EMBO/soda-nel huggingface dataset:
Direct interaction of β-catenin with nuclear ESM1 supports stemness of metastatic prostate cancer (2020)
Figure 7

Figure 7. ESM1 stabilizes the association of TCF4/β-catenin complex.

(A) Prediction of ESM1 and β-catenin interaction using HADDOCK.

(B) HEK293T cells were transfected with the indicated ESM1 point mutant constructs. Cell extracts were pulled down with purified GST-ARM.

(C) Cell extracts of 22Rv1-M transfected with either Scramble-shRNA or ESM1-shRNA were immunoprecipitated with an β-catenin antibody and blotted with anti-TCF4 and anti-BCL9 antibody.

(D) Prediction of the ESM1, β-catenin, and TCF4 interaction using HADDOCK. The surface representation of ARM is colored red and blue for negative and positive charges, respectively, and grey color represents neutral residues.

(E) Upper, Schematic diagram of ESM1-targeting short peptides. Lower, HEK293T cells were transfected with ESM1-HA constructs. Cell extracts were treated with the short peptides, pulled down with purified GST-ARM and blotted with anti-HA and anti-TCF4 antibody.

(F) HEK293T cells were transfected with the indicated ESM1 point mutant constructs. Cell extracts were pulled down with purified GST-ARM and blotted with an anti-TCF4 antibody.

(G) ChIP-qPCR validation of β-catenin binding to the CCND1 and c-Myc promoter in 22Rv1-M cells transfected with either Scramble-shRNA or ESM1-shRNA. Bars are the mean ± SD of three independent experiments. ** P< 0.01 when compared to shScramble cells by two-tailed Student's t test.

(H) Upper, Schematic diagram of ChIP-qPCR primers targeting regions upstream of the CCND1 and c-Myc transcription start sites (TSSs). Bar graphs show β-catenin and ESM1 binding at the CCND1 and c-Myc promoter regions. Differences in DNA levels compared with IgG control are shown as fold changes presented as the mean ± SD of three independent experiments.

(I) The effects of ESM1 targeting peptides on tumor invasion and spheroid formation. Scale bar: 100 μm. Bars are the mean ± SD of three independent experiments. ** P< 0.01 when compared to Pep-con group by two-tailed Student's t test.

(J) The effects of ESM1 point mutants on tumor invasion and spheroid formation. Scale bar: 100 μm. Bars are the mean ± SD of three independent experiments. ** P< 0.01 when compared to vector cells, ## P< 0.01 when compared to ESM1 cells by two-tailed Student's t test.
Modulation of intrinsic inhibitory checkpoints using nanocarriers to unleash NK cell activity (2021)
Figure 1

Figure 1: Inhibition of Cbl-b, c-Cbl and SHP-1 enhance NK cell function.

(A) YTS KIR2DL1 cells were mock-transfected or transfected with N.S siRNA or Cbl-b siRNA, c-Cbl siRNA and SHP-1 siRNA, using Amaxa electroporation. After 48 hours, cells were lysed, and subjected to western blot with anti-Cbl-b, anti-c-Cbl or anti-SHP-1 antibodies. Analysis by ImageJ densitometry is summarized in the graph below. Data are means ± SEM of three independent experiments.(n=3) P values were calculated by One-sample t-test, and are indicated by asterisks *P < 0.05.

(B) YTS KIR2DL1 cells were transfected with either N.S siRNA (blue curve) or with SHP-1 and Cbls siRNA (red curve). After 48 hours, cells were loaded with calcium-sensitive Fluo-3-AM and analyzed for basal intracellular calcium levels for 1 min. The NK cells were then mixed with 721.221 Cw4 target cells at 37°C and monitoring of calcium levels was continued. The data shown are representative of three independent experiments.

(C) YTS KIR2DL1 cells were incubated with either 721.221 Cw4 or Cw7 target cells for 2 hours and analyzed by flow cytometry to determine the expression of CD107a. Expression of CD107a was measured by mean fluorescence Intensity (MFI). Relative expression of CD107a MFI was normalized to the mock-transfected sample following Cw4 incubation. Data are means ± SEM of five independent experiments.(n=5). P values were calculated by One-sample t-tests and One way ANOVA following Tukey's post hoc analysis are indicated by asterisks *P < 0.05, ****P<0.0001 .

(D) Granzyme release. YTS KIR2DL1 cells were mock-transfected or transfected with N.S siRNA or SHP-1 and Cbls siRNA. After 48 hours, cells were incubated with either 721.221 Cw4 or Cw7 target cells, the supernatant was then collected and granzyme B levels were evaluated using ELISA sandwich assay. The levels of granzyme B were quantified using standard recombinant granzyme B concentrations Data are means ± SEM of eight independent experiments;(n=8); P values were calculated by One way ANOVA following Tukey's post hoc analysis, and are indicated by asterisks *P < 0.05..

(E) Killing assay. YTS KIR2DL1 cells were mock-transfected or transfected with N.S siRNA or SHP-1 and Cbls siRNA. After 48 hours, cells were incubated with [35S] Met-loaded 721.221 Cw4 or Cw7 target cells. After 5 hours of co-culture, the supernatant was collected, and the radioactive signal was measured. Data are means ± SEM of three independent experiments. P values were calculated by One-way ANOVA following Tukey's post hoc analysis and are indicated by asterisks *P < 0.05, **P < 0.01 .

Data information: Exact P-values are shown in Appendix Table S1.
Modulation of intrinsic inhibitory checkpoints using nanocarriers to unleash NK cell activity (2021)
Figure 3

Figure 3: Specific and efficient siRNA delivery to NK cells using NKp46 antibody-coated NPs.

(A) NK92-NKp46low, NK92-NKp46high, YTS KIR2DL1, K562 and 721.221 HLA-Cw4 cells were stained with anti-NKp46 monoclonal antibody followed by staining with Alexa568-Fluor goat anti-mouse IgG secondary antibody. Cells were then analyzed using flow cytometry.

(B) NK92-NKp46low, NK92-NKp46+, YTS KIR2DL1, K562, and 721.221 HLA-Cw4 cells were incubated with 50 µg rhodamine-labeled NPs and analyzed using flow cytometry to confirm the internalization of the coated NPs in a NKp46-specific manner.

(C-F) (C) NK92-NKp46low, (D) NK92-NKp46high, (E) YTS KIR2DL1 and (F) K562 cells were incubated with 50 µg rhodamine-labeled NPs and analyzed using confocal microscopy. Upper panel shows wide field images. Lower panel shows single-cell images. The data shown are representative of three independent experiments.

(G) YTS KIR2DL1 were incubated with NKp46 antibody coated-NPs loaded with FITC labeled siRNA. After 24 hours, the cells were stained with anti-NKp46 monoclonal antibody followed by staining with Alexa568-Fluor goat anti-mouse IgG secondary antibody. Then, the cells were analyzed by flow cytometry to determine the incorporation of the rhodamine-labeled NPs loaded with FITC-siRNA and NKp46 antibody staining. Cells that were positive for both rhodamine-labeled NPs and NKp46 antibody staining, were analyzed for the presence of fluorescent FITC-siRNA.
Modulation of intrinsic inhibitory checkpoints using nanocarriers to unleash NK cell activity (2021)
Figure 5

Figure 5: NKp46 antibody-coated NPs do not induce spontaneous non-specific NK cell activation or cytokine secretion.

(A) YTS KIR2DL1 cells were incubated with SHP-1 and Cbls siRNA-loaded NPs (red curve) or empty NPs (purple curve). After 48 hours, cells were loaded with Fluo-3-AM and analyzed for intracellular calcium levels for 20 min at 37°C. YTS KIR2DL1 cells that were treated with PBS served as a negative control (green curve). NK cells that were mixed with 721.221 Cw7 target cells served as a positive control (blue curve). Calcium levels were analyzed by spectrofluorometry. (n=3)

(B) Freshly isolated PBMCs were treated with SHP-1 and Cbls siRNA-loaded NPs, empty NPs, PBS as negative control or PHA as a positive control. Following incubation, supernatant from each sample was collected and the levels of human cytokines TNF-α, IL-6, IL-10 and IFN-γ were determined using ELISA. The data represent four independent experiments (n=4). P values were calculated by One way ANOVA following Tukey's post hoc analysis, and are indicated by asterisks. *P≤0.008. Data are shown as mean ± SEM.

Data information: Exact P-values are shown in Appendix Table S1.
Modulation of intrinsic inhibitory checkpoints using nanocarriers to unleash NK cell activity (2021)
Figure 6

Figure 6: NK cells gene silenced for SHP-1 and Cbls siRNA via NKp46 antibody-coated NPs exhibit therapeutic efficacy in human B-cell lymphoma engrafted mice.

(A) Schematic representation of the experimental timeline.

(B) Tumor volumes measured daily in SCID/NOD mice treated with 3x106 irradiated YTS KIR2DL1 and NPs containing Cbl-b, c-Cbl and SHP-1 siRNA (n=13), or mice treated with 3x106 irradiated YTS KIR2DL1, and NPs containing N.S siRNA that served as a negative control (n=12). P values were calculated using two-tailed Student's t-test and are indicated by asterisks. * P < 0.05. Data are shown as mean ± SEM.

(C) Average growth rates of tumors from the first treatment to the end point from same experimental groups of mice as indicated in B. P value was calculated by two-tailed Student's t-test and is indicated within the graph P =0.001. Data are shown as mean ± SEM.

(D) Images of the tumor bearing mice at the end point of the experiment (upper panel) and excised tumors (lower panels).

(E) Following 24 hours after the last treatment, extracted cells from tumors of either SHP-1 and Cbls or N.S siRNA loaded NKp46-NPs treated mice were analyzed by flow cytometry. Expression of CD107a was measured by mean fluorescence Intensity (MFI). Relative expression of CD107a MFI was normalized to the sample treated with N.S siRNA loaded NPs. The data represent three independent experiments. Data are shown as mean ± SEMn=3; P value was calculated by one sample t-test and is indicated within the graph P =0.009.

(F) Survival analysis in mice treated with NK cells' gene silenced for SHP-1 and Cbls using siRNA-loaded NPs (n=18) or N.S siRNA-loaded NPs (n=18) as demonstrated by Kaplan-Meier survival curve. P values were calculated using the log-rank test and is indicated within the graph P =0.00009. ­­

Data information: Exact P-values are shown in Appendix Table S1.

Expanded View figure legends
A binary effector module secreted by a type VI secretion system (2021)
Figure 2

Figure 2 - VP1388 and VP1390 are required for antibacterial toxicity, not immunity.

A-C Viability counts of the indicated V. parahaemolyticus (A-B) or E. coli (C) prey strains before (0 h) and after (4 h) co-incubation with the indicated V. parahaemolyticus attackers on media containing 3% NaCl at 30°C. In A and B, media also contain 0.1% arabinose to induce expression from plasmids. In A, prey strains contain either an empty plasmid (pEmpty) or a plasmid for arabinose-inducible expression of VP1389 (pVP1389). In B and C, prey strains contain an empty plasmid that provides a selection marker, and the attackers are derivatives of a ∆hns mutant (parental). In B, the attackers contain an empty plasmid, or plasmids for the arabinose-inducible expression of VP1388 (pVP1388) or VP1390 (pVP1390). Data are shown as the mean ± SD, n = 3 technical replicates. Statistical significance between samples at the 4 h timepoint by an unpaired, two-tailed Student's t-test is denoted above. A significant difference was considered as P < 0.05. DL, assay detection limit. ∆hcp1 was used as a T6SS1- control strain. The experiment was performed at least three times with similar results; results of a representative experiment are shown.
A binary effector module secreted by a type VI secretion system (2021)
Figure 3

Figure 3 - VP1388 and VP1390 are loaded together on the T6SS spike and are secreted co-dependently.

A VP1388 binds VP1390. Immunoprecipitation using α-FLAG antibodies from E. coli cells co-expressing the indicated C-terminal FLAG- and Myc-tagged proteins from arabinose-inducible plasmids.

B VP1388 and VgrG1 co-precipitate with VP1390. Immunoprecipitation using α-FLAG antibodies from V. parahaemolyticus ∆hns/∆hcp1/∆vp1390 derivatives harboring plasmids for the arabinose-inducible expression of FLAG-tagged sfGFP or VP1390. Cells were grown in MLB media supplemented with chloramphenicol to maintain the plasmids, and 0.1% arabinose. Endogenous VP1388 and VgrG1 were detected using α-VP1388 and α-VgrG1 antibodies, respectively.

C Expression (cells) and secretion (media) of VP1388, VP1390, and VgrG1 from the indicated V. parahaemolyticus ∆hns-derived strains harboring an empty plasmid (pEmpty) or plasmids for the arabinose-inducible expression of VP1388 (pVP1388) or VP1390 (pVP1390). Samples were grown in media containing 3% NaCl and supplemented with 0.1% arabinose at 30°C. RNA polymerase β (RNAp) was used as a non-secreted protein loading control. Arrows denote the bands at the expected size of VP1388 or VP1390, as indicated in the appropriate panels.

Data information: The experiments were performed at least three times with similar results; results of a representative experiment are shown in each panel.

Source data are available online for this figure.
A binary effector module secreted by a type VI secretion system (2021)
Figure 4

Figure 4 - VP1390 is a periplasm-targeting toxin that leads to cell lysis.

A Toxicity of periplasm-targeted proteins in E. coli. E. coli strains containing plasmids for the arabinose-inducible expression of sfGFP (used as a control), VP1388 or VP1390 fused to an N-terminal PelB signal peptide (perisfGFP, periVP1388, and periVP1390, respectively) were spotted at 10-fold serial dilutions onto LB agar plates supplemented with kanamycin (to maintain plasmids) and either 0.2% glucose, to repress protein expression, or 0.001% arabinose, to induce protein expression. Plates were incubated over-night at 30°C or 37°C, as indicated.

B VP1389 interacts with VP1390. Co-immunoprecipitation of FLAG-tagged VP1390 or BC3020 using Myc-tagged VP1389 when co-expressed in V. parahaemolyticus Δvp1389 (input). Precipitated proteins (output) were detected by immunobotting using α-Myc and α-FLAG antibodies. Source data are available online for this figure.

C VP1390 induces cell lysis in E. coli. Time-lapse microscopy of E. coli cells expressing periplasm-targeted sfGFP, VP1388, or VP1390 (perisfGFP, periVP1388, and periVP1390, respectively) from an arabinose-inducible vector, grown on LB agarose pads supplemented with kanamycin (to maintain the plasmid), 0.2% arabinose (to induce expression), and propidium iodide (PI; pink). Merging of the phase contrast and PI channels are shown. Scale bar = 2 µm.

Data information: The experiments were performed at least three times with similar results; results of a representative experiment are shown in each panel.
A binary effector module secreted by a type VI secretion system (2021)
Figure 5

Figure 5 - Operon-mediated toxicity results in prey cell lysis.

Time-lapse microscopy of competition between V. parahaemolyticus ∆hns (T6SS+/VP1390+), ∆hns/∆hcp1 (T6SS-/VP1390+), or ∆hns/∆vp1390 (T6SS+/VP1390-) attackers and V. parahaemolyticus ∆vp1389 prey that express GFP. Attacker and prey were mixed (2:1 ratio) and spotted on LB agarose pads supplemented with propidium iodide (PI; pink). Merging of the phase contrast, GFP (green), and PI (pink) channels, as well as the PI channel alone are shown. Scale bar = 5 µm. The experiment was performed three times with similar results; results of a representative experiment are shown.
The phyB-dependent induction of HY5 promotes iron uptake by systemically activating FER expression (2021)
Figure 1

Figure 1. The effects of light and photoreceptors on Fe uptake in tomato plants.

A. Fe absorption rates determined every 6 h over a 24 h period during the day (0~6 h and 6~12 h) and at night (12~18 h and 18~24 h) under Fe-sufficient (20 µM, +Fe) conditions. Day and night are indicated by the white and black bars, respectively. Time points 0 and 12 h correspond to 8 AM and 8 PM, i.e. the times for light on and off, respectively.

B. Relative FCR activities during the day (at 6 h and 12 h) and at night (at 18 h and 24 h) under Fe-sufficient (20 µM, +Fe) conditions. Day and night are indicated by the white and black bars, respectively. Time points 0 and 12 h correspond to 8 AM and 8 PM, i.e. the times for light on and off, respectively.

C. Fe contents of the whole plants of wild-type plants and photoreceptor mutants grown under white light with either Fe-sufficient (20 µM, +Fe) or Fe-deficient (2 µM, -Fe) conditions for 7 d.

D. Relative FCR activities in the roots of wild-type plants and photoreceptor mutants grown under white light with either Fe-sufficient (20 µM, +Fe) or Fe-deficient (2 µM, -Fe) conditions for 7 d.

Data information: Values are the means ± SD, n = 4 (biological replicates), P < 0.05 (Tukey's test).
The phyB-dependent induction of HY5 promotes iron uptake by systemically activating FER expression (2021)
Figure 2

Figure 2. Shoot phyB plays an important role in Fe uptake and photosynthetic electron transport in leaves.

A-C. Fe contents of whole plants, FER expression in roots and relative FCR activities in the roots of plants grown under Fe-sufficient (20 µM, +Fe) or Fe-deficient (2 µM, -Fe) conditions for 7 d. The phyB1B2 (phyB) mutants and wild-type (WT) plants were reciprocally grafted and grown hydroponically under white light.

D-F. Fe contents of whole plants, FER expression in roots and relative FCR activities in the roots of plants grown under Fe-sufficient (20 µM, +Fe) or Fe-deficient (2 µM, -Fe) conditions for 7 d. Plants were exposed to high (H-, 2.0) or low (L-, 0.5) red (R) to far red (FR) ratios.

Data information: Values are the means ± SD, n = 4 (biological replicates), P < 0.05 (Tukey's test).
The phyB-dependent induction of HY5 promotes iron uptake by systemically activating FER expression (2021)
Figure 3

Figure 3. HY5 gene expression is influenced by phyB and the movement of HY5.

A. Levels of HY5 transcripts in leaves of reciprocal grafts of phyB1B2 (phyB) and wild-type (WT) tomato plants grown under white light (200 µmol m-2s-1) for 7 d.

B. Levels of HY5 transcripts in roots of reciprocal grafts of phyB1B2 (phyB) and wild-type (WT) tomato plant grown under white light (200 µmol m-2s-1) for 7 d.

C. Accumulation of HY5 protein in leaves and roots of reciprocal grafts of phyB and WT plants grown under white light (200 µmol m-2s-1) for 7 d. Rubisco and β-Actin were used as loading controls for leaves and roots in the western blot analysis, respectively. The values shown above each lane indicate the relative abundance of the HY5 protein.

D. Accumulation of HY5 protein in the roots of WT and phyB plants under high (H, 2.0) or low ratio of (L, 0.5) red (R) to far red (FR) light conditions. β-Actin was used as loading controls in the western blot analysis. The values shown above each lane indicate the relative abundance of the HY5 protein.

E. Accumulation of HY5:3HA protein in leaves and roots of reciprocal grafts involving HY5-OE and WT plants grown under white light (200 µmol m-2s-1) for 7 d. Rubisco and β-Actin were used as loading controls for leaves and roots in the western blot analysis, respectively.

Data information: In (A, B), values are the means ± SD, n = 4 (biological replicates), P < 0.05 (Tukey's test). +Fe: 20 µM Fe-EDTA; -Fe: 2 µM Fe-EDTA.
The phyB-dependent induction of HY5 promotes iron uptake by systemically activating FER expression (2021)
Figure 4

Figure 4. HY5 plays an important role in light-regulated Fe uptake in tomato plants.

A. Fe content of whole plants. Plants with reciprocal grafts of wild-type (WT), HY5-RNAi (hy5) and HY5-over expression (HY5) were hydroponically grown under white light conditions with either Fe-sufficient (20 µM, +Fe) or Fe-deficient (2 µM, -Fe) conditions for 7 d.

B. FER expression in roots. Plants with reciprocal grafts of wild-type (WT), HY5-RNAi (hy5) and HY5-over expression (HY5) were hydroponically grown under white light conditions with either Fe-sufficient (20 µM, +Fe) or Fe-deficient (2 µM, -Fe) conditions for 7 d.

C. Relative FCR activities in roots. Plants with reciprocal grafts of wild-type (WT), HY5-RNAi (hy5) and HY5-over expression (HY5) were hydroponically grown under white light conditions with either Fe-sufficient (20 µM, +Fe) or Fe-deficient (2 µM, -Fe) conditions for 7 d.

D. Fe content of whole plants. WT and hy5 plants were grown under high (H) or low ratios of (L) red (R) to far red (FR) light conditions with either Fe-sufficient (20 µM, +Fe) or Fe-deficient (2 µM, -Fe) conditions for 7 d.

E. FER expression in roots. WT and hy5 plants were grown under high (H) or low ratios of (L) red (R) to far red (FR) light conditions with either Fe-sufficient (20 µM, +Fe) or Fe-deficient (2 µM, -Fe) conditions for 7 d.

F. Relative FCR activities in roots. WT and hy5 plants were grown under high (H) or low ratios of (L) red (R) to far red (FR) light conditions with either Fe-sufficient (20 µM, +Fe) or Fe-deficient (2 µM, -Fe) conditions for 7 d.

Data information: Values are the means ± SD, n = 4 (biological replicates), P < 0.05 (Tukey's test).
The phyB-dependent induction of HY5 promotes iron uptake by systemically activating FER expression (2021)
Figure 5

Figure 5. HY5 acts as a transcriptional regulator of FER expression.

A,B. The ACE-box elements in the promoter of tomato FER gene and oligonucleotides were used in electrophoretic mobility shift assays. Numbering is from predicted transcriptional start sites. The FER probe contained one ACE-box (FER-wt), whereas in the FER-mut probe the ACE-box core sequence was mutated. The wild type (WT) and mutated ACE-box sequences are underlined. The mutated bases were indicated in red.

C. HY5 directly binds to the ACE-box of FER promoter in vitro. Recombinant HY5 was purified from E. coli cells and used for DNA binding assays with probes of FER-wt, and FER-mut. The protein purified from empty vector was used as the negative control. One thousand-fold excess of unlabeled probe was used for competition. WT and HY5-OE plants were exposed to white light (200 µmol m-2s-1) grown under Fe-deficient (2 µM, -Fe) conditions for 7 d.

D. Schematic diagram of the promoters of the indicated genes. Black triangles indicate the ACE-box. Red arrows indicate primers used for ChIP-qPCR assays. The translational start site (ATG) is shown at position +1.

E. Direct binding of HY5 to the FER promoter analyzed using ChIP-qPCR in 35S-HY5-3HA-overexpressing (HY5-OE) tomato plants. WT and HY5-OE plants were exposed to white light (200 µmol m-2s-1) grown under Fe-deficient (2 µM, -Fe) conditions for 7 d. Input chromatin was isolated from root samples at 7 d. The epitope-tagged HY5-chromatin complex was immunoprecipitated with an anti-HA antibody. A control reaction was processed side-by-side using mouse IgG. Input- and ChIP-DNA samples were quantified by RT-qPCR using primers specific for the promoter and exon fragment of the FER gene as indicated in (D). The ChIP results are presented as percentage of the input DNA.

Data information: In (E), values are the means ± SD, n = 4 (biological replicates), P < 0.05 (Tukey's test).
The phyB-dependent induction of HY5 promotes iron uptake by systemically activating FER expression (2021)
Figure 6

Figure 6. Mutations in FER abolish light quality-regulated Fe deficiency-induced responses.

A-C. Fe content of whole plants (A) and the photochemical efficiencies of PSI and PSII [Y(I) and Y(II)] (B and C) in young leaves of grafted tomato plants with grafts between WT/WT and WT/fer grown under H-R/FR (2.0) or L-R/FR (0.5) conditions for 7 d.

D-F. Transcript levels of FER, FRO1 and IRT1 in the roots of grafted tomato plants with grafts between WT/WT and WT/fer grown under H-R/FR (2.0) or L-R/FR (0.5) conditions for 7 d.

Data information: Values are the means ± SD, n = 4 (biological replicates), P < 0.05 (Tukey's test). +Fe: 20 µM Fe-EDTA; -Fe: 2 µM Fe-EDTA.