EMBO/soda-nel huggingface dataset:
A GID E3 ligase assembly ubiquitinates an Rsp5 E3 adaptor and regulates plasma membrane transporters (2022)
Figure 2

Figure 2: Gid4 and Gid10 compete for binding to GIDAnt

Lysates from wildtype, ∆Gid4, and Gid4∆C strains expressing endogenously tagged 3xFLAG-Gid10 that were grown at 42°C for one hour, and then returned to 30°C for the indicated timepoints were run on an SDS-PAGE gel and immunoblotted with αFLAG and αPGK.

Wildtype and Gid10 overexpressing yeast strains expressing endogenously tagged Gid4 were grown for 19 hours in YPE, and transitioned to YPD for the indicated timepoints. Lysates were run on an SDS-PAGE gel and immunoblotted with αFLAG and αPGK. Points represent mean, error bars represent standard deviation (n>3 biological replicates).

Fbp1 protein abundance in wildtype and Gid10 overexpressing yeast strains grown in ethanol medium (YPE) for 19 hours, and following 30 minutes and 2 hours glucose recovery. Bars represent mean, error bars represent standard deviation, significance was determined by one-way ANOVA (unpaired) followed by Tukey's multiple comparison test, * indicates p<0.05, ** indicates p<0.01, (n=3 biological replicates).

In vitro competition assay probing the ability of N-terminally 2xStrep-tagged Gid4 (2xS-Gid4) to exchange with Gid10 in the GIDSR10 complex. Proteins after Strep-Tactin pull-down were visualized by Coomassie-stained SDS-PAGE.
A GID E3 ligase assembly ubiquitinates an Rsp5 E3 adaptor and regulates plasma membrane transporters (2022)
Figure 5

Figure 5. GidSR10 ubiquitinates Art2

Art2-3xFLAG was immunocaptured from yeast cells grown in YPD and incubated with GidSR10, GidSR4, GidAnt, or Gid10 for the indicated timepoints. Progress of the reaction was followed by αFLAG immunoblot.

Mdh2-3xFLAG was immunocaptured from yeast cells following growth in YPE for 19h and incubated with GidSR10, GidSR4, GidAnt, or Gid4 for the indicated timepoints. Progress of the reaction was followed by αFLAG immunoblot.

Art2-3xFLAG or Art2P2S-3xFLAG was immunocaptured from yeast cells grown in YPD and incubated with GidSR10, or GidSR10∆C for the indicated timepoints. Progress of the reaction was followed by αFLAG immunoblot.

In vitro ubiquitination assay probing the ability of Gid10 and Gid4 to promote ubiquitination of Art22-29 N-terminus and its P2S mutant. Progress of the reaction was monitored by fluorescent scan of the gel visualizing the Art2 peptide with FAM appended to its C-terminus (pep*).
Extracellular Vesicles Impose Quiescence on Residual Hematopoietic Stem Cells in the Leukemic Niche (2019)
Figure 2

Figure 2: AML-EV increase the relative frequency of LT-HSC, upregulate P53 expression and confer quiescence. (A) Schematic diagram of methods: the in vivo AML-xenografts (tail-vein injection of 105 Molm-14 cells or vehicle per mouse) and the intra-femoral (IF) injection of AML-EV into one femur with a contralateral control vehicle-injected femur of the same mouse. EV were isolated by serial high-speed centrifugation (at 2x, 10x, 100 x 103 g) from AML cell-line culture media or AML patient plasma. Bone marrow was flushed from long bones at the indicated time-points and immunophenotypic analysis was performed by flow cytometry. (B-C) Flow cytometric analysis showing the frequency of LK and KSL cells in lineage-negative cells (left panel) and LT-HSC in KSL (right panel) in: (B) Molm-14-xenograft (red, n=10) vs. control (black, n=10). Data were obtained from at least two independent experiments. (C) IF injection of EV from Molm-14 cells (red, n=8), AML plasma EV (Orange, n=6) and human CD34 EV (Blue, n=4) relative to the vehicle-injected contralateral femurs. Data were obtained from at least two independent experiments. Statistics: Student's t-test. (* P< 0.05, *** P< 0.001) (D) Cell-cycle histograms of KSL (upper panel) and LT-HSC (lower panel) from Molm-14-xenografts or control using Hoechst-33342 staining. (E) Flow cytometric analysis of the Ki67-ve percentage of LT-HSC representing the G0 phase of cell-cycle in: (left panel) Molm-14-xenografts (red, n=7) vs. control (black, n=9); (right panel) IF injection of Molm-14-EV, U-937 EV, HL-60 EV (red, n=5,4,4) vs. human CD34 EV (blue, n=3) vs. controls (black). Data were obtained from at least two independent experiments Statistics: Student's t-test. (* P< 0.05, ** P< 0.01). (F-G) Flow cytometric analysis of intracellular P53 levels shown in histograms (F) and MFI (G) of LT-HSC in: (left panel) Molm-14-xenograft, n=6 red vs. non-engrafted control, n=4 black, or (right panel) IF injection of EV from Molm-14, U-937, HL-60 (red, n=5,5,3) or human CD34 cells (blue, n=6) normalized to vehicle-injected contralateral femurs. Data were obtained from at least two independent experiments. Statistics: Student's t-test. (* P< 0.05, ** P< 0.01).
Extracellular Vesicles Impose Quiescence on Residual Hematopoietic Stem Cells in the Leukemic Niche (2019)
Figure 4

Figure 4: AML-EV-contained miR-1246 suppresses protein synthesis in LT-HSC via the mTOR pathway. (A-B) qRT-PCR gene expression analysis showing the fold change of Pi3K/mTOR associated gene panel in KSL cells sorted from: (A) Molm-14-xenografted mice or (B) IF injected mice with Molm-14-EV relative to their controls and normalized to Gapdh endogenous control. Data are expressed as mean ± SEM from at least three independent experiments, performed in technical replicates. Statistics: One-way ANOVA with Bonferroni post hoc correction. (* P< 0.05, ** P< 0.01). (C-E) Flow cytometric analysis showing the histograms (C) and MFI of intracellular pS6KRP in LT-HSC in: (D) Molm-14-xenograft (red, n=9) vs. non-engrafted control (black, n=7), or (E) IF injection of EV from Molm-14, U-937, HL-60 (red, n=7,3,3) or human CD34 cells (blue, n=4) normalized to the control contralateral femur with subtraction of background fluorescence. Data were obtained from at least two independent experiments. Statistics: Student's t-test. (* P< 0.05). (F-G) Flow cytometric assessment of protein synthesis showing the MFI of: (F) OPP, or (G) pS6RP in NIH-3T3 cells 72 hours after transfection with the indicated miRNA mimics. The results were calculated relative to control (miR-scramble) with the background fluorescence subtracted and performed with at least three independent experiments, in technical replicates. Statistics: One-way ANOVA with Bonferroni post hoc correction (* P< 0.05, ** P< 0.01). (H) Flow cytometric analysis showing the MFI of Raptor in NIH-3T3 cells 72 hours after transfection with the indicated miRNA mimics. The data were calculated relative to miR-scramble and presented as means bars ± SEM and the background fluorescence was subtracted. Performed with at least three independent experiments, in technical replicates. Statistics: One-way ANOVA with Bonferroni post hoc correction (* P< 0.05, ** P< 0.01). (I) Dual Luciferase reporter assay. NIH-3T3 was transfected with the miRNA mimics. Three hours later the cells were transfected with the Raptor 3′UTR cloned into the psiCheck-2 vector for a total of 48 hours. Data are presented as %RLU (relative luciferase units) of the miR-scramble control as means ± SEM from at least three independent experiments, performed in technical replicates. Statistics: One-way ANOVA with Bonferroni post hoc correction (* P< 0.05). (J-K) Cell-cycle flow cytometric analysis using Ki67/Hoechst-33342 staining of the percentage of LT-HSC in the G0 phase after nucleofection of cKit+ cells using the AmaxaTM P3 Primary Cell 4D-Nucleoeftcor kit, (J) Cells were nucleofected with miR-Scramble (CTRL) or miR-1246 mimic (n=3) for 72 hours, (K) Cells were nucleofected with Anti-miR-Scramble (CTRL) or Anti-miR-1246 (n=5) and 1-hour later co-treatment with Molm-14 EVs for 72 hours. Statistics: Student's t-test. (* P< 0.05, ** P< 0.01).
Extracellular Vesicles Impose Quiescence on Residual Hematopoietic Stem Cells in the Leukemic Niche (2019)
Figure 5

Figure 5: LT-HSC restore protein synthesis and proliferative function upon transplantation to a non-leukemic host. (A) A diagram showing the scheme of repopulation experiments on non-leukemic host. KSL were purified from Molm-14-xenografted male mice (red), or control males (black) and transplanted into 150cGy sub-lethally irradiated female recipients (104 cells per mouse) and used for subsequent repopulation studies described in D-F. (B) CFU-C assay showing the relative number of colonies (left panel) and the colony subtypes (right panel) from KSL cells derived from Molm-14-xenografted mice (red, n=6) vs. non-engrafted control mice (black, n=5) and in vitro cultured for 7 days in methylcellulose media. Experiments were performed in technical triplicates. "G" Granulocyte, "M" Monocyte, "E" Erythrocytes. Statistics: Student's t-test. (* P< 0.05, ** P< 0.01). (C) LT-HSC assessment after repopulation, recipient female mice (n=4 per group) were sacrificed after 21 days and their LT-HSC were compared using P53, pS6RP and OPP flow cytometric assays. (D-E) Evaluation of the in vivo long-term repopulation capacity. (D) For secondary transplantation (n=6 per group), PB samples were collected twice weekly for 12 weeks (WK) and the donor chimerism was measured by qPCR of Sex-determining Region Y (SRY) normalized to Gapdh gatekeeper as shown in the left panel. Sixteen weeks later, mice were sacrificed and BM male chimerism percentage was assessed as shown in the right panel. (E) Tertiary transplantation, unfractionated BM cells from the secondary recipients (n=6 per group, 106 cells per mouse) were injected into tertiary 150cGy sub-lethally irradiated female recipients and blood SRY levels were assessed for 20 weeks. Statistics on D-E: Student's t-test for two samples comparison and regression analysis for longitudinal comparison (NS = not significant, * P< 0.05).
Extracellular Vesicles Impose Quiescence on Residual Hematopoietic Stem Cells in the Leukemic Niche (2019)
Figure 6

Figure 6: AML microenvironment conditioned HSC accrue persistent DDR and gain a proliferative advantage. (A) A schematic diagram demonstrating the serial replating experiment. KSL were sorted from primary recipient control mice (black, n=4), or Molm-14-xenografts (red, n=4) and plated in triplicate (500 cells/plate/mouse) in methylcellulose media. Seven days later, colonies were counted and serially replated in triplicate (5000 cells/plate/mouse). The replating process was repeated twice for a total of three rounds (labeled week "WK" 1, 2 and 3). (B-C) Serial replating CFU-C analysis presented as mean ± SEM showing: (B) the number of colonies per 1000 plated cells, and (C) the number of cells per colony, for each week time-point. Statistics: Student's t-test. (* P< 0.05, ** P< 0.01). (D-I) DNA-damage assessment in KSL from primary recipients and after two turns of replating. (D, F, H) Number of γH2AX foci in control-derived and Molm-14-xenograft-derived: (D) Primary recipient KSL, n=211 and 321, (F) cells from W1 plating, n=218 and 369, and (H) cells from W2 plating, n=770 and 853. All cells were stained with phosphoH2axser139 and the nuclear stain Hoechst (blue), imaged using the GE/API DV widefield microscope (60X objective) to show the γH2AX foci (green) and counted using Imaris software. Scale bars are 2μm. Statistics: Student's t-test. (* P< 0.05, ** P< 0.01, *** P< 0.001). (E, G, I) qRT-PCR gene expression analysis showing the fold-change of select DNA-damage response-genes panel in Molm-14-xenograft-derived: (E) Primary recipient KSL, n=6 (G) Cells from W1 plating, n=4, and (I) Cells from W2 plating n=4. Data indicated by bars of fold-change calculated relative to matching controls, normalized to the endogenous control Gapdh and presented as means ± SEM. Statistics: One-way ANOVA with Bonferroni post hoc correction. (* P< 0.05, ** P< 0.01). (J) Scheme of DDR assessment in KSL and LT-HSC derived from serial in vivo repopulation. (K-L) Number of γH2AX foci, respectively, in control- and Molm-14-xenograft-derived: (K) KSL, n=101 / 204, and (L) LT-HSC, n=33 / 53. Images were processed similar to D-F. Scale bars are 3μm. Statistics: Student's t-test. (** P< 0.01). (M-N) qRT-PCR analysis of the DNA-damage-genes panel in Molm-14-xenograft-derived: (M) KSL, and (N) LT-HSC, relative to matched controls. Data depicted as fold-change relative to matching controls, normalized to the endogenous control Gapdh and presented as mean ± SEM. Statistics: One-way ANOVA with Bonferroni post hoc correction. (* P< 0.05).

Expanded view Figure Legends:
Structure of RyR1 in native membranes (2020)
Figure 1

Figure 1. Structure of RyR1 in native SR membranes.

A. A slice through a filtered tomogram showing SR vesicles in contact with a putative T-tubule. Individual RyR1 molecules are indicated by the red circles. The dense protein coat on the surface of the SR vesicles correspond to the SERCA pump. Areas circled in yellow indicate accumulation of density inside the SR. Scale bar: 20 nm.

B. A slice through the structure of RyR1 in contact with putative TT-membrane.

C. A middle slice through the in situ structure of RyR1 at 12.6-Å resolution. The observed additional transmembrane density is indicated by the red arrows. Scale bars in B, C: 10 nm.

D. Slice through the structures of apoRyR1 with the putative TT-membrane (left) and the standalone apoRyR1 (right, with the flipped handendness and reduced resolution for comparison) 22 Å away from the middle slice (Y = 92 in the volume coordinates). Scale bar: 10 nm.

E. Volume-rendered visualizations of the average structure of RyR1 fit with the RyR1 atomic model (PDB: 5TB2). The left panel is a thin section through the center of the volume.

F. The location of the CaM/S100A1 binding site is marked with red circles in slices through maps of the in situ structure (left) and the single particle structure (EMDB: 8393) in the same orientation as the in situ structure and filtered to 15 Å (right). Scale bar: 10 nm.

G. The in situ structure with an atomic model (PDB: 5TB2) fitted showing an unaccommodated density. Red circles are sites of potential interaction between RyR1 and the regulatory proteins.
Structure of RyR1 in native membranes (2020)
Figure 2

Figure 2. Intra-SR extensions of RyR1.

A. A slice through the average structure of the apo-state at a position 22 Å away from the middle slice shown in Fig. 1C. The intra-SR extension is boxed on the density in red.

B. An enlarged view of a volume-rendered representation fitted with an atomic model (PDB: 5TB2) corresponding to the region boxed in (A). The red arrow points to a density between the bilayer leaflets; this density is at a different location from the one in Figure 1C, D.

C. Representative SR vesicles with different distributions of inner-SR density: segregated, evenly distributed, empty, and empty with filaments. RyR1 particles are circled in red, areas circled in yellow indicate accumulation of density inside the SR.

D. The fractions of vesicles that fall into each density category for each RyR1 sample (EDTA: n=64; 1 mM Ca2+: n = 32; 5 mM Ca2+: n=21, technical replicates). Blue corresponds to the fraction of SR lumen vesicles showing evenly distributed density, orange to segregated density, and grey to empty vesicles.

E. Structures of RyR1 determined in presence of increasing Ca2+ concentration (1 mM and 5 mM), all with intra-SR extensions. Scale bars: 10 nm in (A) and (E), 20 nm in (C).
Structure of RyR1 in native membranes (2020)
Figure 4

Figure 4. Activation of RyR1 in situ.

A. Structure of ryRyR1 at a resolution of 17.5 Å. Two views are through the middle slice (top) of the reconstruction and at the level of sarcoplasmic extentions 22 Å below (bottom panels). The movement of the outer domains is associated with a visible change in membrane curvature as compared to the apo-structure. This movement is highlighted by red arrows in the right panels. Scale bar: 10 nm.

B. A volume-rendered representation of the apoRyR1 (orange) and ryRyR1 (blue) structures with the highlighted conformational changes. The ryRyR1 reconstruction was mirrored in the right panel for a clear comparison to the aporRyR1. The left panel depicts the view down onto the receptor from the cytoplasm. The right panel depicts the section through the isosurface indicated by the dashed line, with red arrows indicating the conformational transition of the SR extensions (dark-orange and blue circles for apoRyR1 and ryRyR1, respectively). Red arrows indicate the conformational changes of the SR extentions.

Expanded View Figure Legends
STAT3 but not STAT4 is critical for γδT17 cell responses and skin inflammation (2019)
Figure 1

Figure 1. STAT3 regulates skin γδT17 cell homeostasis and their response and pathogenicity during imiquimod driven psoriasis.

Flow cytometric analysis of γδ T cells in RORγtCRE-STAT3F/F (Cre+) and littermate control mice (Cre−). In graphs, each symbol represents a mouse and line the median. *p < 0.05, **p < 0.01, ***p < 0.001 using Mann-Whitney test.

(A-B) Numbers of γδT17 cells in the LN (A) and skin (B) before (steady-state) and after (IMQ) IMQ-induced psoriasis. Steady-state: n = 8; 4 experiments, IMQ: n = 11-12; 4 experiments.

(C) Representative micrographs indicating ear skin sections stained for H&E before and after IMQ-induced psoriasis (scale bar = 100μm).

(D) Quantification of epidermal thickening from H&E stained sections. Steady-state: n = 8; 4 experiments, IMQ: n = 11-12; 4 experiments. Each symbol is the average thickening from 5 micrographs measured in μm.
STAT3 but not STAT4 is critical for γδT17 cell responses and skin inflammation (2019)
Figure 2

Figure 2. STAT4 is not required for γδT17 cell homeostasis or their inflammatory response.

Flow cytometric analysis of γδ T cells in STAT4−/− (−/−) and littermate control mice (+/−). In graphs, each symbol represents a mouse and line the median.

(A-B) Numbers of γδT17 cells in the LN and skin (staining as in Fig EV1) before (steady-state) and after (IMQ) IMQ-induced psoriasis. Steady-state: n = 6; 3 experiments, IMQ: n = 11; 4 experiments.

(C) Representative micrographs indicating ear skin sections stained for H&E before and after IMQ-induced psoriasis (scale bar = 100μm).

(D) Quantification of epidermal thickening from H&E stained sections. Steady-state: n = 6; 3 experiments, IMQ: n = 8; 3 experiments. Each symbol is the average thickening from 5 micrographs measured in μm.
STAT3 but not STAT4 is critical for γδT17 cell responses and skin inflammation (2019)
Figure 3

Figure 3. Production of IL-17A and IL-17F is driven by STAT3 during inflammation but not at steady-state.

Flow cytometric analysis of lymph node γδ T cells in RORγtCRE-STAT3F/F (Cre+) and littermate control mice (Cre−). In graphs, each symbol represents a mouse or experiment and line the median. *p < 0.05, **p < 0.01, ***p < 0.001 using Mann-Whitney test (B, E, G, H) or 2-way ANOVA (C, F).

(A-F) (A, D) Representative dot plots showing IL-17A (A) or IL-17F (D) and IFNγ production in γδ T cells before (steady-state) and after IMQ-induced psoriasis. Numbers in gate indicate % positive cells; numbers outside the gate indicate mean fluorescence intensity of IL-17A or IL-17F. (B, E) Frequency of IL-17A+ (B) and IL-17F+ (E) γδ T cells before (steady-state) and after (IMQ) IMQ-induced psoriasis. (C, F) Quantification of mean fluorescence intensity (MFI) of IL-17A (C) and IL-17F (F) staining in γδ T cells after IMQ-induced psoriasis (each color represents a different experiment). In (A-C) Steady-state: n = 8; 4 experiments, IMQ: n = 11-12; 4 experiments. In (D-F) Steady-state: n = 6; 3 experiments, IMQ: n = 8 3 experiments. In (C) ***p < 0.001 with 2-way ANOVA, in (F) *p < 0.05 with Mann-Whitney or ***p < 0.001 with 2-way ANOVA.

(G-H) Frequency of IL-17A+ (G) and IL-17F+ γδ T (H) cells or IL-17A (G) and IL-17F (H) MFI following culture with IL-1β, IL-23 or nothing. Each symbol represents one experiment. Open circles = Cre+. (each color represents a different culture condition).
STAT3 but not STAT4 is critical for γδT17 cell responses and skin inflammation (2019)
Figure 4

Figure 4. STAT3 is necessary for IL-22 production by γδT17 cells.

Flow cytometric analysis of lymph node γδ T cells in RORγtCRE-STAT3F/F (Cre+) and littermate control mice (Cre−). In graphs, each symbol represents a mouse or experiment and line the median. *p < 0.05, **p < 0.01, ****p < 0.0001 using Mann-Whitney test.

(A-B) Representative dot plots showing IL-22 and IL-17A production in γδ T cells (A) and frequency of IL-22+ γδ T cells (B) before (steady-state) and after (IMQ) IMQ-induced psoriasis. Steady-state: n = 6; 3 experiments, IMQ: n = 8-9; 3 experiments.

(C-D) Representative dot plots showing IL-22 and IL-17A production in γδ T cells (C) and frequency of IL-22+ γδ T cells (D) following culture without (−) or with 40ng/ml recombinant IL-23 (n = 9-10, 3 experiments).

(E) Frequency of IL-22+ γδ T cells following culture with IL-1β, IL-23 or nothing. Each symbol represents one experiment. Open circles = Cre+ (each color represents a different culture condition).
STAT3 but not STAT4 is critical for γδT17 cell responses and skin inflammation (2019)
Figure 5

Figure 5. Regulation of cytokine production by STAT4

Flow cytometric analysis of lymph node γδ T cells in STAT4−/− (−/−) and littermate control mice (+/−). In graphs, each symbol represents a mouse and line the median. *p < 0.05, ***p < 0.001 using Mann-Whitney test (A, B, E, F) or 2-way ANOVA (C, D).

(A, ) Frequency of IL-17A+ (A) γδ T cells before (steady-state) and after (IMQ) IMQ-induced psoriasis. .

(B) Frequency of IL-17F+ γδ T cells before (steady-state) and after (IMQ) IMQ-induced psoriasis.

(C) Quantification of mean fluorescence intensity (MFI) of IL-17A staining in γδ T cells after IMQ-induced psoriasis

(D) Quantification of mean fluorescence intensity (MFI) of IL-17F staining in γδ T cells after IMQ-induced psoriasis.(E) Frequency of IL-22+ γδ T cells before (steady-state) and after (IMQ) IMQ-induced psoriasis.

(F) Frequency of IL-22+ γδ T cells following culture without (−) or with 40ng/ml recombinant IL-23

Data information: (A, B and E) Steady-state: n = 7; 4 experiments, IMQ: n = 11; 4 experiments. (C, D) IMQ: n = 11; 4 experiments. (F) n = 8; 3 experiments. (B, D) Each color represents a different experiment.