EMBO/soda-nel huggingface dataset:
Nitrate triggered phosphoproteome changes and a PIN2 phosphosite modulating root system architecture (2021)
Figure 8

Figure 8. PIN2 phosphorylation at S439 is important for modulation of protein localization in response to nitrate treatments.

Three different genotypes were used in these experiments. eir1-1 mutant background complemented with PIN2::PIN2wt-GFP (PIN2wt), PIN2::PIN2S439D-GFP (PIN2S439D phospho-mimic point mutation) or PIN2::PIN2S439A-GFP (PIN2S439A, phospho-null point mutation). All genotypes were grown hydroponically as described in Methods and treated with 5 mM nitrate for 2 hours.

(A) Confocal microscopy using Zeiss Airyscan microscope and Zeiss-blue3.1 software was used to visualize PIN2 in Arabidopsis roots. "e" denotes epidermis and "c" cortex. Scale bar = 20 µm.

(B-D) Tuckey box plots show PIN2-GFP fluorescence intensity quantification (number of roots analyzed per experimental conditions = 8-10; arbitrary units, a.u.) at the total cell membrane in roots (B), and from epidermal (C) and cortex (D) plasma membrane in the different genotypes. The box plot shows the data within the interquartile range (25th and 75th percentiles) and a solid black line represents the median. Whiskers show maximum and minimum values no further than 1.5x IQR (interquartile range). Outlier data are plotted individually. Letters denote statistically significant difference between means as determined by one-way ANOVA analysis followed by Tukey HSD post hoc test (p < 0.05).
Targeting human CALR mutated MPN progenitors with a neoepitope-directed monoclonal antibody (2022)
Figure 2

Figure 2: 4D7 disrupts interaction with TpoR and downstream signalling

A. Cell extracts blotted for phospho-STAT5, total STAT5, phospho-ERK, total ERK and actin from TF-1 TpoR cells after incubation with 10 or 20 µg/mL 4D7 or IgG for 4 or 8 hours, in presence of TPO. The last line indicates TPO withdrawal.

B. Similar experiment using TPO-independent TF-1 TpoR CALRdel61 cells.

C. Similar experiment using TPO-independent TF-1 TpoR CALRdel52 cells at 8 hours.

D. Similar experiment using PBMNCs from CALRdel52 PMF primary cells at 8 hours. Additionally, cells were treated with 280 nM of ruxolitinib as a positive control.

E. Fraction of apoptotic sub-G0 population of TF-1 TpoR CALRdel61 cells after 48 hours of 4D7 or IgG treatment (n=3 biological replicates, bars represent standard deviation for 3 replicates, normalised to IgG, with a students unpaired t-test used to determine statistical significance).

F. Western blot showing caspase 3 cleavage occurring within 24 hours of TPO withdrawal in TPO dependent TF-1 TpoR cells. An increase in cleaved caspase 3 is observed after 48 hours of treatment with 20 µg/mL 4D7 in TF-1 TpoR CALRdel61 and TF-1 TpoR CALRdel52 cells.

G. Western blot of TpoR immunoprecipitation under non-reducing conditions showing associated CALR 50 kDa monomers and 100 kDa dimers (red arrowheads) present only in TF-1 TpoR CALRdel61 disrupted by 8-hour treatment with 20 µg/mL 4D7 but not PBS or 20 µg/mL IgG. CALR monomers & dimers are detectable by polyclonal anti-wild type CALR or anti-mutant CALR monoclonal antibodies. Red arrowheads, detected mutant CALR protein; brown arrowheads, detected wildtype CALR protein, asterisk, non-specific bands.
Targeting human CALR mutated MPN progenitors with a neoepitope-directed monoclonal antibody (2022)
Figure 3

Figure 3: 4D7 monoclonal antibody specifically inhibits primary megakaryocyte differentiation of mutated CALR myelofibrosis samples.

A. PCR amplification of CALR exon 9 from patients confirming heterozygous del52 mutation in sorted CD34+ cells obtained from CALR mutated myelofibrosis samples.

B. Graph showing the decreased fold change of CD41+CD61+ megakaryocytes cultured in 4D7 compared to IgG. FACS-sorted CD34+ from myelofibrosis patients with CALR or JAK2 mutation were cultured over 12 days without TPO in the presence of SCF, IL-6 and IL-9. Black columns show JAK2V617F mutation positive samples (n= 4 technical replicates of samples from different patients).

C. Number of CD41+ megakaryocytes derived from isolated CD34+ progenitors from myelofibrosis patients. Number of CD41/CD61+ cells counted on day 12 using trypan blue exclusion (n=1 technical replicate).

D. Summary of fold change reduction of CD41/CD61+ megakaryocytes by 4D7 in all tested CALR mutated patient samples compared to CALR wild type, normalised to IgG (n=11 samples from different patients, with 4 technical replicates per sample).

E Number of CD41+ megakaryocyte colonies from patient samples after 4D7 treatment. CD34+ from patients with myelofibrosis with CALRdel52 or CALRins5 were plated on collagen-based matrix in presence of 20 µg/mL 4D7 or IgG control (n=3 biological replicates).

F. Representative micrographs showing CD41+ colonies in pink and CD41- colonies in blue after treatment with 4D7 or IgG at 100 X or 40X magnification. Scale bar indicates 100µm.

G. Summary of fold change reduction of CD41+ megakaryocytes in CALR mutated samples cultured MegaCult treated with 4D7 compared to IgG (n= 4 biological replicates).

H. Number of megakaryocyte colony forming units grouped according to colony cell number after 4D7 treatment in a mutant CALRins5 sample (n=3 biological replicates).

Data information: Bars represent standard error of means in D and G. Unpaired students t-test used to determine statistical significance in B, D, E, G, H. Bars represent standard deviations in B, E and H. *, P = 0.05 - 0.01, **, P = 0.01 - 0.001, ***, P = 0.001 - 0.0001, ****, P <0.0001, n.s, not significant.
Targeting human CALR mutated MPN progenitors with a neoepitope-directed monoclonal antibody (2022)
Figure 4

Figure 4: 4D7 has no effect on normal progenitor cells and shows activity against ruxolitinib-resistant cells without haematological toxicity.

A. Graph showing effect of ruxolitinib on megakaryocyte differentiation at 50 and 100 nM in healthy CD34+ cells (n= 4 different cords collected with 3 technical replicates).

B. Effect of 4D7 on TPO-dependent megakaryocyte differentiation of healthy CD34+ cells cultured with 20 µg/mL 4D7 or IgG control (n= 2 biological replicates, with 2 technical replicates).

C. Total numbers of hematopoietic colonies plated in MethoCult from healthy cord blood after treatment with 4D7 or IgG. CFU-GM, colony forming unit- granulocyte macrophage; BFU-E, blast forming unit-erythroid; CFU-GEMM, colony forming unit granulocyte, erythroid, monocyte, megakaryocyte (n=3 biological replicates).

D. CD41+ megakaryocyte colonies from healthy CD34+ cells observed on collagen matrix after 4D7 treatment. Colonies were cultured in TPO, SCF, IL-9 and IL-3. Scale bar indicates 100µm.

E. Number of megakaryocyte colony forming units observed in MegaCult assay after treatment of healthy cord blood CD34+ cells with either IgG or 4D7 (n=3 biological replicates).

F. Western blot showing signalling in ruxolitinib-resistant TF-1 TpoR CALRdel61 compared to ruxolitinib sensitive TF-1 TpoR CALRdel61 cells after treatment with 100nM ruxolitinib for 16 hours or 30 minutes and blotted for phospho-STAT5, total STAT5, phospho-ERK, total ERK and actin as indicated. Ruxolitinib-sensitive cells were non-viable after 16 hours of treatment.

G. Comparison of cell growth after DMSO, 20 µg/mL 4D7, IgG or 100 nM ruxolitinib treatment over 4 days of ruxolitinib-resistant TF-1 TpoR CALRdel61. Cells counted using Trypan Blue exclusion (n=3 biological replicates with 3 technical replicates).

H. Number of colonies observed from cells plated in MethoCult following 72 hours of treatment with DMSO, 20 µg/mL 4D7, IgG or 100nM ruxolitinib performed in ruxolitinib-resistant TF-1 TpoR CALRdel61 cells (n=3 biological replicates with 3 technical replicates).

Data information: Error bars on A, B, C and E indicate standard error of mean. Error bars on G and H represent standard deviation. All P values are unpaired Student's unpaired t-test.
Ythdf is a N6-methyladenosine reader that modulates Fmr1 target mRNA selection and restricts axonal growth in Drosophila (2020)
Figure 2

Figure 2. Ythdf and Fmr1 binding behavior to methylated RNA probes

(A) Results of m6A RNA pulldown in BG3 protein cell extracts. Pulldown were performed using biotinylated probes containing four m6A consensus sites (GGACU), with or without the methylation. The p-value is calculated from a two-sided t-test with unequal variants (Welch t-test). Dashed lines represent -log10 p-value =1 as well as log2 enrichment =1. m6A-enriched proteins (-log10 p-value > 1 and log2 enrichment >1) are depicted by bigger dots.

(B, C) Western Blot showing the protein enrichments upon pulldown of the same probes as in (A) incubated with increasing concentrations of purified recombinant GST-Ythdf (B) and His-NT-Fmr1 proteins (C). Quantification of the m6A/A signal intensity is shown below the blot as median +/- SEM of the triplicates. Both proteins bind more efficiently upon methylation.

(D) Results of fluorescence polarization (FP) assay using Cy5 labelled RNA probes containing four AAACU m6A consensus sites, with or without the methylation, incubated with GST-Ythdf and His-NT-Fmr1. The relative FP values from three independent experiments including standard deviation were plotted using GraphPad Prism 8. Binding constants (kd values) were determined by fitting a Michaelis-Menten non-linear regression onto the relative FP values in GraphPad Prism 8, if applicable. The respective binding constants are given in the table next to the graphs.
Ythdf is a N6-methyladenosine reader that modulates Fmr1 target mRNA selection and restricts axonal growth in Drosophila (2020)
Figure 4

Figure 4. Fmr1 and Ythdf physically interact and share common targets

(A) Heatmap indicating the normalized forward versus inverted reverse experiment enrichments on a log2 scale of quantitative proteomics upon pulldown of Flag-tagged Ythdf in S2R+ cells. The threshold was set to a 1-fold enrichment. Fmr1 is co-purified with Ythdf. Known Fmr1 interacting proteins are highlighted in bold with asterisk.

(B) Co-immunoprecipitation experiment in S2R+ cells co-expressing FlagMyc-tagged Ythdf and Myc-tagged Fmr1. Ythdf was used as a bait via its Flag tag. The lysate was treated with Benzonase as indicated to remove interactions enabled by RNA.

(C) GST pulldown of recombinant GST-Ythdf or GST alone mixed with recombinant 6xHis-NT-Fmr1. 2 µM of recombinant proteins were using during the pulldown. The pulldown of the proteins was analyzed by CBB staining.

(D) Overlap of miCLIP dataset generated in S2R+ cells defining m6A modified transcripts with Fmr1 and Ythdf mRNA targets determined by TRIBE in S2R+ cells. The indicated P-value was determined by hypergeometric test.
Ythdf is a N6-methyladenosine reader that modulates Fmr1 target mRNA selection and restricts axonal growth in Drosophila (2020)
Figure 5

Figure 5. Ythdf modulates Fmr1 target selection

(A-D) Pulldown of biotinylated RNA probes of (A) repetitive GGACU sequences incubated with 4 pmol His-NT-Fmr1 and increasing concentrations of GST-Ythdf and (B) the respective quantification plotted as the median of the m6A/A signal intensity +/- SEM of three replicates. The indicated P-values were determined by unpaired, two-tailed Student's t-test. (C) AAACU sequences RNA probes incubated with either 32 pmol recombinant GST-Ythdf or/and 6xHis-NT-Fmr1. Pulled down proteins were analyzed by immunoblotting using α-Ythdf and α-Fmr1 antibodies. (D) EMSA assay using purified GST-Ythdf and His-NT-Fmr1 proteins and RNA probes in the AAACU sequence context.

(E) Intersection of Fmr1 RNA immunoprecipitation target genes for wildtype and Ythdf KO brains, displaying a considerable number of genes gained and lost with Ythdf loss.

(F) Intersection of genes lost (i.e., bound in wild type but not in Ythdf KO) with Ythdf target genes determined by a TRIBE assay. P-value calculated with hypergeometric test.
Ythdf is a N6-methyladenosine reader that modulates Fmr1 target mRNA selection and restricts axonal growth in Drosophila (2020)
Figure 6

Figure 6. Fmr1 and Ythdf regulate CNS axonal growth and Profilin levels

(A) Representative Western blot analysis of protein extracts of late pupae heads (85-95h) from control, Ythdf#0/ Ythdf#0 and Fmr1∆50/Fmr1∆50 flies. The membranes were probed with anti-Profilin or anti-Actin antibodies.

(B) Quantification of Profilin protein levels (mean ± SD) obtained from three independent protein-extraction and Western blot analysis as in (A). Quantification was performed using Fiji. p<0.005 = **; p<0.05 = *, measured with unpaired t-test.

(C) Quantification of chic mRNA levels (mean ± SD) obtained from six independent RNA extractions from control, Ythdf#0/Ythdf#0 and Fmr1∆50 late pupae heads, via real-time PCR analysis. Statistical analysis using unpaired t-test show no significant difference between the samples.

(D) Immunofluorescence analysis of adult chic/+ and chic/+; Ythdf#0/Ythdf#0 using anti-FasII antibody. Scale bar: 50 μm. Pink dashed lines highlight the normal and fused β-lobes.

(E) Quantification of the penetrance of β-lobe fusion phenotype for the indicated genotypes. Bars are labelled with the number of replicates.
Ythdf is a N6-methyladenosine reader that modulates Fmr1 target mRNA selection and restricts axonal growth in Drosophila (2020)
Figure 7

Figure 7. Fmr1 and Ythdf regulate CNS axonal growth and inhibit Futsch translation

(A) Representative confocal images of muscle-6/7 NMJ synapses of abdominal hemisegment A2 for the indicated genotypes labelled HRP (red) and with α-futsch (green) to reveal the neuronal membrane and Futsch protein level. Scale bar: 20 μm.

(B) Quantification of normalized Futsch protein level at NMJ 6/7 in A3 of the indicated genotypes (n = 20). Bars show mean ± s.e.m. P values were determined with a Student's t-test. (p<0.05 = *; p<0.01 = **; p<0.0001=****).

(C) Western Blot analysis of Futsch protein level in isolated third instar larval brains of different mutants compared to control. Numbers indicate normalized Futsch protein level in comparison to Tubulin levels.

(D) Quantification of futsch mRNA levels obtained from isolated 3rd instar larvae brains of the indicated genotypes via real-time PCR analysis. Bars show average ± SD of biological triplicates.

(E) Translating Ribosome affinity purification assay. Quantification of futsch mRNA levels upon immunoprecipitation of GFP-tagged Rpl10Ab or GFP in wildtype and Ythdf#0 3rd instar larvae with Real-time qPCR. Bars show average ± SD of technical triplicates.

(F) Quantification of normalized bouton number (total number of boutons/muscle surface area (μm2 × 1,000)) of NMJ 6/7 in hemisegment A3 of the indicated genotypes. Bars show mean ± s.e.m. Multiple comparisons were performed using one-way ANOVA with a post-hoc Sidak-Bonferroni correction (n.s. = not significant; p<0.01 = **; p<0.0001=****).

Bars are labelled with the number of replicates.
Ythdf is a N6-methyladenosine reader that modulates Fmr1 target mRNA selection and restricts axonal growth in Drosophila (2020)
Figure 8

Figure 8. Ythdf recruits Fmr1 and repress Futsch translation via m6A

(A) futsch genomic region (X1,408,435 - 1,454,00) and corresponding browser tracks from miCLIP-seq experiments in S2R+ cells. futsch transcripts contain two m6A peaks in its 5´ UTR, as indicated.

(B) Schematic of GFP reporter constructs containing an actin promoter and either wildtype 5´ UTR region of futsch or a mutated version (futsch**) containing two-point mutations (A - T) at the identified m6A sites. mCherry is under the control of a same actin promoter.

(C, D) RNA immunoprecipitation assay. Quantification of GFP RNA levels upon immunoprecipitation of Myc-tagged Fmr1 or Flag-tagged Ythdf. Bars show average ± SD of technical triplicates (C) or Flag-tagged Fmr1 in the presence of overexpressed HA-tagged Ythdf. Bars show median ± S.E.M of biological triplicates. P-values were determined with an unpaired, two-tailed Student's t-test. (n.s. = not significant; p<0.01 = **) (D). Rpl15 levels were used as a normalization control and the enrichment of mCherry is shown as a negative control for background binding.

(E-G) Representative Western blot analysis of the protein level (E) and quantification of the translation efficiency of S2R+ cells transfected with the wildtype or mutated futsch reporter constructs upon control or Mettl3 KD (F) or Ythdf KD (G). LacZ dsRNA was used for the control KD. The translation efficiency was calculated as the ratio of the relative GFP protein level and GFP mRNA level. mCherry served as a normalization control for both the protein and mRNA level. Bars show mean ± s.e.m of biological triplicates. P values were determined with a Student's t-test. (n.s. = not significant; p<0.05 = *; p<0.01 = **).

(H) Model of the interplay between Ythdf and Fmr1 in translation control.

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TMBIM5 is the Ca2+/H+ antiporter of mammalian mitochondria (2022)
Figure 1

Figure 1 LETM1 and TMBIM5 interact

A: Scheme illustrating workflow for minaturized AP-MS experiments, left to right: whole cells or isolated mitochondria were lysed or solubilized, respectively. The cell/mitochondrial lysates were used for affinity purification (AP) using the STREP tag and tandem affinity purification (TAP) using STREP and HA tag found on the bait protein. Eluates of the AP and control experiments were reduced, alkylated, and digested by trypsin. Peptides are purified on a C18 stage tip and then run on a LTQ Orbitrap Velos. Protein identifications were made by internal tools using MASCOT and Phenyx and removal of non-specific interactors done using the CRAPome. Created with Biorender.com.

B: MCU was selected as a model protein, the functional complex consists of the 5 proteins above (MCU, MCUb, MICU1, MICU2, EMRE). Note that an additional tissue-specific tertiary interaction partner (MICU3) is only expressed at very low levels in HEK293 cells (Diego De Stefani, personal communication). Illustration adapted from (Sancak et al., 2013).

C: All high confidence interaction partners of LETM1 are shown as nodes. Node color indicates SAINT score, a probability-based measure of interaction confidence. See also Appendix Fig S1A-C.

D: Co-immunoprecipitation of TMBIM5 and LETM1 protein in tandem in the left 3 panels. The input represents the mitochondrial crudely isolated from HEK293 cells and was used as input for the co-IP, LETM1 was immunoprecipitated (left panel, IP: LETM1) using a LETM1 monoclonal antibody and Protein G magnetic beads (ProtG). ProtG beads alone were used as a negative control for binding, immunoprecipitates were immunoblotted (IB) for the indicated proteins to demonstrate interaction. 10% of the input was used for immunoblotting. Prohibitin (PHB) was used as a control to illustrate no nonspecific binding of inner mitochondrial membrane proteins complexes. The middle and right panel of the coIPs illustrates the converse experiment, in the middle in TMBIM5WT and right TMBIMKO, using TMBIM5 as bait (right panel, IP: TMBIM5). The last two right panels show blots from BN-PAGE conducted in TMBIM5WT and KO.
TMBIM5 is the Ca2+/H+ antiporter of mammalian mitochondria (2022)
Figure 2

Figure 2 TMBIM5KO causes mitochondrial matrix swelling and cristae disorganization

A: Western blot analysis of TMBIM5 in control and targeted HeLa and HEK293 clones.

B: Proliferation assay of HEK293 cells in function of TMBIM5. Graph shows the mean ± SD of three individual counts, One-way ANOVA with Dunnett's multiple comparisons test performed against TMBIM5WT *p=0.0155.

C: Live imaging of HEK293 TMBIM5WT and KO cells stained with MitoTracker Green FM. Scale bars: 10 μm.

D: Alteration of the mitochondrial ultrastructure shown by transmission electron microscopy, red arrow pointing to dilated matrix. Wider mitochondria in middle and right panel compared to controls, a middle panel showing the strongest phenotype of matrix width and cristae forms. Scale bars: 1 µm.

E: Isolated mitochondria from three independent replicates of HEK293 TMBIM5WT and TMBIM5KO1 and KO2 were analyzed by immunoblotting using the indicated antibodies, HSP60 and TOM40 served as mitochondrial loading controls.

F: Densitometric analysis of the bands in (E) normalized to loading control, bar graph of three individual experiments (biological replicates), mean ± SD, One-way ANOVA with Bonferroni's multiple comparisons test performed against TMBIM5WT *p<0.05, **p<0.008, Two-way ANOVA with Bonferroni's multiple comparisons test performed for the OPA1 statistics against TMBIM5WT, ***p=0.0009, ****p<0.0001.
TMBIM5 is the Ca2+/H+ antiporter of mammalian mitochondria (2022)
Figure 7

Figure 7 TMBIM5 proteoliposomes mediate Ca2+ and Ca2+-dependent H+ transport

(A) Western blot analysis of purified and reconstituted hTMBIM5 for evaluating the incorporation of hTMBIM5 into proteoliposomes prepared as described in materials and methods. (B) Sketch illustrating the reconstitution of hTMBIM5 in proteoliposomes (red), and the empty liposomes (blue) prepared with Ca2+-5N or pyranine for (C-F).

(C-F) Transport of Ca2+ by hTMBIM5 reconstituted in proteoliposomes containing 10 μM Ca2+-5N at the pH indicated in the panels (C-E) or 20 μM pyranine at pH 7.0 (F). After reconstitution, the fluorescence measurement was started by diluting 200 µL proteoliposomes (red trace) up to 3 mL with transport buffer prepared as described in materials and methods at the indicated pH (C, E) or at pH 7.0 (D, F). After 100 sec, as indicated by the arrow, 7 mM Ca2+ was added to the sample and fluorescence change was recorded. As a control, the same measurement was performed diluting 200 µL liposomes (without incorporated protein, blue trace) up to 3 mL with the same transport buffer. See also Appendix Fig S7 for TMBIM5 optimization, induction and structure overview.

(G-H) Transport of Ca2+ by hTMBIM5 reconstituted in proteoliposomes containing 10 μM Ca2+-5N at pH 7.0. recorded as in (D). (G) and (H) left: sketch illustrating the assay. In (G), after reconstitution 200 μL proteoliposomes were diluted up to 3 mL with transport buffer prepared as described in Materials and Methods. Delta pH was generated adding Ca2+ at pH 6.0 (light green trace), 7.0 (red trace) or 8.0 (deep green trace). In (H), after reconstitution 200 μL proteoliposomes were diluted up to 3 mL with transport buffer prepared as described in materials and methods in the presence of 20 mM K+ and ethanol (red trace) or 10 μM nigericin (orange blue) to generate delta pH. Fluorescence intensity is indicated as Arbitrary Units (AU). Results are representative of three independent experiments (biological replicates).

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