EMBO/soda-nel huggingface dataset:
Rac1-Rab11-FIP3 regulatory hub coordinates vesicle traffic with actin remodeling and T-cell activation (2016)
Figure 1

Figure 1: Rac1 is associated to Rab11-positive recycling endosomes.

A-C: Jurkat T cells were transfected with control, FIP3.1 or FIP3.2 siRNA oligonucleotides. A: Intracellular distribution of endogenous Rab11 and Rac1 was assessed by immunofluorescence. C: FIP3 expression was analyzed by Western blot. Quantification of bands was performed with ImageJ and normalized to a non-specific band of 35 kDa used as loading control. D: Schematic representation of FIP3 structural features: proline-rich region, EF-hand domain, coiled-coil domain (CC), Arf binding domain (ABD) and Rab binding domain (RBD). E, F: Jurkat cells were transfected with expression vectors encoding GFP, GFP-FIP3-WT or GFP-FIP3-I738E. Endogenous Rac1 was detected by immunofluorescence. G: Jurkat cells were transfected with expression vectors encoding GFP, Rab11WT-GFP, or Rab11Q70L-GFP (constitutively active) or Rab11S25N-GFP (dominant negative) mutants. Endogenous Rac1 was detected by immunofluorescence.

A, E, G: Three-dimensional (3D) confocal images were post-treated by deconvolution. A 0.4-µ-thick medial stack is shown. The pericentrosomal vesicular compartment is zoomed at the bottom right-hand corner.

B, F, H: Population analysis of the co-localization volume between Rab11 and Rac1 within the percentriolar vesicular compartment. Each dot represents one cell. Horizontal bars represent the mean ± SEM, Mann-Whitney test was used. Images are representative of three experiments. Bar, 5 µm.
Rac1-Rab11-FIP3 regulatory hub coordinates vesicle traffic with actin remodeling and T-cell activation (2016)
Figure 2

Figure 2. Rac1 interaction with FIP3 and effect of FIP3 silencing on Rac1 expression

A, B: Effect of FIP3 silencing on Rac1 expression. Jurkat cells were transfected with control, FIP3.1 or FIP3.2 siRNA. Three days later cells were lysed and the expression of FIP3, Rac1 and β-tubulin analyzed by western blot. The intensity of bands corresponding to FIP3 and to Rac1 was quantified by ImageJ and normalized to the β-tubulin band used as loading control. The mean ± SD of the percentage of band intensity in silenced cells with respect to controls of three independent experiments is shown.

B: Rac1 interaction with FIP3: HEK293T cells were transfected with GFP, GFP-FIP3 WT or GFP-FIP3I738E expression vectors. 24 h later, cells were lysed and immunoprecipitated with anti-GFP Ab. Immunoprecipitates were analyzed by electrophoresis and western blot using anti-GFP Ab (top panel) or anti-Rac1 Ab (bottom panel). Total cell lysates were analyzed (100,000 cells). The position of Rac1 (20 kDa) and Ig light chain (25 kDa) polypeptides is depicted on the right side. Arrowheads at the bottom point the bands corresponding to Rac. Quantification of bands was performed with ImageJ as the percentage of intensity with respect to the total lysate bands on the left, shown below. A representative experiment out of three carried out is shown.
Rac1-Rab11-FIP3 regulatory hub coordinates vesicle traffic with actin remodeling and T-cell activation (2016)
Figure 3

Figure 3: Rab11-FIP3 controls Rac1 clustering at the immunological synapse. Jurkat T cells were transfected with expression vectors encoding: A, C: GFP, GFP-FIP3WT, or GFP-FIP3I738E. B, C: Rab11WT-GFP, Rab11Q70L-GFP (constitutively active), or Rab11S25N-GFP (dominant negative). D, E: or transfected with control, or FIP3.1 siRNA. Cells were allowed to form immunological synapses with SEE-pulsed Raji cells for 30 min. Intracellular distribution of endogenous Rac1 and surface distribution of CD3 were detected by immunofluorescence. 3D confocal images were post-treated by deconvolution. A 0.4-µm-thick medial stack is shown. C, E: Population analyses of the amount of Rac1 at the immunological synapse relative to the total cellular Rac1. Each dot corresponds to one cell. Horizontal bars represent the mean ± SEM. Mann-Whitney test. Images are representative of three experiments. Bar, 5 µm.
Rac1-Rab11-FIP3 regulatory hub coordinates vesicle traffic with actin remodeling and T-cell activation (2016)
Figure 4

Figure 4 : Rab11-FIP3 regulates immunological synapse symmetry and size. Jurkat T cells were transfected with either control or FIP3.1 siRNA. A: Cells were allowed to form immunological synapses with SEE-pulsed Raji cells for 30 min and endogenous Lck was detected by immunofluorescence. 3D confocal images were post-treated by deconvolution. A 0.4-µm-thick medial stack is shown. B, C: The minimal angle formed by the axis connecting the synapse edges and the axis connecting the mass centers of the T cell and the APC nuclei was measured, as schematized in B. D: Transfected Jurkat T cells were allowed to spread on anti-CD3-coated coverslips for the indicated time, and F-actin was detected with FITC-Phalloidin. A single optical section at the level of the contact surface is shown. E, F: Population analyses of the spreading area of cells at the level of the contact surface. Each dot corresponds to one cell. Horizontal bars represent the mean ± SEM. Mann-Whitney test. Images are representative of three experiments. Bar, 5 µm. G: FIP3 expression was analyzed by Western blot. Quantification of bands was performed with ImageJ and normalized to a non-specific band of 35 kDa used as loading control.
Rac1-Rab11-FIP3 regulatory hub coordinates vesicle traffic with actin remodeling and T-cell activation (2016)
Figure 5

Figure 5: Rab11-FIP3 regulates T cell spreading and cortical rigidity independently of TCR engagement.

A, B, C: Jurkat T cells transfected with siRNA control, FIP3.1 or FIP3.2 were allowed to spread on poly-lysine-coated coverslips for the indicated times. Intracellular actin was detected with FITC-coupled Phalloidin. A: A single optical section at the level of the contact surface is shown. Bar, 5 µm. B: Population analyses of the spreading area of cells at the level of the contact surface. Each dot represents one cell. Horizontal bars represent the mean ± SEM. C:Time course representation of B, showing mean ± SEM. D-F: Primary human CD4 T cells were transfected with control or FIP3.1 siRNA. D, E: Cells were allowed to spread on poly-lysine-coated coverslips for 5 min. Intracellular Lck was detected by immunofluoresecence. D: A single optical section at the level of the contact surface is shown. Bar, 10 µm. E: Population analyses of the spreading area of cells at the level of the contact surface. F: Cells were stained with CMFDA dye, suspended in a paraformaldehyde solution and submitted to centrifugation at 3,724 xg onto poly-lysine-coated coverslips. Confocal optical sections separated by 0.2 µm were acquired. X-Z images were built with ImageJ. The maximal sections in ‘x’ (width) and ‘z’ (thickness) were determined for each cell (upper panel). Plots represent the deformability index (x/z). Each dot corresponds to one cell. Horizontal bars represent the mean ± SEM. Mann-Whitney test. Images are representative of three experiments. G: FIP3 expression was analyzed by Western blot. Quantification of bands was performed with ImageJ and normalized to a non-specific band of 35 kDa used as loading control.
Rac1-Rab11-FIP3 regulatory hub coordinates vesicle traffic with actin remodeling and T-cell activation (2016)
Figure 6

Figure 6: Rab11-FIP3 regulates T cell spreading via a Rac1-dependent mechanism.

Jurkat T cells were transfected with control or FIP3.1 siRNA, then transfected again with empty vector or with expression vectors encoding Myc-tagged-Rac1WT, -Rac1G12V (constitutively active mutant), or -Rac1Rac1T17N (dominant negative mutant). Cells were allowed to spread on poly-lysine-coated coverslips for the indicated time and the spreading area of cells measured. A-C, H: Population analyses of the spreading area of cells at the level of the contact surface. A-C: Time course plots representing the mean ± SEM of the data shown as in I. D-G: Staining for nucleus (blue = Dapi), F-actin (green = phalloidin) and Rac1-Myc (red = anti-Myc) is shown. A single optical section at the level of the contact surface is shown. Bar, 5 µm. H: Dot plots corresponding to measurements at 15 min of spreading: Each dot represents one cell. Horizontal bars represent the mean ± SEM. Mann-Whitney test. Data are representative of three experiments.
Rac1-Rab11-FIP3 regulatory hub coordinates vesicle traffic with actin remodeling and T-cell activation (2016)
Figure 7

Figure 7: Rac1 inhibitor reverts FIP3-silencing-induced effects on T cell spreading and synapse symmetry.

A: Jurkat T cells transfected with control or FIP3.1 were treated 1 h with the Rac1 inhibitor NSC23766 at 100 µM and allowed to spread on poly-lysine-coated coverslips for 15 min. F-actin was detected with FITC-Phalloidin and the spreading area of cells at the contact surface was measured. B: Transfected cells were allowed to form immunological synapses with SEE-pulsed Raji cells for 30 min and intracellular distribution of Lck was detected by immunofluorescence. Synapse angle was measured as in figure 4. Each dot corresponds to one cell. Horizontal bars represent the mean ± SEM. Mann-Whitney test.
Rac1-Rab11-FIP3 regulatory hub coordinates vesicle traffic with actin remodeling and T-cell activation (2016)
Figure 8

Figure 8: FIP3 silencing enhances IL2 production.

A: Jurkat T cells transfected with control or FIP3.1 were activated on anti-CD3-coated plastic plates in the presence of soluble anti-CD28 during 16 h. Concentration of IL2 released in the supernatant was measured by ELISA. Histograms represent the mean ± SD of three experiments. Mann-Whitney test. B: T cells were activated with soluble anti-CD3 and Erk1/2 phosphorylation was analyzed by Western blot. Graph represents the percentage of each band intensity with respect to that of cells transfected with control siRNA and left non-stimulated (t=0). The intensity each band from Western blot was normalized with respect to the intensity of β-actin for each time point. Representative experiment out of three performed.
Amyloid Precursor Protein Maintains Constitutive and Adaptive Plasticity of Dendritic Spines in Adult Brain by Regulating D-serine Homeostasis (2016)
Figure 1

Figure 1. Constitutive plasticity of dendritic spines is decreased in the cortex of adult APP-KO mice.

(A) Consecutive in vivo imaging of the same apical dendrites from layer V pyramidal neurons in the somatosensory cortex over 7 weeks reveals formation and elimination of dendritic spines (white and empty arrowheads, respectively) in WT and APP-KO mice. All mice were housed in standard conditions. Scale bar - 10 μm. (B-E) Graphical representations of the relative spine density, spine turnover rate (TOR), elimination and formation over the imaging period. Non-linear regression (F test) has been used for fitting the data points and comparing the difference between groups. N=4 mice per group ** p<0.01.
Amyloid Precursor Protein Maintains Constitutive and Adaptive Plasticity of Dendritic Spines in Adult Brain by Regulating D-serine Homeostasis (2016)
Figure 2

Figure 2. Adaptive plasticity of dendritic spines is impaired in APP-KO mice

(A) Consecutive in vivo imaging of the same apical dendrites from layer V pyramidal neurons in the somatosensory cortex over 46 days reveals formation and elimination of dendritic spines (white and empty arrowheads, respectively) in WT and APP-KO mice. Prior to the exposure to environmental enrichment (EE), all mice were housed in standard conditions. Scale bar - 10 μm. (B-C) Graphical representations of the relative spine density and spine elimination over the period of the exposure of mice to EE. Non-linear regression (F test) has been used for fitting the data points. Repeated one-way ANOVA was performed followed by Dunnett test. WT n=5 mice and APP-KO n=6 mice; * p<0.05, ** p<0.01, NS - no significant difference.
Amyloid Precursor Protein Maintains Constitutive and Adaptive Plasticity of Dendritic Spines in Adult Brain by Regulating D-serine Homeostasis (2016)
Figure 3

Figure 3. Dendritic spine morphology is altered in APP-KO mice.

(A) Typical confocal images of apical dendrites with spines (z-projections) from layer V pyramidal neurons in the somatosensory cortex of WT and APP-KO mice (top and bottom) housed in standard and enriched environments (left and right). For classification of spine types, 3D reconstructions by Imaris have been applied. Thin, mushroom and stubby spines are encoded in blue, green and red, respectively. Scale bar represents 2 μm. (B-C) Summary plots of thin and mushroom spine fractions in WT and APP-KO mice exposed to standard (SE) and enriched environments (EE). . Two-tailed Student t-test was used and n=6 mice in all experimental groups; * p<0.05, ** p<0.01, NS - no significant difference.
Amyloid Precursor Protein Maintains Constitutive and Adaptive Plasticity of Dendritic Spines in Adult Brain by Regulating D-serine Homeostasis (2016)
Figure 4

Figure 4. Quantification of D-serine concentrations in WT and APP-KO mice.

(A) Extracellular D-serine concentrations in cortex were detected by enzyme-based microelectrode biosensors. (B, C) High performance liquid chromatography was performed to quantify total D-serine and L-serine contents in mouse brain of two genotypes. APP-KO mice were fed with D-serine over one month. One-way ANOVA was used for analysis followed by Bonferroni’s multiple comparisons test and n>6 mice per group; * p<0.05, ** p<0.01 as compared to WT.
Promoters of ASCL1- and NEUROD1-dependent genes are specific targets of lurbinectedin in SCLC cells (2022)
Figure 1

Figure 1: Overexpression of ASCL1 and NEUROD1 promotes the expression of their responsive genes.

(A) Western blot showing the protein expression of ASCL1 and/or NEUROD1 in NCI-H69, NCI-H82, NCI-H146, NCI-H510A, NCI-H526, SHP-77 and DMS-53 SCLC cells (lanes 4-10); A549 and NCI-H460 NSCLC cells and a IMR-90 HFL cell lines (lanes 1-3). Tubulin (α-Tub) is shown as loading control. Each blot is representative from at least three independent experiments.

(B, C) Venn diagrams showing the overlap between DMS-53 RNA-seq (Blue circle) and either ASCL1 ChIP-seq (Orange circle) (B) or NEUROD1 (Green circle) (C) ChIP-seq promoter annotated peaks in untreated conditions (t=0). Top panels. Tables showing the common and unique features from Venn diagram. According to RNA-seq data, a gene has been considered transcribed, if it has a coverage (number of reads per base of the gene) of more than 1 and a cut off value of TPM (transcript per million reads) of 4. DMS53 RNA-seq: genes being transcribed; ASCL1- and NEUROD1-Chip-seq: genes targeted byASCL1 or NEUROD1; DMS53 RNA-seq and either ASCL1- or NEUROD1-seq: genes bound by either ASCL1 or NEUROD1 and being transcribed; DMS-RNA-seq only: transcribed genes not targeted by ASCL1 or NEUROD1; ASCL1- and NEUROD1-Chip-seq only: non-transcribed genes in our experimental conditions. Lower panel : Venn diagram showing the overlap between ASCL1 and NEUROD1 (Dark green Circle) targeted and downregulated genes (3470 genes) in untreated conditions.

(D) Picture of ASCL1 (above) and NEUROD1 (below) ChIP-seq associated motifs around the genome (P=1e-6800 and P=1e-60, respectively). 13254 RNAseq filtered genes by mean coverage >1 and mean tpm>= 4. 10657 had 1 or more ASCL1 motif in their custom promoter region. 8925 Genes had 1 or more NEUROD1 motif in their custom promoter region.
Promoters of ASCL1- and NEUROD1-dependent genes are specific targets of lurbinectedin in SCLC cells (2022)
Figure 2

Figure 2: Promoter-TSS occupancy of ASCL1 and NEUROD1 targeted genes:

(A-E) ChIP-seq experiments indicates the localization of (A) ASCL1, (B) NEUROD1 and (E) RNAPII. ATAC-seq (C) and (D) the presence of H3K27Ac mark indicate the chromatin accessibility around the TSS at t=0 (blue) and t=4h (green) after lurbinectedin treatment; -2.0 and +2.0 kb to TSS coordinates of all hg19 genes.

(F) Structure of Bio-lur. The Biotin moiety is highlighted in dotted circle.

(G) Chem-seq (Green) experiments indicates the localization of lurbinectedin around TSS. All ChIP-seq profiles are representative of two independent experiments.
Promoters of ASCL1- and NEUROD1-dependent genes are specific targets of lurbinectedin in SCLC cells (2022)
Figure 3

Figure 3: Lurbinectedin binds transcriptionally active regions in SCLC:

(A-D) Chip-seq genome track of ASCL1/NEUROD1, RNAPII, and H3K27Ac on ASCL1, BCL2; INSM1 and MYB (hg19), in untreated (dark blue) and in lurbinectedin treated (green) conditions respectively; to be noticed the location of CGG rich motifs (lower part of each panel) which parallel the presence of lurbinectedin (red).

(E) Summary of the localization of lurbinectedin ASCL1-, NEUROD1-, RNAPII-, H3K27Ac-ChIP-seq, ATAC-seq and lurbinectedin-Chem-seq.

(F, G) Histogram showing the distance (in bp) between TSS and either (F) lurbinectedin or (G) RNAPII bound to the DNA. Only peaks located in region between -2.0 and +2.0kb on each side of TSS were taken into consideration. The y axis on every Chip-seq profile represents the Coverage Score.

(H) histogram showing the absolute distance between RNAPII and lurbinectedin peaks from nearest TSS.
Promoters of ASCL1- and NEUROD1-dependent genes are specific targets of lurbinectedin in SCLC cells (2022)
Figure 4

Figure 4: Lurbinectedin primes Ubiquitination and degradation of RNAPII:

(a) Western blot showing RNAPII across the panel of; A549 and NCI-H460 NSCLC cells; IMR-90 HFL cells; NCI-H146, NCI-H82, DMS-53, NCI-H510A, NCI-H526 and SHP-77 SCLC cells before (-) and after (+) treatment with lurbinectedin. α-Tubulin (α-Tub) is shown as loading control; lower panel, Histogram showing the average of RNAPII before (dark blue) and after (light Blue) lurbinectedin treatment in arbitrary units (a.u.) normalized against α-Tub. Data is presented as Mean ± SEM as determined by Two Way RM ANOVA and Šídák's multiple comparisons test (n=3 biological replicas). ****P ≤0.0001.

(B and C) Immunofluorescence of RNAPII (red) before (Ctrl) and after lurbinectedin treatment. DAPI (blue) was used to stain nuclei; scale bar is 65 μm. Data is presented as Mean ± SEM. ****p ≤0.0001*P ≤ 0.00332, **P ≤ 0.0021, as determined by Student's t-test (n = 3 biological replicates).

(D) Affinity purification of Bio-lur fractions containing elongating RNAPII Ser2 (lane 4). Input (lanes 1-2) represents 10% of the starting material. Respective molecular weights are shown on the right (kDa).

(E) Upper panel: GST-pulldown of ubiquitinated proteins showing hypo- (IIA), -hyper (II0) phosphorylated RNAPII (lanes 4-5 upper panel) and Ser2 Phosphorylated RNAPII (Ser2P) (lanes 4-5 lower panel) after lurbinectedin treatment. Empty beads (Mock) (lane 3) as a negative control; Lower Panel: GST-pulldown of ubiquitinated proteins Ser2P) (lanes 4-5). Representative smear of successful ubiquitinated proteins pulldown (Lanes 4 and 5) as compared to input (lanes 1 and 2). Empty beads (Mock) (lane 3) as a negative control. Each Western blot is representative of three independent experiments.

(F) Density profile of annotated genes for RNAPII-Ser2 P ChIP seq. Lines representing ChIP seq profile at 0h (blue) and 4h (green) after lurbinectedin treatment -10.0 and +10.0 kb to TSS coordinates (P=0.005476). Decrease in density between 0h and 4h is highlighted in yellow.
SOX9 maintains human fetal lung tip progenitors by enhancing WNT and RTK signalling (2022)
Figure 1

Figure 1. A CRISPRi screen identified crucial factors regulating human fetal lung progenitor cell self-renewal.

(A) Schematic of introducing inducible CRISPRi system into the organoid cells using serial lentiviral infection.

(B) Schematic of the inducible CRISPRi system. The inducible CRISPRi system was controlled by both Dox inducible system at mRNA level and TMP stabilisation of DHFR destabilising domain at protein level to achieve a tight control of CRISPRi function as previously reported (Sun et al, 2021).

(C) Representative flow cytometry results showing CD71, a cell surface marker, can be efficiently knocked-down in the majority of organoid cells after 5 days of Dox and TMP treatment. N = 3 different organoid lines and 3 different gRNAs for CD71 were used. Mean ± SEM is labelled.

(D) Workflow for a focused library CRISPRi screen for transcription factors regulating human fetal lung tip progenitor cell self-renewal. Parental organoid lines with inducible CRISPRi system were established by lentiviral transduction and expanded. A CRISPRi gRNA library of 300 gRNAs was packaged into lentivirus and was used to infect single cells from two independent parental inducible CRISPRi organoid lines with an infection efficiency of ~15%. TagRFP+EGFP+ double-positive organoid cells were collected. A fraction of cells was frozen for analysis of gRNA abundance in the starting population. The rest were seeded in Matrigel, given 8 days to recover and grown into small organoid colonies before treatment with Dox and TMP. Organoids were cultured in self-renewing medium with Dox and TMP for 2 weeks before harvest. Organoids were physically broken into pieces for passaging twice during this period. TagRFPhighEGFP+ and TagRFPlowEGFP+ fractions were collected separately for downstream genomic DNA isolation and analysis.

(E) Volcano plot summarising gRNA abundance changes for each target gene. Strong depletion hits (-log10(FDR) > 2 and log2(FC) < 0) dark blue; strong enrichment hits (-log10(FDR) > 2 and log2(FC) > 0) red; intermediate depletion hits (1 < -log10(FDR) < 2 and log2(FC) < 0) light blue; unchanged genes grey. FDR, false discovery rate.

(F) Schematic of the experimental design for serial passaging assay to validate gene knockdown effects on self-renewal. ~3000 TagRFPhighEGFP+ double positive cells were harvested for each condition and seeded in Matrigel. Organoid cells were given 8 days to recover and grown into small colonies before treating with Dox and TMP. Organoids were then maintained in Dox and TMP and serially passaged by breaking into pieces every 3-4 days.

(G) Summary of serial passaging assay results for different knockdowns. 3 independent organoid lines each were transduced with 2 different gRNAs against the gene targets, making 6 organoid lines altogether.

(H) Representative wide field images to show organoid growth of the indicated gene knockdown organoids at the indicated passage number. Scale bars denote 1 mm.