85β (Y655A) is less stable than wild-type p85β. The experiment was performed as described in c except that p85β(Y655A) was used. In the graph, the amount of p85β (wild-type or mutant) is represented relative to the amount at time 0. Uncropped images of blots are shown in Supplementary Fig. S8.
FBXL2- and PTPL1-mediated degradation of p110-free p85β regulatory subunit controls the PI(3)K signalling cascade (2013)
figf4

(a) Lysates from cells expressing the indicated FLAG-tagged F-box proteins (FBPs) were used in binding reactions with beads coupled to either a peptide or a phospho-peptide (sequences shown on top of the panel). Beads were washed with lysis buffer, and bound proteins were eluted and subjected to SDS–PAGE and immunoblotting. (b) Denatured extracts from HEK293T cells stably expressing either FLAG-tagged wild-type p85β or FLAG-tagged p85β(Y655A) were immunoprecipitated (IP) with either an anti-FLAG resin or a 4G10 platinum resin and immunoblotted (IB) as indicated. Immunoblots of whole-cell lysates (WCL) are also shown in the last two lanes. (c) Denatured extracts from HEK293T cells expressing either FLAG-tagged wild-type p85β or the indicated FLAG-tagged p85β mutants were immunoprecipitated (IP) with an anti-FLAG resin and immunoblotted as indicated. Uncropped images of blots are shown in Supplementary Fig. S8.
FBXL2- and PTPL1-mediated degradation of p110-free p85β regulatory subunit controls the PI(3)K signalling cascade (2013)
figf5

(a) The CaaX motif of FBXL2 is not required to bind PTPL1. HEK293T cells were transfected with either FLAG-tagged wild-type FBXL2 or FLAG-tagged FBXL2(CaaX/SaaX). 24 h post-transfection, cells were collected and whole-cell lysates (WCL) were immunoprecipitated (IP) and immunoblotted as indicated. (b) Schematic representation of p85β mutants. Binding of p85β to PTPL1 and p110α is indicated with the symbol +. (c) PTPL1 stimulates the binding of FBXL2 to wild-type p85β, but not p85β(Y655A). HEK293T cells were transfected with GFP-tagged FBXL2 and either FLAG-tagged p85β or FLAG-tagged p85β(Y655A). Where indicated, HA-tagged PTPL1 or HA-tagged PTPL1(C/S) were also transfected. The experiment was performed as described in a. (d) PTPL1 silencing inhibits the FBXL2–p85β interaction. HeLa cells stably expressing FLAG-tagged FBXL2 were transfected with either an siRNA targeting PTPL1 or a non-silencing siRNA (NS). Forty-eight hours post-transfection, cells were collected and whole-cell lysates (WCL) were immunoprecipitated (IP) and immunoblotted as indicated. (e) During a 72-h serum starvation, NHFs were transfected twice with either siRNAs targeting FBXL2 or PTPL1, or a non-silencing siRNA (NS). Cells were subsequently re-stimulated with media containing serum and collected at the indicated time points for immunoblotting. SR, serum re-addition. (f) HEK293T cells were transfected twice with either siRNAs targeting FBXL2 or PTPL1, or a non-silencing siRNA (NS). Cells were transfected with p85β(Y655A) and 16 h after cells were incubated with cycloheximide (CHX) for the indicated times, collected and analysed by immunoblotting as indicated. (g) During a 48-h serum starvation, U2OS cells stably transfected with a doxycycline-inducible p85β construct were transfected twice with either an siRNA targeting PTPL1 or a non-silencing siRNA (NS). During the last 16 h before collection, p85β expression was stimulated with doxycycline. Cells were subsequently re-stimulated with media containing serum and collected 30 min later. Denatured cell lysates were immunoprecipitated with an anti-FLAG resin and immunoblotted as indicated. The asterisk indicates a non-specific band. Uncropped images of blots are shown in Supplementary Fig. S8.
FBXL2- and PTPL1-mediated degradation of p110-free p85β regulatory subunit controls the PI(3)K signalling cascade (2013)
figf6

(a) During a 72-h serum starvation, NHFs were transfected with either siRNAs targeting FBXL2, p85β, both FBXL2 and p85β, or a non-silencing siRNA (NS). Cells were subsequently stimulated with media containing serum and collected at the indicated time points for immunoblotting. The graph at the right shows FBXL2 mRNA levels in the different samples analysed using real-time PCR in triplicate measurements. The values represent the ratios between FBXL2 and GAPDH mRNAs. SR, serum re-addition. (b) Cells were treated as in a, except that 3 h after serum re-addition, cells were lysed, immunoprecipitated (IP) with an anti-IRS1 antibody and immunoblotted as indicated. Uncropped images of blots are shown in Supplementary Fig. S8.
FBXL2- and PTPL1-mediated degradation of p110-free p85β regulatory subunit controls the PI(3)K signalling cascade (2013)
figf7

(a) During a 72-h serum starvation, NHFs were transfected with either siRNAs targeting FBXL2 (no. 1 or 2) or a non-silencing siRNA (NS). Cells were subsequently stimulated with media containing serum and collected at the indicated time points for immunoblotting. SR, serum re-addition. (b) During a 72-h serum starvation, NHFs were transfected with either siRNAs targeting FBXL2, both FBXL2 and p85β, or a non-silencing siRNA (NS). Cells were subsequently stimulated with media containing serum and collected at the indicated time points for immunoblotting. SR, serum re-addition. (c) U2OS cells stably transfected with a doxycycline-inducible p85β construct were serum starved for 48 h. During the last 16 h before collection, p85β expression was stimulated with doxycycline. Cells were subsequently stimulated with media containing serum and collected at the indicated time points for immunoblotting. SR, serum re-addition. (d) Cells constitutively expressing GFP–LC3 were transfected with HA-tagged p85β, fixed and incubated with an anti-HA p85β antibody (red). Arrows in the top panels point to the enhanced autophagic response (LC3-II vesicles) in cells expressing high levels of p85β, as shown in the bottom panels. Uncropped images of blots are shown in Supplementary Fig. S8. Scale bars, 10 μm.
Iron-induced NCOA4 condensation regulates ferritin fate and iron homeostasis (2022)
Figure 1

Figure. 1: NCOA4 forms detergent-insoluble condensates in iron-replete cells.

A. Results of CRISPR screening analyzed by the MAGeCK algorithm.

B. Wild type (WT) and NCOA4 KO MEFs were pretreated with 25 μg/ml ferric ammonium citrate (FAC) for 16 h and then chased with 20 μg/ml cycloheximide (CHX) for the indicated times. Soluble ferritin expression levels were determined by immunoblotting with the indicated antibodies.

C. WT MEFs untreated or treated with 10 μg/ml FAC for 12 h were lysed with SDS-containing sample buffer or Triton buffer, and analyzed by immunoblotting with the indicated antibodies.

D. Schematic of fractionation procedures.

E. Subcellular fractions of MEFs treated with 20 μM Dfo for 12 h, 10 μg/ml FAC for 12 h, or 100 μM Deferoxamine (Dfo) for 6 h after treatment with 10 μg/ml FAC for 12 h were prepared by differential centrifugation as described in (D). Each fraction was subjected to SDS-PAGE and analyzed by immunoblotting with the indicated antibodies.

F. WT MEFs were untreated, treated with 10 μg/ml FAC for 12h or treated with 10 μg/ml FAC, 10 μM E64d and 10 μg/ml pepstatin A (pep A) for 12h. The cells were immunostained with anti-NCOA4 and anti-LAMP1. Scale bar, 10 μm. Quantitative data are shown as the means ± SD of three biological replicates. At least 30 cells were quantified in each replicate. P=0.033 (Welch Two Sample t-test).

G. NCOA4 puncta were photobleached in MEFs stably expressing NCOA4-GFP after treatment with 10 μg/ml FAC for 12 h, and then fluorescent recovery was monitored. p62 puncta were photobleached in MEFs stably expressing GFP-human p62, and then fluorescent recovery was monitored. Representative images are shown. Time 0 indicates the start of recovery after photobleaching. Scale bars, 2 μm.

H. Quantitative data of fluorescence recovery in (G) shown as means ± SD. 26 dots (NCOA4) or 25 dots (p62) were quantified from three biological replicates.

I. Time-lapse imaging of MEFs stably expressing NCOA4-GFP. Representative fusion and fission images are shown. Red arrowheads indicate the fission event. Scale bars, 5 μm.
Iron-induced NCOA4 condensation regulates ferritin fate and iron homeostasis (2022)
Figure 2

Figure. 2: Multivalent interactions are required for NCOA4 condensation under iron-replete conditions

A. Schematic diagram of NCOA4 variants used in this study.

B. NCOA4 KO MEFs reconstituted with NCOA4 variants were treated or not treated with 10 μg/ml FAC for 12 h and then fixed for imaging. Cells were immunostained with anti-myc antibody. Representative images are shown. Scale bar, 10 μm.

C. Number of puncta per cell in (B) are shown as the means ± SEM of at least 40 cells in each condition from two biological replicates.

D. NCOA4 KO MEFs reconstituted with NCOA4 variants were treated or not treated with 10 μg/ml FAC for 12 h, fractionated, and analyzed by immunoblotting with the indicated antibodies.

E. DISOPRED3 disorder score of human NCOA4.

F. WT MEFs stably expressing NCOA4 IDR (aa 167-334) fused with tandem FKBP were treated with 10 μg/ml FAC or 20 μM Dfo for 12 h and then cultured with 0.1 nM FKBP ligand (AP20187) for 6h. The cells were fixed for imaging and immunostained with anti-myc to analyze puncta of the fusion protein. Scale bar, 10μm.

G. Number of puncta in (F) are shown as means ± SEM of at least 35 cells in each condition from two biological replicates. P<0.0001 (Dfo+ligand vs FAC+ligand) and P=0.048 (FAC vs FAC+ligand) (Kruskal-Wallis ANOVA with Dunn's multiple comparison test). P values were adjusted using the Bonferroni method.

H. Coomassie-stained SDS-PAGE gel of purified proteins.

I. The amount of co-purifying iron in purified proteins in (H) was measured by inductively coupled plasma mass spectrometry (ICP-MS). Data are shown as means ± SD of three biological replicates. P=0.032 (Welch Two Sample t-test).
Iron-induced NCOA4 condensation regulates ferritin fate and iron homeostasis (2022)
Figure 3

Figure. 3: Iron induces NCOA4 condensation.

A. Purified NCOA4 proteins were incubated with FeSO4 for 1 h under aerobic conditions and fractionated. Samples were subjected to SDS-PAGE and stained with Coomassie Brilliant Blue. B. Quantitative results in (A) are shown as means ± SEM of three biological replicates.

C. Purified FlAsH-NCOA4 (20 nM) and 10 μM FeSO4 were incubated at 37°C under aerobic conditions and observed at the indicated times. Scale bar, 10μm.

D. Phase diagram of the formation of FlAsH-NCOA4 condensates at the indicated protein and FeSO4 concentrations.

E. Purified FlAsH-NCOA4 (20 nM) and the indicated metals were incubated at 37°C under anaerobic conditions for 1 h and observed. Scale bar, 10μm.

F. Electron micrograph of NCOA4 KO MEFs reconstituted with myc-hNCOA4. Cryosections were labeled with anti-myc antibody. Scale bar, 200nm.

G. Quantification of the diameter of NCOA4 condensates. Red line shows the mean. The dot plot represents the diameter of 36 condensates from two biological replicates.

H, I. Representative energy-dispersive X-ray spectra of (H) cytosol and (I) condensate. Black arrowheads indicate Fe (Kα) peaks.

J. Quantification of iron count in cytosol and condensates. Data are shown as the means ± SEM of at least 10 cytosols or condensates from two biological replicates. P<0.0001 (Welch Two Sample t-test).
Iron-induced NCOA4 condensation regulates ferritin fate and iron homeostasis (2022)
Figure 4

Figure. 4: NCOA4 condensates fine-tune ferritin levels via two distinct mechanisms.

A. MEFs were treated with 10 μg/ml FAC for the indicated times and harvested. Cell lysates were fractionated and analyzed by SDS-PAGE with the indicated antibodies. Asterisks indicate a non-specific band. TfR1; Transferrin receptor 1

B. MEFs were cultured for 12 h in medium containing 10 μg/ml FAC supplemented with DMSO or 10 μM E64d and 10 μg/ml pepstatin A (pepA). Samples were analyzed by immunoblotting with the indicated antibodies. The black arrowhead indicates an intact ferritin band, and the white arrowhead indicates a partial degradation band of ferritin.

C. NCOA4 KO MEFs reconstituted with hNCOA4-myc were cultured with 10 μg/ml FAC for the indicated times and fixed for imaging. Cells were immunostained with ferritin and myc antibodies. Scale bar, 10μm.

D. Myc (NCOA4) puncta were extracted, and average signal intensities of myc and ferritin in the puncta in (C) were plotted.

E. MEFs expressing hNCOA4-TurboID were treated with 10 μg/ml FAC for 6 h or 24 h and then cultured with DMSO or 50 μM biotin for 30 min. Cells were lysed with Triton buffer, and lysates were pulled down by streptavidin beads. Inputs and pulldown samples were analyzed with immunoblotting by indicated antibodies.

F. WT MEFs pretreated with 10 μg/ml FAC for 6 h or 24 h were chased with 20 μg/ml CHX and analyzed by immunoblotting with the indicated antibodies.

G. NCOA4 KO MEFs reconstituted with myc-hNCOA4 were pretreated with 10 μg/ml FAC and then cultured with 20 μg/ml CHX or 200 nM bafilomycin A1 (BafA). Cells were fixed for imaging and immunostained using myc and LAMP1 antibodies. Scale bar, 10μm.
Iron-induced NCOA4 condensation regulates ferritin fate and iron homeostasis (2022)
Figure 5

Figure. 5: TAX1BP1 is essential for clearance of NCOA4 condensates and ferritin via recruitment of FIP200.

A. WT, ATG7 KO, TAX1BP1 KO, and FIP200KO MEFs were treated or not treated with 10 μg/ml FAC for 12 h. Cell lysates were fractionated and analyzed by immunoblotting with the indicated antibodies.

B. Schematic diagram of TAX1BP1 variants used in this study. NB, NCOA4 binding

C. HEK293T cells were transfected with GFP-NCOA4 and the indicated FLAG-His-TAX1BP1 variants. Soluble lysates extracted from transfected cells were immunoprecipitated with anti-FLAG antibody. Inputs and immunoprecipitated samples were analyzed by immunoblotting with the indicated antibodies.

D-F. WT MEFs and TAX1BP1 KO MEFs reconstituted with the indicated TAX1BP1 variants were untreated or treated with 10 μg/ml FAC for 12 h. (D, F) Cell lysates were fractionated and analyzed by immunoblotting with the indicated antibodies. (E) Cells were fixed for imaging and immunostained to analyze ferritin localization. Scale bar, 10μm.
Iron-induced NCOA4 condensation regulates ferritin fate and iron homeostasis (2022)
Figure 6

Figure. 6: The properties of NCOA4 affect the TAX1BP1 dependence of ferritin degradation and interaction with ferritin.

A. MEFs were pretreated with 10 μg/ml FAC for 12 h and then cultured with 100 μM Dfo for the indicated time. Cells were lysed with SDS-containing sample buffer or Triton buffer. Lysates were analyzed by immunoblotting with the indicated antibodies.

B. MEFs were treated with 20 μM Dfo for 12 h, 10 μg/ml FAC for 12h, or 100 μM Dfo for 6 h after treatment with 10 μg/ml FAC for 12 h. Triton-soluble lysates were subjected to glycerol gradient ultracentrifugation analysis.

C. WT and TAX1BP1 KO MEFs were pretreated with 25 μg/ml FAC for 16 h and then cultured with 100 μM Dfo. Lysates were fractionated and then analyzed by immunoblotting with the indicated antibodies.

D. Soluble FTL bands in (C) were quantified by densitometry. Data are shown as the means ± SD of three biological replicates.

E. TAX1BP1 KO MEFs (sg1 clone) stably expressing FLAG-OsTIR1 (F74G) and FLAG-mAID-mTAX1BP1 were pretreated with 10 μg/ml FAC for 12 h, cultured with DMSO or 1 μM 5-Ph-IAA for 2 h, and then treated with 100 μM Dfo. Soluble lysates were analyzed by immunoblotting with the indicated antibodies. 5-Ph-IAA, a derivative of Auxin; mAID, mini auxin-inducible degron

F. TAX1BP1 KO MEFs (clone sg1) in which ATG7 was knocked down were pretreated with 10 μg/ml FAC for 12 h and then cultured with 100 μM Dfo. Soluble lysates were analyzed by immunoblotting with the indicated antibodies.

G. TAX1BP1 KO MEFs (clone sg1) stably expressing myc-hNCOA4 pretreated with 25 μg/ml FAC for 16 h were cultured with 100 μM Dfo, and soluble lysates were analyzed by immunoblotting with the indicated antibodies.

H, I. NCOA4 KO MEFs reconstituted with NCOA4 variants were pretreated with 10 μg/ml FAC for 12 h and then chased with (H) 20 μg/ml CHX or (I) 100 μM Dfo for the indicated times. Soluble lysates were analyzed by immunoblotting with the indicated antibodies.

J. MEFs expressing hNCOA4-TurboID were treated with 10 μg/ml FAC for 3 h (indicated as F) or 100 μM DFO for 3 h after treatment with 10 μg/ml FAC for 12 h (indicated as D), and then cultured with DMSO or 50 μM biotin for 30 min. Triton-soluble cell lysates were pulled down by streptavidin beads. Inputs and pulldown samples were analyzed by immunoblotting with the indicated antibodies. The black and white arrowheads indicate hNCOA4-TurboID and endogenous NCOA4, respectively.
The IL-6 signaling axis is a critical driver and therapeutic target in diffuse large B-cell lymphoma (2019)
Figure 1

Figure 1. DLBCL cell lines engraft and form orthotopic lymphomas in MISTRG mice that can be traced by luciferase expression.

A-C 10x106 ZsGreen- and luciferase-expressing U-2932, RC-K8 and RIVA cells were intravenously injected into six-week-old male (M) and female (F) MISTRG mice and monitored weekly using IVIS for at least four and up to six weeks. The color scales on the right indicate the radiance, i.e. the sum of the photons per second from each pixel inside the ROI/number of pixels (photons/sec/cm2/sr).

D The frequency of involvement of the indicated tissues is shown for one cohort of mice and is representative of two independently analyzed cohorts per cell line.

E Gating strategy for the identification of U-2932 cells residing in spleen and bone marrow, among all leukocytes in these organs. Cells were stained for human and mouse CD45. Very few hCD45+ U-2932 cells were detectable in the spleen, whereas their frequencies were much higher in the bone marrow (BM). In BM, >90% of U-2932 cells were positive for ZsGreen. The green curve shows U-2932 cells recovered from BM, overlayed with unlabelled U-2932 cells grown in vitro (grey curve).

F, G Frequencies of human lymphoma cells, as judged by hCD45 expression, among all (human and mouse, h+m) CD45+ leukocytes in the spleen (S), femoral bone marrow (BM) and jaw bone marrow (JB) at the study endpoint of the mice shown in D (four weeks post injection for U-2932, five weeks p.i. for RIVA and six weeks p.i. for RC-K8).

Data information: Data in A-G are representative of at least two independently conducted studies per cell line. In the study shown, n was 7 (RIVA), 8 (U-2932) and 9 (RC-K8) mice. Statistics do not apply here.