EMBO/soda-nel huggingface dataset:
Quantifying persistence in the T cell signaling network using an optically controllable antigen receptor (2021)
Figure 4

Figure 4. Activation of FOS transcription factor remains dependent on proximal signaling

(A) Representative fluorescent Western blot showing the dynamics of FOS phosphorylation (p-FOSS32) on light-modulated control. Conjugated OptoCAR‑T cells were stimulated for a defined period after dimerizer addition (denoted above blots) before either being left in the dark (dark line) or illuminated (blue line). Full blot image with total protein normalization control shown in Figure EV3A.
(B) Representative fluorescent Western blot showing the dynamics of FOS expression (FOSTotal) on light-modulated control, from the same dataset as in (A). Full blot image with total protein normalization control shown in Figure EV3B.
(C) Quantification of FOS expression detected by fluorescent Western blots at different timepoints prior to illumination. Datasets were corrected for background fluorescence and scaled to FOS level at 3 hours. Bars show mean ± SEM (n=3).
(D) Quantification of the magnitude of phosphorylated FOS (p-FOSS32) as a fraction of total FOS, detected by fluorescent Western blots at different timepoints prior to illumination. Datasets were scaled relative to phosphorylated FOS level at 3 hours. Bars show mean ± SEM (n=3).
(E) Distributions of phosphorylated FOS molecular mass at different timepoints after illuminating OptoCAR‑T cell conjugates to disrupt signaling, quantified from blot in (A). The molecular mass distributions on initial illumination after 3 hours of activation (0 min) and end of measurement (15 min) are shown as grey and blue lines, respectively. Distributions in other plots are shown as red lines that vary between these extremes.
(F) A schematic to show how the active (higher molecular mass) fraction was estimated from the distributions in (E), with a cutoff of 0.4 defined by the 0 min distribution and used to analyze the datasets.
(G) The data from three independent experiments (blue, red and yellow) are shown either when FOS active fraction was measured in the dark (dashed lines) or under illumination (solid lines).
(H) The light state datasets shown in (G) were combined and rescaled between the active fraction at 0 and 15 minutes. Indicated half-life was calculated from the time taken for the activated fraction to decrease to 50% of maximum. Bars show mean ± SEM (n=3).
(I) The dark state datasets shown in (G) were similarly combined and rescaled, as in (H). Bars show mean ± SEM (n=3).
Quantifying persistence in the T cell signaling network using an optically controllable antigen receptor (2021)
Figure 6

Figure 6. Short-term signal persistence can be directly observed in gene expression output

(A) Schematic showing the relationship between a signal (grey) and the corresponding output (red). A continuous signal pulse leads to maximal output (top left) whereas well-separated pulses independently increase output (top right). When pulses are separated by shorter periods when signals persist, the combined output is increased between these two bounds (bottom row).
(B) Single pulses of varying duration were applied to OptoCAR‑T cells and the geometric mean of GFP expression was measured for all pulse lengths 24 hours after initiation of signaling, with illumination started once the signal pulse had ended. All values have been corrected for background GFP fluorescence from non-activated OptoCAR‑T cells and shown relative to the output at 24 hours. A representative dataset is provided in Figure EV4F. Bars show mean ± SEM (n=3)
(C) Equivalent datasets as in (B) for the geometric mean of CD69 upregulation. A representative dataset is provided in Figure EV4F. Bars show mean ± SEM (n=3)
(D) Schematic illustrating the pulse-frequency modulation of signal pulses in OptoCAR‑T cells. For a given pulse duration, the length of the light interval between pulses was varied between duty cycles of 100% (continuous) and 25%. The output after 24 hours was measured for all samples.
(E) Series of experiments showing how NFAT-mediated GFP expression is modulated by pulsatile trains of signaling. A combined signaling period of 6 hours was broken into signal pulses ranging from 15 minutes to 2 hours and the light-induced 'off' interval between these pulses varied, denoted as x in the schematic above each plot. Geometric mean of GFP intensity is shown plotted as a function of light-induced 'off' interval, relative to a single, continuous 6-hour output. Bars show mean ± SEM (n=3) of biological replicates.
(F) Series of experiments showing how CD69 upregulation is modulated by pulsatile trains of signaling, from same dataset as in (E). Bars show mean ± SEM (n=3).
Quantifying persistence in the T cell signaling network using an optically controllable antigen receptor (2021)
Figure 7

Figure 7. anti-CD19 CAR‑T cell activation can be enhanced by pulsatile signaling

(A) Schematic showing the anti-CD19 OptoCAR engineered to be light-responsive. Binding of the OptoCARCD19 expressed in T cells to CD19-expressing B cells drives signaling through an equivalent intracellular sequence used in the OptoCAR, where illumination with blue light causes the reversible disruption of receptor signaling.
(B) Stimulating primary CD4+ T cells expressing OptoCARCD19 over 24 hours in the dark drives expression of CD137 (4‑1BB), a robust activation marker. Continuous illumination completely abolishes this to CD137 levels equivalent to unstimulated T cells.
(C) Plot of geometric mean of CD137 expression when the length of an individual signaling pulse is varied. Single pulse ranged from 0 to 24 hours, with sample illumination initiated at the end of the pulse and CD137 expression measured for all samples 24 hours after initiation of signaling. Bars show mean ± SEM of biological replicates (n=4).
(D) The cRQ values from RT-qPCR of indicated cells. Total RNA was extracted and CD137 mRNA levels measured relative to that from HEK293T cells. Conjugated OptoCARCD19‑T cells were also assayed either before or after 24 hours stimulation in the dark state. Individual values from three biological replicates are shown, with mean depicted by bar plot.
(E) Series of experiments showing how CD137 expression is modulated by pulsatile trains of signaling in primary CD4+ T cells activated through OptoCARCD19. A combined signaling period of 6 hours was broken into pulses ranging from 15 minutes to 2 hours and the refractory period between these pulses varied (x-axis), shown in the schematic above each plot. Geometric mean of CD137 intensity is shown plotted as a function of inter-pulse interval, scaled to continuous 6-hour output. Bars show mean ± SEM (n=5).
(F) Representative dataset from (E), demonstrating how pulsatile stimulation of OptoCARCD19‑T cells drives more efficient activation. Dotted and gray histograms show CD137 expression in resting cells and 24-hour activation, respectively. The filled histogram shows either a continuous 6‑hour stimulation (left) or cumulative pulsed signals over 24 hours driving substantially more potent activation.

SUPPLEMENTAL INFORMATION LEGENDS
Structure of cytosine transport protein CodB provides insight into nucleobase-cation symporter mechanism (2022)
Figure 1

Figure 1: Cytosine binding and transport by CodB.

A) In E coli, CodB is found in an operon with CodA with overlapping genes. Transcription is regulated by the Nitrogen Assimilation Control protein (NAC) in response to low nitrogen levels (Danielsen et al., 1992; Muse et al, 2003; Santos-Zavaleta et al, 2019).

B) Binding affinity of CodB for cytosine as measured using the thermostability assay. Cytosine was titrated into detergent solubilised membranes from cells overexpressing CodB. The Kd was estimated to be 51 ± 9 µM. The measurements are the average of 4 independent titrations with error bars of the s.e.m.

C) Time course of 3H-5-cytosine uptake by CodB. Experiments were done, either in the presence or absence of an inwardly-directed sodium ion gradient or with choline chloride. Lemo21 (DE3) cells were used as a background measurement, with Lemo21(DE3) expressing CodB. Values reported are the averaged mean ± s.e.m. from n = 3 independent cultures.
Pervasive compartment-specific regulation of gene expression during homeostatic synaptic scaling (2021)
Figure 3

Figure 3: Validation of compartment-specific regulations.

(A) Real-time quantitative PCR (RT-qPCR) of transcripts changing differentially between compartments using the compartmentalized cultures after 48 hours PTX-treatment. n=8 independent biological replicates (except for Plk2 (n=7), where one measurement couldn't be performed due to insufficient cDNA); PTX-effect was assessed by three-way ANOVA followed by Tukey's post-hoc multiple comparison test; *p < 0.05; **p < 0.01.

(B) Representative images of single-molecule FISH (smFISH) in either control or 48 h PTX-treated rat hippocampal neurons (DIV20) using probes specific for Add2, Dnajc6 and Sort1 (green). MAP2 immunostaining (red) was used to visualize neuronal somata and dendrites. Inserts at higher magnification illustrate PTX-dependent changes in dendritic RNA puncta. Scale bar = 10 μm.

(C) Quantification of B. (n=3-4 independet biological replicates with 8-10 cells averaged per condition and replicate; two-sample Student's t-test; *p < 0.05; **p < 0.01).

Boxplots: central line: median; box: 25th to 75th percentile; whiskers: until last data point within 1.5x interquartile range (IQR).
Pervasive compartment-specific regulation of gene expression during homeostatic synaptic scaling (2021)
Figure 5

Figure 5: PTX-mediated regulation of local translation.

(A) Representative pictures of nascent Camk2a-peptides in mock- and PTX-treated neurons. Upper Panels: Representative overview images with merged channels (grey: MAP2-staining, red: Duolink-PLA signal, blue: Hoechst nuclear staining). Middle Panels: Magnifications of boxed areas marked in (A). MAP2 signal was used to outline somata and dendritic processes (white lines). Lower Panels: Negative control of puromycin-untreated cells with representative Duolink-signal in dendrite and soma, respectively. (Scale bars: in overview images 20 μm, in inserts of higher magnification 5 μm).

(B) Same as A with nascent Synapsin1-peptides.

(C) Quantification of Duolink-particle density of Figure 5A and B assessing PTX-treatment effect. n=3 independent experiments with 10 images averaged per condition and experiment; Three-way ANOVA followed by Tukey's post-hoc multiple comparison test; *p < 0.05; **p < 0.01).

(D) Quantification of Duolink-particle density of Figure 5A and B assessing Puromycin-treatment effect. n=3 independent experiments for Puromycin-Minus and n=6 datapoints for Puromycin-Plus conditions (PTX- and mock-conditions combined together across n=3 independent experiments, taken into account in ANOVA-model) with 10 images averaged per condition and experiment; Three-way ANOVA followed by Tukey's post-hoc multiple comparison test; **p < 0.01, ***p < 0.001).

Boxplots: central line: median; box: 25th to 75th percentile; whiskers: until last data point within 1.5x interquartile range (IQR).
Defective autophagy is a key feature of cerebral cavernous malformations. (2015)
Figure 1

KRIT1-ablated cells display autophagy suppression

Immunoblot analysis of p62 and LC3 I/II in KRIT1 wt and KRIT1-KO endothelial cells. Actin was used as a loading control. Quantification of total LC3 on actin is reported (*P = 0.02712). The results are representative of three independent experiments.

Representative images of p62 dots in KRIT1 wt and KRIT1-KO endothelial cells. Scale bar, 20 μm. Magnifications in insets. Right, quantitative analysis of p62 distribution of dots is reported (four independent experiments; n = 35 cells per group). *P = 0.00542 (dotted); *P = 0.00014 (nuclear).

Immunoblot analysis of p62 and LC3 I/II in KRIT1-KO and KRIT1-KO re-expressing KRIT1 (KO+KRIT1) MEFs. Left, immunoblot showing KRIT1 levels in KRIT1-KO and KO+KRIT1 cells. Right, immunoblots for p62 and LC3 I/II. Actin was used as a loading marker. Quantification of total LC3 on actin is reported (*P = 0.01248). The results are representative of three independent experiments.

Representative images of p62 dots in KO+KRIT1 (top) and KRIT1-KO cells (bottom). Scale bar, 50 μm. Magnifications in insets. Right, quantitative analysis of the number of p62 dots per cell is shown (four independent experiments; n = 50 cells per group). *P = 7.18e−14.

Immunoblot analysis of hBMECs transiently transfected with control siRNA or KRIT1 siRNA. Left, evaluation of siRNA efficiency with antibody directed against KRIT1. Right, immunoblots for p62 and LC3 I/II. Actin was used as a loading marker. Quantification of total LC3 on actin is reported (*P = 0.03071). The results are representative of three independent experiments.

Immunoblot analysis of EA.hy926 cells transiently transfected with control siRNA or KRIT1 siRNA. Left, evaluation of siRNA efficiency with antibody directed against KRIT1. Right, immunoblot for p62 and LC3 I/II. Actin was used as a loading marker. Quantification of total LC3 on actin is reported (*P = 0.02527). The results are representative of three independent experiments.

Immunofluorescence analysis of p62 (green) and ProteoStat Aggresome staining detection reagent (red) in KRIT1 wt and KRIT1-KO lung endothelial cells. The yellow signal in the merged images represents an overlapping spatial relationship between green and red fluorescence. Magnification in insets. Scale bar, 50 μm. The images are representative of four independent experiments.

Immunofluorescence analysis of p62 (green) and ProteoStat Aggresome staining detection reagent (red) in KRIT1-KO re-expressing KRIT1 (KO+KRIT1) and KRIT1-KO MEFs. The yellow signal in the merged images represents an overlapping spatial relationship between green and red fluorescence. Magnification in insets. Scale bar, 50 μm. The images are representative of four independent experiments.

Source data are available online for this figure.
Defective autophagy is a key feature of cerebral cavernous malformations. (2015)
Figure 3

Defective autophagy underlies major phenotypic signatures of CCM disease

Cd44, PAI1 (also known as Serpine1), and Id1 mRNA expression levels in KRIT1 wt and KRIT1-KO endothelial cells were assessed by quantitative real-time PCR. Where indicated, KRIT1 wt and KRIT1-KO endothelial cells were treated with 100 nM Torin1 or 500 nM rapamycin for 16h. The data are expressed as the mean ± s.e.m. Cd44: *P = 0.02848 (KO ctrl vs. KO Rapa); *P = 0.02605 (KO ctrl vs. KO Tor1). PAI1: *P = 0.04446 (KO ctrl vs. KO Rapa); *P = 0.03996 (KO ctrl vs. KO Tor1). Id1: *P = 0.00266 (KO ctrl vs. KO Rapa); *P = 0.01554 (KO ctrl vs. KO Tor1). n = 3 independent experiments.

Immunoblot analysis of CD31/Pecam-1, vascular endothelial cadherin (VE-cadherin), and actin in KRIT1-KO endothelial cells that were treated with 100 nM Torin1 or 500 nM rapamycin for 24 h. The results are representative of three independent experiments.

Immunoblot analysis of CD31/Pecam-1, vascular endothelial cadherin (VE-cadherin), N-cadherin, alpha-smooth muscle actin (alpha-SMA), and actin in HUVECs transfected with control siRNA or ATG7 siRNA.

Formation of capillary-like structures measured by tube formation assays. HUVECs were transfected with control siRNA or ATG7 siRNA for 72 h. Representative phase-contrast (Scale bar, 100 μm) and calcein-fluorescent (Scale bar, 50 μm) images were reported. All data are presented as percentage ± s.e.m from three different experiments performed in duplicate. *P = 1.29e−11.

Immunoblot analysis of p62, LC3 I/II, and actin in CCM3 wt and CCM3-KO endothelial cells treated with 100 nM Torin1 or 500 nM rapamycin for 4 h. The results are representative of three independent experiments.

Representative immunostaining of retina sections from wt and a model of inducible and endothelial-specific CCM3-KO (CCM3-ECKO) at postnatal day 14. Endothelium was stained with isolectin B4 (ISOB4) (blue). A, artery; V, vein. p62 aggregates can be observed in endothelial cells forming retinal lesions in CCM3-ECKO animals (scale bar: 200 μm). Scale bar of magnifications: 100 μm.

Representative immunostaining of brain sections from wt and a model of inducible and endothelial-specific CCM3-knockout mice (CCM3-ECKO) at postnatal day 9. p62 aggregates can be observed in the proximity of CCM lesions (arrows). Cell nuclei (DAPI) are in blue. Scale bar, 30 μm. Smaller panel shows the magnifications of blood vessels (green). Scale bar, 10 μm.

Source data are available online for this figure.
Defective autophagy is a key feature of cerebral cavernous malformations. (2015)
Figure 4

Accumulation of p62 in endothelial cells lining human CCM lesions

p62 immunohistochemical (IHC) staining in human brain tissue.

A, B Normal vascular endothelium of autoptic brain parenchyma samples is lacking the typical autophagic p62 granules as shown by the negative staining for p62. Scale bars: (A) 200 μm; (B) 100 μm.

C–F Two different representative samples of CCM lesions with a thin, single layer brain endothelium displaying either moderate (C, D) or marked (E, F) positive perinuclear “pearl necklace-like” immunostaining for p62 granules. (C, D), case n° 4 (p62++), and (E, F), case n° 8 (p62+++) are representative of CCM cases listed in Appendix Table S1. Scale bars: (C, E) 200 μm; (D, F) 100 μm. Arrows indicate endothelial p62 positive staining.

G–I Hematoxylin and eosin (H&E) staining (G) and p62 immunohistochemical analysis (H, I) of a CCM surgical sample (case n° 6 in Appendix Table S1) containing normal vessels in the peri-lesional area, which served as an internal control. Notice marked p62-positive staining in endothelial cells lining a CCM lesion (H, arrows) and p62-negative staining in endothelial cells lining a normal peri-lesional vessel (I, arrows). Scale bars: (G) 300 μm; (H, I) 100 μm. Background staining in brain parenchyma surrounding CCM lesions may be attributed to either cell debris or p62 immunoreactivity in neuronal and glial cells.
Phospholipid-transfer function of PTPIP51 at mitochondria-associated ER membranes (2021)
Figure 3

Figure 3. Phospholipid binding and phospholipid transfer functions of PTPIP51.

(A) PIP strip lipid binding assay with recombinant PTPIP51_ΔTM, PTPIP51_TPR, and PTPIP51_ΔTPR.

(B) PA binding of PTPIP51 (PTPIP51_ΔTM and PTPIP51_TPR) was monitored by a PA precipitation assay and a liposome flotation assay. The POPC:POPE:POPA ratios in each liposome were 50:50:0, 50:40:10, 50:30:20, and 50:20:30. Diagrams of the liposome precipitation and flotation assays are illustrated separately.

(C) The PA transfer activity of PTPIP51 (PTPIP51_ΔTM and PTPIP51_TPR) was monitored by a FRET-based lipid transfer assay. A schematic cartoon of the FRET-based in vitro lipid transfer assay is presented on the left. a.u.: arbitrary unit.

(D) The alternative bead pulldown-based lipid transfer assay with PA and MLCL. A schematic cartoon representation of the fluorescent lipid transfer assay is shown on the left. The y-axes in the graphs show the percentage of acceptor liposome fluorescence calculated using the following formula: 100×Facceptor/(Facceptor+Fdonor).

Data information: The data are presented as the mean ± SD of technical triplicate (C) or quadruplicate (D) from one representative experiment (n = 2), and p-values calculated using one-way ANOVA are shown.
Phospholipid-transfer function of PTPIP51 at mitochondria-associated ER membranes (2021)
Figure 4

Figure 4. The interaction between the FFAT-like motif of PTPIP51 and VAPB.

(A) A GST pulldown assay was performed to monitor the interaction between 6×His tag-fused VAPB_ΔTM and GST-fused PTPIP51_TPR (or GST-fused PTPIP51_ΔTM). VAPB_ΔTM pulled down by PTPIP51_ΔTM is marked with an asterisk and highlighted with a red arrow.

(B) Schematic representation of the FFAT-like motif of PTPIP51 and amino acid sequence alignment of the FFAT-like motifs of human, mouse, and chicken proteins.

(C) Results of fluorescence polarization (FP) experiments to monitor the interaction between the FFAT-like motif-containing peptide of PTPIP51 and VAPB_ΔTM. The Kd value of the interaction determined in the FP experiment was ~53 µM. Peptides with mutation of conserved phenylalanine and tyrosine residues in the FFAT motif were also tested. The data are presented as the mean ± SD of technical quadruplicate experiments.
Phospholipid-transfer function of PTPIP51 at mitochondria-associated ER membranes (2021)
Figure 5

Figure 5. Mitochondrial CL levels mediated by PTPIP51.

(A) CL levels after PTPIP51 depletion and reconstitution were monitored. A doxycycline-induced shRNA expression system was used to deplete the PTPIP51 protein in HeLa cells. Left, western blot analysis of HeLa cells in which PTPIP51 was depleted and restored. β-ACTIN was used as the loading control. Right, the mitochondrial CL level (nmol/mg) according to PTPIP51 expression. To reconstitute PTPIP51, the full-length PTPIP51 gene was transiently expressed. The data are presented as the mean ± SD of technical triplicate experiments from one representative experiment (n = 3), and p-values calculated using Student's t-test are shown.

(B) Quantification of CL in mitochondria using lipidomic analysis after PTPIP51 depletion and reconstitution. Color codes for each sample were presented in the lower right subpanel. Experiments were technically repeated three times, and the results are represented as the means ± SD of technical replicates (n = 3). *p < 0.05; **p < 0.01 (Student's t-test). Quality control (QC) samples, which were pooled identical aliquots of the mitochondria samples, were measured four times throughout the run for data reproducibility. Upper right, western blot analysis of HeLa cells in which PTPIP51 WT or ∆FFAT mutant were depleted and restored. β-Actin was used as a loading control.
FYCO1 is a Rab7 effector that binds to LC3 and PI3P to mediate microtubule plus end-directed vesicle transport. (2010)
Figure 1.

FYCO1 is a novel LC3B-interacting protein. (A and B) Endogenous FYCO1 binds to GST-LC3B. GST-LC3B bound to glutathione–Sepharose beads was incubated with HeLa cell lysate, and copurified proteins were identified by Coomassie blue staining and mass spectrometry (A) or immunoblotting with anti-FYCO1 antibody (B). (C) GST-LC3B but not GST-p62 or GST alone binds to FYCO1 in a GST pull-down assay. GST, GST-LC3B, or GST-p62 bound to glutathione–Sepharose beads was incubated with [35S]methionine-labeled myc-FYCO1. Formed protein complexes were isolated and visualized by autoradiography (top) or Coomassie blue staining (bottom). (D) GFP-LC3B immunoprecipitates endogenous FYCO1 from HeLa cell lysate. HeLa cells expressing GFP or GFP-LC3B were lysed and processed as in B. IP, immunoprecipitation; MM, molecular mass; WB, Western blot.