EMBO/soda-nel huggingface dataset:
Space exploration by dendritic cells requires maintenance of myosin II activity by IP3 receptor 1 (2014)
Figure 2

IP3 receptors are required for dendritic cell fast migration

Relative gene expression of IP3R1 (blue), IP3R2 (red), and IP3R3 (green) in IP3R‐silenced DCs. DCs were infected with two lentiviruses encoding for different shRNAs for IP3R isoforms. ShScramble‐infected DCs were used as a control. Gene expression was determined by a quantitative PCR. It was calculated with respect to GAPDH expression and normalized to the levels observed in the shScramble. The median plus standard deviation of three independent experiments are shown.
Immunoblot for IP3R type 1 and IP3R type 3 in IP3R‐silenced DCs. Actin was used as a control. IP3R3 appears as two bands as previously described (22).
Immunoblot for IP3R type 1 (top) and IP3R type 3 (bottom) in DCs transduced with lentivirus encoding for different IP3R shRNAs. Actin was used as a control.
Relative IP3R2 expression in ShScramble‐infected DCs (gray), shIP3R1 (blue), and IP3R3 (green)‐silenced DCs. The experiment was performed as described in (A).
Table showing the nomenclature for shRNAs related to their isoform‐specific silencing. The shRNAs chosen to pursuit this study are highlighted in red.
Transmigration assay of shScramble‐, IP3R(1,3)A (blue)‐, IP3R(2,3)B (red)‐, and IP3R(1,3)C (green)‐expressing DCs. Cells were loaded in the upper chamber of a 5‐μm pore collagen‐coated transwell assay, recovered from the upper and lower chambers after 16 h, and counted. The median from three independent experiments is shown.
Quantification of the mean cell velocity of shIP3R(1,3)A (blue)‐, shIP3R(2,3)B (red)‐, and shIP3R(1,3)C (green)‐silenced immature DCs migrating in micro‐channels. shScramble‐infected DCs were used as a control (gray) (n > 100 cells from three independent experiments for shIP3R(1,3)A and shIP3R(1,3)C and two independent experiments for shIP3R(2,3)B). Boxes illustrate 10–90 percentiles of values, and whiskers represent the range of values. P‐values were calculated using a Kruskal–Wallis test.
Quantification of the mean cell velocity of shIP3R(1,3)C (green)‐silenced immature DCs migrating in micro‐channels in the presence of 2 mM BAPTA. shScramble‐infected DCs were used as a control (gray) (n > 70 cells from two independent experiments). P‐values were calculated using a Kruskal–Wallis test.
Space exploration by dendritic cells requires maintenance of myosin II activity by IP3 receptor 1 (2014)
Figure 5

IP3R1 regulates myosin IIA activity and dynamics in migrating immature DCs

A Immunoblot showing the intracellular levels of phosphorylated MLC in DCs transduced with the shScramble or shRNAs for IP3Rs (shIP3R(1,3)A′, shIP3R(2,3)B′, and shIP3R(1,3)C′). Actin was used as a control.
B Inverted sequential epifluorescence images (20×) showing myosin IIA‐GFP localization of a representative shScramble (left)‐ and shIP3R(1,3)C‐expressing DC (right) migrating in micro‐channels. Myosin IIA‐GFP knock‐in DCs were imaged every 1 min (1 out of 2 images is shown). Asterisks highlight changes of direction during locomotion.
C, D Myosin IIA‐GFP localization (red) versus velocity fluctuations (blue, ΔV/V0) in a representative shScramble‐transduced DC (C) or shIP3R(1,3)C‐silenced (D) migrating in micro‐channels. DCs were recorded as in (B).
E Quantification of the interval between curves (highlighted in D) obtained for myosin IIA positioning and velocity from shScramble‐ and shIP3R(1,3)C‐expressing DCs. Cells were recorded as in (B) (n = 20 cells from two experiments). The Mann–Whitney test was applied for statistical analysis.
Space exploration by dendritic cells requires maintenance of myosin II activity by IP3 receptor 1 (2014)
Figure 6

IP3R1 controls the migration persistence of DCs through the Ca2+/calmodulin‐dependent myosin light chain kinase (MLCK)

A Immunoblot showing the intracellular levels of phosphorylated MLC in DCs treated overnight with increasing amounts of the MLCK inhibitor, ML7.
B, C Analysis of immature DCs migrating in micro‐channels in the presence of ML7 or DMSO as a control (n > 170 cells from two independent experiments). Velocity fluctuations (ΔV/V0) of immature DCs migrating in micro‐channels (B). Whiskers show 10–90 percentiles plus the median. P‐values were calculated using a Kruskal–Wallis test. Frequency of changes (C) in direction of DCs migrating in micro‐channels. Mean plus SD are shown. A Kruskal–Wallis test was applied for statistical analysis.
D, E Analysis of shScramble (gray)‐ and shIP3R(1,3)C (green)‐expressing DCs migrating in micro‐channels in the presence of 20 μM ML7 (n > 70 cells per condition from 3 independent experiments). Velocity fluctuations (ΔV/V0) of immature DCs migrating in micro‐channels (D). Boxes illustrate 10–90 percentiles of values, and whiskers represent the range of values. P‐values were calculated using a Kruskal–Wallis test. Frequency of changes (E) in direction observed in DCs migrating in micro‐channels. Means plus SD are shown. A Kruskal–Wallis test was applied for statistical analysis.
Palmitoylation of γb protein directs a dynamic switch between Barley stripe mosaic virus replication and movement (2022)
Figure 1

Figure 1. Identification of palmitoylation sites within γb protein.

A, B Detection of γb palmitoylation through the biotin-switch assay in N. benthamiana. The γb-3xFlag and γb3CS-3xFlag proteins were treated with (Hyd+) or without (Hyd-) hydroxylamine, the thioester cleavage reagent. Lanes labelled ' Palmitoylation ' show γb-3xFlag or γb3CS-3xFlag amounts recovered from the neutravidin beads. The loading controls indicate sample loading onto the neutravidin beads.

C Systemic symptoms of N. benthamiana plants inoculated with BSMV and BSMV3CS at 10 dpi.

D Western blot detection of viral accumulation in BSMV- and BSMV3CS-infected N. benthamiana plants with anti-TGB1 or anti-γb antibodies. SL8, eighth systemically infected leaf; SL9, ninth systemically infected leaf. Ino, agroinfiltrated leaves. Rubisco large subunit (RbcL) was used as a loading control.

E Symptoms in the first (SL1) and second (SL2) systemically infected leaves of barley inoculated with BSMV or BSMV3CS at 12 dpi.

F Western blot analysis of protein accumulation in BSMV- and BSMV3CS- systemically infected barley leaves with anti-TGB1 or anti-γb antibodies. RbcL was used as a loading control.

Data information: In (A, B, D and F), representative data are shown and three biological replicates had similar results.

Source data are available online for this figure.
Palmitoylation of γb protein directs a dynamic switch between Barley stripe mosaic virus replication and movement (2022)
Figure 2

Figure 2. Non-palmitoylated γb enhancement of BSMV replication by localization in close proximity to chloroplasts.

A Effects of γb palmitoylation on BSMV replication. Movement-deficient BSMV (RNAα + RNAγ/RNAγ3CS) was agroinfiltrated into N. benthamiana leaves. At 3 dpi, total protein and RNA were extracted for Western and Northern blot analyses. Antibodies and probes used for molecular analyses are indicated on the right of each panel. Actin was used to monitor equal protein loading, and methylene blue-stained rRNAs served as RNA loading controls.

B Western blot analyses of chloroplast localization of γb and γb3CS. Transiently expressed αa-GFP in intact chloroplasts isolated from BSMVγb-3xFlag- or BSMV3CS-3xFlag-infected N. benthamiana leaves at 4 dpi. Detection of γb-3xFlag and γb3CS-3xFlag with an anti-Flag antibody; TGB1 analysis with an anti-TGB1 antibody; αa-GFP detection with an anti-GFP antibody. NTRC was a loading control for intact chloroplasts (Wang et al., 2021). Phosphoenolpyruvate carboxylase (PEPC) (Kai et al, 2003) was used to assess cytosolic contamination of isolated chloroplasts.

C Subcellular localization of γb-GFP and γb3CS-GFP in N. benthamiana epidermal leaf cells. The first and second rows show subcellular localization of γb-GFP and γb3CS-GFP transient expression in leaves. The third and fourth rows show localization of γb-GFP and γb3CS-GFP in BSMVγb-GFP- and BSMV3CS-GFP-infected leaves at 3 dpi. Figures on the right indicate the normalized fluorescence intensities of the GFP (green) or the chloroplast autofluorescence (red) channels along the dashed white lines shown in the merged images of the left panel. Scale bars, 20 μm.

D Subcellular localization of γb-GFP or γb3CS-GFP in isolated N. benthamiana protoplasts harvested at 3 dpi. The fifth and sixth columns show γb localization in BSMVγb-GFP- (movement-deficient) N. benthamiana protoplasts after treatment with 200 μM 2-BP inhibitor. The released protoplasts were collected after 48 h incubation in DMSO or 2-BP followed by confocal microscopy visualization. Figures at the bottom indicate the normalized fluorescence intensities of the GFP (green) and the chloroplast autofluorescence (red) channels along the dashed white lines in the merged images above. Scale bars, 20 μm.

Data information: In (A-D), representative data are shown and three biological replicates had similar results.

Source data are available online for this figure.
Palmitoylation of γb protein directs a dynamic switch between Barley stripe mosaic virus replication and movement (2022)
Figure 3

Figure 3. Palmitoylation of γb facilitates BSMV cell-to-cell movement.

A Effects of γb palmitoylation on virus cell-to-cell movement at 3 dpi with the BSMV duplex fluorescence (dfBSMV) reporter system (Li et al., 2018b). The dfBSMVmγb mutant control contains an ATG substitution of the γb initiation codon (Jiang et al., 2020). Red fluorescence produced from the mCherry expression cassette illustrates the primary infection foci, whereas GFP fluorescence outside the red region shows invaded cells surrounding the primary infection foci. Fluorescence was evaluated by confocal microscopy. Scale bars, 100 μm.

B Quantification of virus movement in Panel A. To determine the movement capacity of the viruses depicted in each panel, the mCherry and GFP fluorescent intensities were first determined by ImageJ software. Then, the intensities within the secondarily invaded cells were divided by the fluorescent mCherry plus the GFP intensities in the primary infection foci (n = 10). The Y-axis indicates the relative sizes of the green areas in comparison to that of the red-colored areas.

C Confocal microscopy analysis of BSMV movement at different timepoints. The dfBSMV derivatives were infiltrated into N. benthamiana leaves, which were harvested at 48 hpi, 60 hpi, and 72 hpi, and analyzed by confocal microscopy. Scale bars, 100 μm.

D Biotin-switch assay to access palmitoylation of γb in BSMV-infected N. benthamiana leaves at different timepoints. BSMVγb-3xFlag was treated with the hydroxylamine (Hyd+) thioester cleavage reagent. Equal sample amounts used for palmitoylation analyses were monitored by Western blot analysis with an anti-Flag antibody.

E Subcellular localization of BSMVγb-GFP and BSMV3CS-GFP in N. benthamiana leaf epidermal cells. The mCherry-HDEL and mTalin-RFP reporters were used as markers to indicate the ER and actin localization (Jiang et al., 2020). Figures at the bottom indicate the normalized fluorescence intensities of the GFP and mCherry channels along the white dashed lines in the merged images above. Scale bars, 10 μm.

F, G Western blot analysis of subcellular fractions extracted from N. benthamiana leaf tissues at 3 dpi. S3 indicates the supernatant separated by centrifugation at 3,000 g; and S30 is the 30,000 g supernatant; P30 is the 30,000 g pellet. The association of γb with membranes was determined by 1% Triton X-100 extraction.

Data information: In (A, C, D and E), representative data are shown and three biological replicates had similar results. In (B), the box boundaries indicate the upper (25th percentile) and lower (75th percentile) quartiles, whiskers indicate minimum and maximum values, central band indicates the median. Letters in the chart denote statistically significant differences among groups according to the Duncan's multiple range test (p < 0.05).

Source data are available online for this figure.
Palmitoylation of γb protein directs a dynamic switch between Barley stripe mosaic virus replication and movement (2022)
Figure 5

Figure 5. Palmitoylation of γb by NbPAT15 and NbPAT21.

A Transcriptome analysis of NbPATs in BSMV- or BSMVmγb-infected N. benthamiana plants. The BSMVmγb mutant has a UUG substitution for the AUG initiation codon (AUG→UUG) that prevents translation of the γb protein (Jiang et al., 2020; Zhang et al., 2017). The heat map shows the differential expression of NbPATs during BSMV or BSMVmγb infections compared to plants infiltrated with the empty vector (EV) control. The numbers shown in the graph represent adjusted log2 transformed transcription levels. The three rows of values represent different expression amounts of the NbPATs in two-by-two comparisons of the EV, BSMV, and BSMVmγb. Minima and maxima as error bar.

B Analysis of γb palmitoylation in NbPAT15- or NbPAT21-silenced N. benthamiana plants. NbPAT15 and NbPAT21 were silenced by intron-spliced hairpin RNA-mediated RNAi approaches (Gao et al, 2022). The γb-3xFlag plasmid was agroinfiltrated into the leaves, followed by biotin-switch assays at 3 dpi.

C Effects of γb palmitoylation by NbPAT15 or NbPAT21 on BSMV cell-to-cell movement. Infiltrated leaves were observed by confocal microscopy at 3 dpi. Red fluorescence produced from the mCherry expression cassette shows the primary infection foci, and GFP fluorescence outside the red regions shows virus spread into secondary infected cells. Scale bars, 100 μm.

D Quantification of BSMV movement. The green and red fluorescent areas were measured by ImageJ software (n = 10).

E RT-PCR analysis of BSMV RNA accumulation in NbPAT15- and NbPAT21-silenced N. benthamiana leaves. EV-treated leaves served as the control.

Data information: In (B and C), representative data are shown and three biological replicates had similar results. In (D), the box boundaries indicate the upper (25th percentile) and lower (75th percentile) quartiles, whiskers indicate minimum and maximum values, central band indicates the median. Letters in the chart denote statistically significant differences among groups according to the Duncan's multiple range test (p < 0.05). In (E), error bars indicate mean ± SEM from three independent experiments, the letters indicate statistically significant differences (p < 0.05) from Student's t-test.

Source data are available online for this figure.
Palmitoylation of γb protein directs a dynamic switch between Barley stripe mosaic virus replication and movement (2022)
Figure 6

Figure 6. NbPAT15 and NbPAT21-mediated palmitoylation of γb to convert BSMV replication to cell-to-cell movement.

A, B BiFC assays to identify interactions of γb with NbPAT15 and NbPAT21 during BSMV infection in N. benthamiana at 3 dpi. PAT15-YFPn and PAT21-YFPn were co-expressed with BSMVγb-YFPc. Chloroplast autofluorescence is depicted as a false red color and mCherry-HDEL provides the ER marker. Figures on the right show the normalized fluorescence intensities of GFP, mCherry and the chloroplast autofluorescence channels along the white dashed lines in the merged images. Scale bars, 20 μm.

C Co-localization analyses of αa-GFP, RFP-TGB1, and CFP-γb in N. benthamiana leaves at 3 dpi. Mixtures of A. tumefaciens containing αa-GFP, RFP-TGB1, and CFP-γb were infiltrated into NbPATs-silenced N. benthamiana leaves or the non-silenced TRV:GUS control. Chloroplast autofluorescence is displayed as a false pink color. Figures on the right show the normalized fluorescence intensities of GFP, RFP, CFP and the chloroplast autofluorescence channels along the white dashed lines in the merged images. Scale bars, 10 μm.

D Co-IP assays of interactions between γb and TGB1 in NbPAT15- and NbPAT21-silenced N. benthamiana leaves, TRV:GUS and TRV:GUS combined with the 2-BP inhibitor provided positive and negative controls, respectively. The silenced leaves were inoculated with BSMV3xFlag-TGB1/γb-GFP mixtures (RNAα + RNAβ3xFlag-TGB1 + RNAγγb-GFP) and harvested at 3 dpi. Total proteins were immunoprecipitated with anti-Flag beads. Input and IP proteins were analyzed by Western blotting with anti-GFP or anti-Flag antibodies.

E Average amounts of RFP-TGB1 foci co-localizing with αa-GFP and CFP-γb at the chloroplast peripheries shown in panel C (n = 12).

Data information: In (A, B, C and E), representative data are shown and three biological replicates had similar results. In (E), the box boundaries indicate the upper (25th percentile) and lower (75th percentile) quartiles, whiskers indicate minimum and maximum values, central band indicates the median. Letters in the chart denote statistically significant differences among groups according to the Duncan's multiple range test (p < 0.05).

Source data are available online for this figure.
Palmitoylation of γb protein directs a dynamic switch between Barley stripe mosaic virus replication and movement (2022)
Figure 8

Figure 8. Inhibition of callose accumulation during interactions of palmitoylated γb with NbREM1.

A BiFC analyses of interactions between NbREM1 and γb or γb3CS. Combinations of different constructs are indicated on the left. Scale bars, 20 μm.

B Co-IP analyses to analyze interactions of NbREM1 with γb or γb3CS in N. benthamiana leaf tissues infiltrated with A. tumefaciens mixtures containing movement-deficient BSMV plasmids (RNAα + RNAγγb-GFP or RNAγ3CS-GFP) and NbREM1-3xFlag. Total proteins were extracted at 3 dpi and immunoprecipitated with anti-Flag beads. Input and immunoprecipitated proteins (IP) were analyzed by Western blot analyses with an anti-GFP or anti-Flag antibodies.

C NbREM1 effects on BSMV cell-to-cell movement. A. tumefaciens containing NbREM1 or NbREM1 mutants were first agroinfiltrated into N. benthamiana leaves. After an additional 24 h, the dfBSMV reporter system was agroinfiltrated into the preinfiltrated regions of the leaves, and confocal microscopy was carried out 3 days later. Scale bars, 100 μm.

D Analysis of the cleaved 5(6)-carboxyfluorescein (CF) movement in N. benthamiana leaves. RNAα + RNAγ/RNAγ3CS were co-infiltrated into transiently overexpressed NbREM1 N. benthamiana leaves, and 1 μL CFDA was loaded onto the adaxial leaf blade surface and incubated for 5 min followed by confocal analysis at 3 dpi. The areas of dye diffusion are marked by dashed white circles. Scale bars, 100 μm.

E Quantification of CF movement. Dye diffusion areas identified by the dashed white circles in panel D were measured by image J software (n = 6).

F Aniline blue staining of callose in N. benthamiana leaf tissues. Leaves were infiltrated with mixtures of different A. tumefaciens constructs indicated beside the panels. At 3 dpi, the 0.1% aniline blue fluorochrome was infiltrated into N. benthamiana leaves. After incubation at 5 min in the dark, the leaves were observed by confocal microscopy at 405 nm. Scale bars, 20 μm.

G Quantification of callose staining intensity as shown in panel F. The relative callose staining intensity was set to 1 in the REM samples (n = 10).

H Aniline blue staining of callose in leaves with different treatments above the panel in NbPAT15 and NbPAT21 RNAi plants. Scale bars, 20 μm.

I Quantification of callose staining intensity as shown in panel H. The relative callose staining intensity was set as 1 in the samples of EV (n = 8).

Data information: In (A, B, D, F and H), representative data are shown and three biological replicates had similar results. In (E,G and I), the box boundaries indicate the upper (25th percentile) and lower (75th percentile) quartiles, whiskers indicate minimum and maximum values, central band indicates the median. Letters in the chart denote statistically significant differences among groups according to the Duncan's multiple range test (p < 0.05).

Source data are available online for this figure.
MAPK Signaling Regulates c-MYC for Melanoma Cell Adaptation to Asparagine Restriction (2020)
Figure 1

Figure 1. Asparagine Restriction Upregulates ATF4 and c-MYC.

A. Immunoblotting of c-MYC and ATF4 in melanoma cells treated with NT-siRNA (-) or siASNS (#1 or #2) for 72 hr.

B and C. Reverse transcription with quantitative PCR (RT-qPCR) of the indicated transcripts in melanoma cells 36 hr after treatment with siASNS (#1 or #2) (B), or siASNS, siATF4, or both (C).

D. Immunoblotting of ATF4 and ASNS in melanoma cells 72 hr after treatment with siASNS, siATF4, or both.

E. Proliferation of melanoma cells over indicated times after treatment with siASNS, siATF4, or both.

F. RT-qPCR analysis of indicated transcripts in melanoma cells 36 hr after treatment with siASNS (#1 or #2).

G and H. Immunoblotting of c-MYC in melanoma cells treated for 72 hr with siASNS, L-Asn, or both (G), or in A375 cells treated as indicated for 72 hr (H).

Data information: Data are shown as the mean ± SD, n = 3 biological replicates. **p ≤ 0.01, ***p ≤ 0.001, ****p ≤ 0.0001. Unpaired Student's t-test was used in (B), (C), and (F); two-way ANOVA was utilized in (E). In (E), the statistical significance denoted in black and orange corresponds to the comparison of siASNS+siATF4 vs. siASNS and siATF4 respectively.
MAPK Signaling Regulates c-MYC for Melanoma Cell Adaptation to Asparagine Restriction (2020)
Figure 2

Figure 2. MAPK-GSK3-β Signaling Pathway is Required for AAR Signaling Activation in Asparagine-Restricted Melanoma Cells.

A. Immunoblotting of c-MYC in A375 cells treated with NT-siRNA or siASNS for 24 hr followed by cycloheximide treatment for indicated timepoints (left). Plot showing relative protein levels of c-MYC (y-axis) as a function of time lapsed (x-axis) in the left panel (right).

B-D. Immunoblotting of indicated proteins in melanoma cells 72 hr after treatment with NT-siRNA (-) or siASNS (#1 or #2) (B and C), or siASNS, PLX-4032, or both (D).

E. Immunoblotting of indicated proteins in A375 cells cultured in L-Asn and treated with L-A'ase, PLX-4032, or both for 72 hr.

F. A375 cells were treated with siASNS, SCH772984, or both for 72 hr followed by immunoblotting of phosphorylated and total GSK3-β.

G. A375 cells were treated with siASNS, HA-GSK3-β (S9A), or both for 72 hr followed by immunoblotting of indicated proteins.

H. RT-qPCR analysis of indicated transcripts in melanoma cells 36 hr after treatment with siASNS, HA-GSK3-β (S9A), or both.

I. A375 cells were treated as indicated for 72 hr followed by immunoblotting of c-MYC.

J. Immunoblotting of c-MYC and HA proteins in A375 cells treated as indicated for 72 hr.

Data information: Data are shown as the mean ± SD, n = 3 biological replicates. **p ≤ 0.01, ***p ≤ 0.001, ****p ≤ 0.0001. Unpaired Student's t-test was used in (H).
MAPK Signaling Regulates c-MYC for Melanoma Cell Adaptation to Asparagine Restriction (2020)
Figure 3

Figure 3. c-MYC is Critical for the Induction of ATF4 in Asparagine-Restricted Melanoma Cells.

A. Immunoblotting of indicated proteins in melanoma cells treated with siASNS, sicMYC (#1 or #2), or a combination of siASNS and sicMYC (#1 or #2) for 72 hr.

B. RT-qPCR analysis of indicated transcripts in melanoma cells 36 hr after treatment with siASNS, sicMYC, or both.

C. Immunoblotting of indicated proteins in melanoma cells 72 hr after treatment with siASNS, sicMYC, PLX-4032, or a combination of siASNS and sicMYC, or siASNS and PLX-4032.

D. Immunoblotting of indicated proteins in A375 cells 72 hr after treatment with siASNS, HA-GSK3-β (S9A), or both.

E. RT-qPCR analysis of indicated transcripts in melanoma cells after 36 hr treatment with siASNS, HA-GSK3-β (S9A), or both.

F. A375 cells were treated as indicated for 72 hr followed by immunoblotting of indicated proteins.

G and H. Proliferation of melanoma cells over indicated times after treatment with siASNS, sicMYC, or both (G), or siASNS, HA-GSK3-β (S9A), or both (H).

Data information: Data are shown as the mean ± SD, n = 3 biological replicates. **p ≤ 0.01, ***p ≤ 0.001, ****p ≤ 0.0001. Unpaired Student's t-test was used in (B) and (E); two-way ANOVA was utilized in (G) and (H). The statistical significance denoted in black and orange corresponds to the comparison of siASNS+sicMYC vs. siASNS and sicMYC respectively (G) and the comparison of siASNS+GSK3-β (S9A) vs. siASNS and GSK3-β (S9A) respectively (H).