EMBO/soda-nel huggingface dataset:
The Golgi microtubules regulate single cell durotaxis (2020)
Figure 1

Figure 1 - Microtubules are essential for single cell durotaxis

Left: Schematic diagram of manufacturing polyacrylamide gels with stiffness gradient. The gel containing photoinitiator is solidified by ultraviolet light irradiation. Mask with a constant speed is used to control the irradiation time for a hardness gradient. Right: Representative stiffness gradient of polyacrylamide gels measured by atomic force microscope was plotted (34 kPa/mm and 29 kPa/mm respectively). All error bars are s.e.m (n=5 regions, replicates are biological).

Representative images of fluorescently labeled fibronectin at the gradient gel surface and the quantification of fluorescence intensity. 1-4 in turn represent hard to soft areas. Scale bar: 100 μm. All error bars are s.e.m (n=10 regions, replicates are biological), ns, not significant, by one-way ANOVA.

Left: Representative time-lapse images showing the migration of single hRPE cells on the surface of polyacrylamide gels with uniform stiffness (top) and stiffness gradient (bottom), referring as random migration and durotaxis migration respectively. Yellow line is the cell outline and blue line is the cell migration trajectory. Scale bar: 50 μm. Middle: Angular displacement of hRPE cells on the uniform stiffness gels (top) and stiffness gradient gels (bottom). Right: y/x FMI (forward migration index), velocity and persistence of hRPE cells with random migration and durotaxis. All error bars are s.e.m (n=50 cells, replicates are biological), ns, not significant, by Student's t-test.

Left: Representative immunofluorescent staining of F-actin in control and CK-666 (100 μM, 3 h) treated hRPEs. Scale bar: 20 μm. Right: Y FMI of control and CK-666 (100 μM, pre-treated for 1h and incubated for the whole experiment) treated hRPEs moving on stiffness gradient gel. All error bars are s.e.m (n=50 cells, replicates are biological).

Left: Representative immunofluorescent staining of α-tubulin in control and nocodazole (8 μM, 30 min) treated hRPEs. Scale bar: 20 μm. Right: Y FMI of control and nocodazole (8 μM, pre-treated for 10 min and incubated for the whole experiment) treated hRPEs moving on stiffness gradient gel. All error bars are s.e.m (n=50 cells, replicates are biological).
The Golgi microtubules regulate single cell durotaxis (2020)
Figure 2

Figure 2 - Single cell durotaxis is under the regulation by Golgi-microtubules

Representative immunofluorescent staining of γ-tubulin (yellow) and DAPI (blue) in control and centrinone-B (500 nM, 7 days) treated hRPEs. Scale bar: 20 μm.

Angular displacement and y FMI of control and centrinone-B (500 nM, 7 days) treated hRPEs moving on stiffness gradient gels. All error bars are s.e.m (n=50 cells, replicates are biological).

Western blot showing the protein level of CAMSAP2 in negative control and CAMSAP2 siRNA transfected hRPEs. GAPDH was used as loading control. Bar chart shows quantification of protein levels normalized to GAPDH in each condition. All error bars are s.e.m (n=3 independent experiments, replicates are biological), ****P < 0.0001, by Student's t-test.

Angular displacement and y FMI of negative control and CAMSAP2 siRNA transfected hRPEs moving on stiffness gradient gels. All error bars are s.e.m (n=50 cells, replicates are biological).

Western blot showing the protein level of AKAP450 in negative control and AKAP450 siRNA transfected hRPEs. α-tubulin was used as loading control. Bar chart shows quantification of protein levels normalized to α-tubulin in each condition. All error bars are s.e.m (n=3 independent experiments, replicates are biological), ****P < 0.0001, by Student's t-test.

Angular displacement and y FMI of control and AKAP450 siRNA transfected hRPEs moving on stiffness gradient gels. All error bars are s.e.m (n=50 cells, replicates are biological).

Left: Representative immunofluorescent staining of α-tubulin in control, CAMSAP2 siRNA transfected and centrinone-B (500 nM, 7 days) treated hRPEs. Scale bar: 20 μm. Right: Quantification of microtubule fluorescence intensity. All error bars are s.e.m (n=20 cells, replicates are biological), ns, not significant, by one-way ANOVA.

Western blot showing the protein level of α-tubulin in control, CAMSAP2 siRNA transfected and centrinone-B (500 nM, 7 days) treated hRPEs. GAPDH is used as loading control. Bar chart shows quantification of protein levels normalized to GAPDH in each condition. All error bars are s.e.m (n=3 independent experiments, replicates are biological), ns, not significant, by one-way ANOVA.

Left: Representative live-cell fluorescence imaging of EGFP-EB1 in control, CAMSAP2 siRNA transfected and centrinone-B (500 nM, 7 days) treated hRPEs. Scale bar: 20 μm. Right: Quantification of EB1 growth speed obtained by plusTipTracker. Vinlin plot center lines denote the median, and dashed lines represent the 25th and 75th percentiles. N=10 cells, replicates are biological, ns, not significant, by one-way ANOVA.
The Golgi microtubules regulate single cell durotaxis (2020)
Figure 3

Figure 3 - Loss of Golgi microtubules does not affect RhoA activity or cell contraction

Western blot showing the protein level of active RhoA, total RhoA in negative control and CAMSAP2 siRNA transfected hRPEs. GAPDH is used as loading control. Bar chart shows quantification of protein levels normalized to GAPDH in each condition. All error bars are s.e.m (n=3 independent experiments, replicates are biological), ns, not significant, by Student's t-test.

Left: Representative live-cell fluorescence imaging of GFP-AHPH in negative control and CAMSAP2 siRNA transfected hRPEs. Scale bar: 20 μm. Right: Quantification of GFP-AHPH fluorescence intensity. All error bars are s.e.m (n=20 cells, replicates are biological), ns, not significant, by Student's t-test.

Left: Representative immunofluorescent staining of pMLC (yellow) and DAPI (blue) in negative control and CAMSAP2 siRNA transfected hRPEs. Scale bar: 20 μm. Right: Quantification of pMLC fluorescence intensity. All error bars are s.e.m (n=20 cells, replicates are biological), ns, not significant, by Student's t-test. Below: Western blot showing the protein level of pMLC in negative control and CAMSAP2 siRNA transfected hRPEs. α-tubulin is used as loading control

Left: Images showing collagen gel deformation at 0 h, 8 h and 24 h. HRPEs transfected with negative control, CAMSAP2 and AKAP450 siRNA were seeded in 1% collagen gel (yellow dashed lines). Scale bar: 5 mm. Right: Quantification of gel area. All error bars are s.e.m (n=3 independent experiments, replicates are biological), ns, not significant, by one-way ANOVA.
The Golgi microtubules regulate single cell durotaxis (2020)
Figure 4

Figure 4 - Golgi microtubules regulate focal adhesion dynamics

Left: Representative live-cell fluorescence imaging of RFP-paxillin in control and nocodazole (8 μM, 30 min) treated hRPEs. Scale bar: 20 μm. Right: Quantification of FA area obtained by FAAS. All error bars are s.e.m. N=10 cells, replicates are biological, ****P < 0.0001, by non-parametric test.

Top: Representative live-cell fluorescence imaging of RFP-paxillin in negative control and CAMSAP2 siRNA transfected hRPEs. Time-lapse fluorescence imaging zoomed from the yellow box showed the assembly and disassembly stage of a single FA. Scale bar: 20 μm. Bottom: Quantification of FA area, assembly rate and disassembly rate obtained by FAAS. All error bars are s.e.m. Vinlin plot center lines denote the median, and dashed lines represent the 25th and 75th percentiles. N=10 cells, replicates are biological, ****P < 0.0001, by non-parametric test.

Top: Schematic diagram of FA-MT contact analysis. Position of EB1 and paxillin were extracted from live-cell images, followed by automated co-localization identification. Scale bar: 20 μm. Bottom: Representative time-lapse fluorescence imaging of EGFP-EB1 and RFP-paxillin zoomed from the yellow box above showed the targeting of MT to FA. FA-MT contact ratio was calculated by the ratio of co-localization events to the total EB1 number.

FA-MT contact ratio of negative control, CAMSAP2, AKAP450 siRNA transfected and centrinone-B (500 nM, 7 days) treated hRPEs. Vinlin plot center lines denote the median, and dashed lines represent the 25th and 75th percentiles. N=10 cells, replicates are biological, ns, not significant, ****P < 0.0001, by non-parametric test.

Western blot showing the protein level of ELKS in negative control and ELKS siRNA transfected hRPEs. α-tubulin was used as loading control. Bar chart shows quantification of protein levels normalized to α-tubulin in each condition. All error bars are s.e.m (n=3 independent experiments, replicates are biological), ****P < 0.0001, by Student's t-test.

FA-MT contact ratio of negative control and ELKS siRNA transfected hRPEs. Vinlin plot center lines denote the median, and dashed lines represent the 25th and 75th percentiles. N=10 cells, replicates are biological, ns, not significant, by non-parametric test.

Y FMI of negative control and ELKS siRNA transfected hRPEs moving on stiffness gradient gels. All error bars are s.e.m (n=50 cells, replicates are biological).
The Golgi microtubules regulate single cell durotaxis (2020)
Figure 5

Figure 5 - The Golgi apparatus is oriented in response to mechanical cues to maintain cell polarity

Left: Representative immunofluorescent staining of GM130 (yellow), F-actin (gray) and DAPI (blue) in negative control and CAMSAP2 siRNA transfected hRPEs at the edge of the wound after 8 h. Scale bar: 50 μm. Middle: Schematic diagram of Golgi-nucleus orientation towards the front (45°-135°) or the rear (-45°- -135°). Right: Quantification of the percentage of Golgi-nucleus orientation towards the front or rear of the cell. All error bars are s.e.m (n=100 cells, replicates are biological), ***P<0.001, ****P<0.001, by Student's t-test.

Left: Representative immunofluorescent staining of GM130 (yellow) and DAPI (blue) in negative control and CAMSAP2 siRNA transfected hRPEs on the stiffness gradient gel. Scale bar: 20 μm. Middle: Schematic diagram of Golgi-nucleus orientation towards the stiff side (45°-135°) or the soft side (-45°- -135°). Right: Quantification of the percentage of Golgi-nucleus orientation towards the stiff or soft side. All error bars are s.e.m (n=100 cells, replicates are biological), *P<0.05, **P<0.01, by Student's t-test.

Left: Schematic diagram of manufacturing polyacrylamide gels with islands of higher stiffness by 405 nm laser irradiation. Middle: Modulus of polyacrylamide gels with stiff islands measured by atomic force microscope. Right: Representative live-cell fluorescence imaging of cells marked with the Golgi (green) and nucleus (blue) on the soft region and the stiff island. Scale bar: 50 μm.

Representative time-lapse fluorescence images showing the migration of hRPE cell to the stiff island and the reorientation of Golgi in this process. Scale bar: 20 μm.

Quantification of the percentage of cells having a stiff-end biased Golgi positioning. N=29 cells, replicates are biological.
Rewired signaling network in T cells expressing the chimeric antigen receptor (CAR) (2020)
Figure 1

Figure 1 - CAR forms microclusters that are signaling-active.

A. Domain structure of CAR.

B. Schematic of the system for stimulating CAR T cells and imaging CAR dynamics. Supported lipid bilayers (SLBs) were coated with the extracellular domain of CD19 and an integrin ligand ICAM-1.

C. TIRF microscopy revealed time courses of the CAR-GFP microcluster formation as CAR T cells spread on the CD19 and ICAM-1-coated SLBs.

D. Enrichment of phosphor-tyrosine (pY) at CAR microclusters. CAR T cells were fixed 20 min after being plated on the SLB, followed by staining with an anti-pan phosphor-tyrosine antibody. No CD19 was included in the control experiment (only ICAM-1). Scale bar: 2 μm.
Rewired signaling network in T cells expressing the chimeric antigen receptor (CAR) (2020)
Figure 2

Figure 2 - CAR induces non-typical immunological synapses.

T cells expressing GFP-CAR were plated on CD19 and ICAM-1 coated SLBs. TIRF microscopy was implemented to visualize CAR localization.

A. CAR microclusters underwent centripetal movement to form cSMAC (upper panel) or remained separated (lower panel).

B. Quantification of the categories regarding the formation of CAR cSMAC. Unstable cSMACs are defined by a cSMAC duration below 12 min. Data are shown as mean ± SD. n = 3 independent experiments. 37, 38, and 47 cells were quantified in individual experiments.

C. Quantification of time from cell landing to the start of retrograde flow. Data are shown as mean ± SD. n = 33 (stable), 31 (unstable), 10 (kinapse), and 41 (no cSMAC) cells. Results are pooled from three independent experiments.

D. Localization of CAR and CD45 in the immunological synapse. CAR T cells were fixed 20 min after being plated on the SLB, followed by staining with an anti-CD45 antibody.

E. Line scan of CAR and CD45 across the long axis of the immunological synapse. Shown are the normalized fluorescence intensity.

Data information: Scale bar: 2 μm.
Rewired signaling network in T cells expressing the chimeric antigen receptor (CAR) (2020)
Figure 3

Figure 3 - LAT is not required for CAR microcluster formation.

A. WT or LAT-deficient Jurkat cells (Jcam2.5) expressing CAR-GFP were stimulated on SLB-coated with OKT3 (anti-TCR antibody) or CD19 (CAR antigen); in both conditions, the lipid bilayers were also coated with ICAM-1 to facilitate cell attachment. TIRF microscopy revealed clustering of Ax647-labeled streptavidin-Biotin-OKT3 or CD19, which serves as a probe for TCR or CAR respectively. Scale bar, 2 µm.

B. Quantification of clustering as normalized variance. n = 100 cells. Shown are the mean ± SD. Statistical test: unpaired two-tailed t-test. **** p < 0.0001. ** 0.005 < p < 0.05.
Rewired signaling network in T cells expressing the chimeric antigen receptor (CAR) (2020)
Figure 4

Figure 4 - The 1st generation CAR forms microclusters and immunological synapses in LAT-deficient Jurkat T cells.

A. Schematics of the 3rd and 1st generation of CAR.

B. WT or LAT-deficient (Jcam2.5) cells expressing the 3rd or 1st generation CAR were stimulated on supported lipid bilayers coated with CD19 and ICAM-1. TIRF microscopy revealed clustering of CAR and Ax647-labeled streptavidin-Biotin-CD19. Scale bar, 2 µm.

C. Quantification of clustering level of Ax647-labeled streptavidin-Biotin-CD19. Shown are means ± SD. Clustering is quantified as normalized variance. N = 50 cells for CAR 3rd WT and 1st WT, 63 cells for CAR 3rd lat-, and 51 cells for CAR 1st lat-. **: 0.0005 < p < 0.005. Statistical test: unpaired two-tailed t-test.
Rewired signaling network in T cells expressing the chimeric antigen receptor (CAR) (2020)
Figure 5

Figure 5 - CAR microclusters activate Gads-SLP76-actin pathway in the absence of LAT.

CAR (3rd generation) T cells were plated on supported lipid bilayers coated with CD19 and ICAM-1 and imaged by TIRF microscopy.

A. Gads was recruited to CAR microclusters in both the wild-type and LAT-deficient (Jcam2.5) cells. n = 33 or 34 cells. Shown are the means ± SE. n.s., p = 0.58 by unpaired two-tailed t-test. As a control, Gads is not recruited to LAT-deficient cells activated by OKT3.

B. Phosphorylated SLP76, recognized by an anti-SLP76 pY145 antibody, was enriched in CAR microclusters in both the wild-type and LAT-deficient cells. In contrast, phosphorylated SLP76 was not enriched in TCR microclusters in LAT-deficient cells. n = 50 cells. Shown are the means ± SE. n.s., p = 0.95 by unpaired two-tailed t-test.

C. Actin remodeling was induced in LAT-deficient cells by CD19-, but not OKT3-coated SLB. No ICAM-1 was supplemented to SLB in this experiment to rule out the costimulation of actin via the LFA-1/ICAM-1 pathway. Actin network was revealed by F-tractin-mScarlet. n = 6 cells. Shown are the means ± SD. n.s., p = 0.68, and 0.98 by unpaired two-tailed t-test.

Data information: Scale bar: 2 µm.
Rewired signaling network in T cells expressing the chimeric antigen receptor (CAR) (2020)
Figure 6

Figure 6 - LAT promotes but is not required for Raji B cell-induced CAR T activation.

A. Cell-cell conjugation assay. Wild-type or Lat-null (J.LAT) Jurkat T cells expressing CAR-GFP were mixed with Raji cells expressing mCherry-CAAX at a 1:1 ratio for 1 hr before being fixed. Three independent experiments were performed and over 90 cells were scored in each experiment. Scale bar: 5 µm.

B. CAR T cells were incubated with Raji B cells at a 1:1 ratio for 24 hrs and IL-2 from the supernatant were measured by ELISA.

C. CAR T cells were incubated with Raji B cells at a 1:1 ratio and fixed at 24hr, stained with an anti-CD69 antibody, and analyzed by FACS. CD69 positive cells were scored within the CAR-GFP+ population.

Data information: Shown are the means ± SD. N = 3 experiments. n.s. p > 0.05, * 0.005 < p < 0.05, ** 0.0005 < p < 0.005, *** 0.0001 < p < 0.0005, **** p < 0.0001. Unpaired two-tailed t-test was applied.
Rewired signaling network in T cells expressing the chimeric antigen receptor (CAR) (2020)
Figure 7

Figure 7 - LAT is not required for CAR microcluster formation or SLP76 activation following CD19 stimulation in human primary T cells.

A. Western blot showed that LAT is efficiently depleted in human primary T cells using CRISPR. Shown are the means ± SD. n = 4 independent experiments.

B. CAR T cells were stimulated on SLBs coated with biotin-OKT3 (left) or biotin-CD19 (right) supplemented with ICAM-1. TIRF microscopy revealed clustering of Alexa647-labeled streptavidin-Biotin-OKT3 or CD19. Scale bar, 5 µm.

C. Quantification of clustering of OKT3 or CD19 as normalized variance. n > 200 cells from two independent donors were scored for each condition. Central band: mean; Box: quartiles; Whisker: rest of the distribution. ****, p < 0.0001; n.s. p = 0.48.

D. Immunofluorescence analysis revealed membrane-localized phosphorylated SLP76. Cells were plated on supported lipid bilayer coated with ICAM-1 only (control) or ICAM-1 supplemented with CD19 or OKT3 as indicated. The white dashed circle indicated cell boundary. Scale bar, 5 µm.

E. Quantification of pSLP76 level on the membrane. n > 200 cells were scored, and the cells were isolated from two independent donors. Central band: mean; Box: quartiles; Whisker: rest of the distribution. ****, p < 0.0001; n.s. p = 0.11.

F. T cells were incubated with Raji B cells at a 4:1 ratio and IL-2 production was measured by ELISA at 24 hr. Data were pooled from three donors. *, 0.005 < p < 0.05; **, 0.0005 < p < 0.005. Unpaired two-tailed t-test was applied.
K63-linked polyubiquitination of LGP2 by Riplet regulates RIG-I-dependent innate immune response (2022)
Figure 1

Figure 1. Polyubiquitination of LGP2

(A-E) Wild-type and LGP2-KO HEK293 cells in 24-well plates were transfected with 200 ng/ml of short poly I:C or infected with SeV. IFN-β and IP-10 mRNA expression was determined by RT-qPCR. The data represent the mean ± SD (n = 3, t-test, *p < 0.05). LGP2 KO2 and KO3 are independently isolated LGP2 KO clones.

(F) HEK293 cells in 24-well plates were transfected with LGP2 (0.1, 0.3, or 0.6 μg/well) and RIG-I (0.1 μg/well) expression vectors as indicated. 24 h after transfection, cells were stimulated with 200 ng of short poly I:C for 4 hr. Total RNAs were extracted, and the expression of IFN-β mRNA was determined by RT-qPCR and normalized to GAPDH (n = 3, t-test, *p < 0.05).

(G) HEK293FT cells were transfected with FLAG-tagged LGP2 and HA-tagged ubiquitin expression vectors. Whole cell extracts (WCE) were prepared 24 h after transfection. Immunoprecipitation was performed with anti-FLAG antibody (IP: FLAG) and the proteins were detected by western blot analysis with the indicated antibodies.

(H) FLAG-tagged LGP2 was transfected into HEK293 cells. Cells were then infected with SeV at MOI = 10 for 24 h. Immunoprecipitation was performed with anti-FLAG antibody, and the proteins were detected with the indicated antibodies.

Data information: All data are representative at least two independent experiments, and "n" represents the number of samples (biological replicates). Error bars represent standard deviation from the mean.
K63-linked polyubiquitination of LGP2 by Riplet regulates RIG-I-dependent innate immune response (2022)
Figure 2

Figure 2. Kinetics of LGP2 ubiquitination

A) HEK293 cells were transfected with FLAG-RIG-I or FLAG-LGP2 expressing vector. Cells were then infected with SeV, and whole cell extracts were collected at indicated time points. Immunoprecipitation was performed with anti-FLAG antibody. The proteins were subjected to SDS-PAGE and detected by western blotting.

B) A549 cells transfected with HA-tagged ubiquitin were stimulated with 200 ng/ml of short poly I:C for 6 h. PLA was performed with anti-HA, anti-RIG-I, and anti-LGP2 antibodies. The number of PLA signals were counted (t-test, *p < 0.05, n = 20).

C) HEK293 cells were transfected with empty and LGP2 expression vectors for 24 hr. PLA was performed with anti-LGP2 and anti-K63-Ub antibodies. The number of PLA signals were counted (t-test, *p < 0.05, n = 33).

D-F) HEK293 cells were transfected with 200 ng/ml of short poly I:C. Cells were fixed at the indicated time points, and PLA was performed with anti-RIG-I, anti-LGP2, and anti-K63-Ub antibodies (D). The number of PLA signals were counted (E, F) (t-test, *p < 0.05, n = 30).

Each dot represents the number of PLA signals of each cell.

Data information: Scale bars represent 10 μm. All data are representative at least two independent experiments, and "n" represents the number of samples (biological replicates). Error bars represent standard deviation from the mean.
K63-linked polyubiquitination of LGP2 by Riplet regulates RIG-I-dependent innate immune response (2022)
Figure 3

Figure 3. Riplet mediates K63-linked polyubiquitination of LGP2

A) HEK293FT cells were transfected with FLAG-tagged LGP2 and HA-tagged Riplet expression vectors. 24 h after transfection, cells were lysed, and WCE were prepared. Immunoprecipitation was performed with anti-FLAG antibody, and the proteins were detected by western blotting with the indicated antibodies.

B) HEK293FT cells were transfected with FLAG-tagged LGP2 expression vector. 24 h after transfection, immunoprecipitation was performed with anti-FLAG antibody and control IgG. Immunoprecipitates were subjected to SDS-PAGE, and the proteins were detected by western blotting with indicated antibodies.

C) HEK293FT cells were transfected with FLAG-tagged LGP2, Riplet, and HA-tagged ubiquitin expression vectors, and immunoprecipitation was performed with anti-FLAG antibody.

D) HEK293FT cells were transfected with FLAG-tagged LGP2, Riplet, and myc-tagged K63-only-Ub expression vectors. 24 h after transfection, immunoprecipitation was performed with anti-FLAG antibody.

E) WT and Riplet KO HEK293 cells were transfected with FLAG-tagged LGP2 and myc-tagged K63-only-Ub expression vectors, and immunoprecipitation was performed with anti-FLAG antibody.

(F, G) WT and Riplet KO HEK293 cells were transfected with 200 ng/ml of short poly I:C for 6 h, and PLA was performed with anti-LGP2 and anti-K63-Ub antibodies. The number of PLA signals were counted (t-test, *p < 0.05, n = 100). Each dot represents the number of PLA signals in each cell. Scale bars represent 10 μm.

Data information: All data are representative at least two independent experiments, and "n" represents the number of samples (biological replicates). Error bars represent standard deviation from the mean.