EMBO/soda-nel huggingface dataset:
Plant E3 ligases SNIPER1 and SNIPER2 broadly regulate the homeostasis of sensor NLR immune receptors (2020)
Figure 1

Figure 1. SNIPER1 negatively regulates snc1-mediated autoimmunity and basal defense.

A. Morphology of four-week-old soil-grown plants of Col-0, snc1, and two independent transgenic lines of SNIPER1 OE into snc1 background. OE stands for overexpression of SNIPER1. Scale bar = 1 cm.

B SNIPER1 gene expression in the indicated plants as determined by RT-PCR. Error bars represent mean ± SD (n = 3). Two independent experiments were carried out with similar results.

C Quantification of H.a. Noco2 sporulation in the indicated genotypes 7 days post inoculation (dpi) with 105 spores per ml water. Error bars represent mean ± SD (n=4). Three independent experiments were carried out with similar results.

D Growth of P.s.m. ES4326 on four-week-old leaves of the indicated genotypes at 0 and 3 dpi with bacterial inoculum of OD600 = 0.001. Error bars represent mean ± SD (n=4 for day 0; n=5 for day 3). Three independent experiments were carried out with similar results.

E. Morphology of four-week-old soil-grown plants of two independent transgenic lines of SNIPER1 OE in Col-0 background. Scale bar = 1 cm. No difference in growth and development was observed.

F. SNIPER1 gene expression in the indicated plants as determined by RT-PCR. Error bars represent mean ± SD (n = 3). Two independent experiments were carried out with similar results.

G. Quantification of H.a. Noco2 sporulation in the indicated genotypes 7 days post inoculation (dpi) with 105 spores per ml water. Error bars represent mean ± SD (n=4). Three independent experiments were carried out with similar results.

H. Growth of P.s.t. DC3000 on four-week-old leaves of the indicated genotypes at 0 and 3 dpi with bacterial inoculum of OD600 = 0.001. Error bars represent mean ± SD (n=4 for day 0; n=5 for day 3). Three independent experiments were carried out with similar results.

Data information: Color squares shown on the right of panel D and panel H are for B-D and F-H, respectively. One-way ANOVA followed by Tukey's post hoc test were performed for panel B, C, D, F, G and H. Statistical signiﬁcance is indicated by different letters (p < 0.01).
Plant E3 ligases SNIPER1 and SNIPER2 broadly regulate the homeostasis of sensor NLR immune receptors (2020)
Figure 2

Figure 2. Overexpression of SNIPER1 suppresses NLR type autoimmune mutants, but not an RLP autoimmune mutant.

A-B. Morphology of four-week-old soil-grown plants of Col-0, chs1-2 (A), chs2-1 (B) and two independent transgenic lines of SNIPER1 OE into chs1-2 (A) and chs2-1 (B) backgrounds. Plants were grown at 16 °C under long day conditions. Scale bar = 1 cm.

C-D. Morphology of four-week-old soil-grown plants of Col-0, chs3-2D (C), snc2-1D eds5 npr1 (D) and two independent transgenic lines of SNIPER1 OE into chs3-2D (C) and snc2-1D eds5 npr1 (D) backgrounds. Scale bar = 1 cm.

Data information: For all panels, fresh weights of plants are shown on the right. One-way ANOVA followed by Tukey's post hoc test were performed for all panels. Statistical signiﬁcance is indicated by different letters (p < 0.01). Error bars represent mean ± SD (n = 5).
Plant E3 ligases SNIPER1 and SNIPER2 broadly regulate the homeostasis of sensor NLR immune receptors (2020)
Figure 3

Figure 3. SNIPER1 is a functional E3 ligase and overexpression of SNIPER1 fully suppresses mekk1-5 ndr1-mediated autoimmunity.

A. Morphology of four-week-old soil-grown plants of Col-0, mekk1-5 ndr1, and two independent transgenic lines of SNIPER1 OE into mekk1-5 ndr1 background. Scale bar = 1 cm. The mekk1 ndr1 double mutant was used instead of the mekk1-5 single mutant is to overcome the sterility.

B. SNIPER1 gene expression in the indicated plants as determined by RT-PCR. Error bars represent mean ± SD (n = 3). Two independent experiments were carried out with similar results.

C. Quantification of H.a. Noco2 sporulation in the indicated genotypes 7 days post inoculation (dpi) with 105 spores per ml water. Error bars represent mean ± SD (n=4). Three independent experiments were carried out with similar results.

D. Growth of P.s.m. ES4326 on four-week-old leaves of the indicated genotypes at 0 and 3 dpi with bacterial inoculum of OD600 = 0.001. mn stands for mekk1-5 ndr1 double mutant. Error bars represent mean ± SD (n=4). Three independent experiments were carried out with similar results.

E. In vitro ubiquitination assay using E. coli-expressed SNIPER1-myc (E3), AtUBC8 (E2), AtUBA1 (E1) and/or HIS-FLAG-Ubiquitin. The molecular mass markers are indicated on the left (kDa). Asterisk indicates SNIPER1-myc. Arrow points to His-FLAG-Ub and straight lines on the right indicate self-ubiquitinated SNIPER1.

Data information: Color squares shown on the right of panel D are for B-D. One-way ANOVA followed by Tukey's post hoc test were performed for panel B, C and D. Statistical signiﬁcance is indicated by different letters (p < 0.01).
Plant E3 ligases SNIPER1 and SNIPER2 broadly regulate the homeostasis of sensor NLR immune receptors (2020)
Figure 4

Figure 4. SNIPER1 reduces sNLRs protein levels upon overexpression.

A-G. Immunoblot analysis of SNC1 (A-B), SUMM2-3HA (C), RPS4HA (D), RPP4myc (E), RPS2HA (F) and RPM1myc (G) protein levels in the indicated genotypes. For all panels, equal loading is shown by Ponceau S staining of a non-specific band. The numbers below represent the normalized ratio between the intensity of the protein band and the Ponceau S band ± SD (n=3). Molecular mass marker in kilo Daltons is indicated on the left. Three independent experiments were carried out with similar results.
Plant E3 ligases SNIPER1 and SNIPER2 broadly regulate the homeostasis of sensor NLR immune receptors (2020)
Figure 5

Figure 5. SNIPER1 OE lines show enhanced disease susceptibility to avirulent pathogens.

A-C. Growth of P.s.t. avrRps4 (A), avrRpt2 (B) and avrRpm1 (C) on four-week-old leaves of the indicated genotypes at 3 dpi with bacterial inoculum of OD600 = 0.0001. Error bars represent mean ± SD (n=5). Three independent experiments were carried out with similar results. Appendix Figure S4 shows the growth of P.s.t. avrRps4, avrRpt2 and avrRpm1 at 0 dpi.

D. Quantification of H.a. Emwa1 sporulation in the indicated genotypes 7 days post inoculation (dpi) with 105 spores per ml water. Error bars represent mean ± SD (n=4). Three independent experiments were carried out with similar results.

Data information: Color squares shown below are for all panels. One-way ANOVA followed by Tukey's post hoc test were performed for all panels. Statistical signiﬁcance is indicated by different letters (p < 0.01).
Plant E3 ligases SNIPER1 and SNIPER2 broadly regulate the homeostasis of sensor NLR immune receptors (2020)
Figure 6

Figure 6. Loss-of-function of both SNIPER1 and SNIPER2 leads to an EDS1-dependent autoimmunity and lethality.

A. Morphology of four-week-old soil-grown plants of Col-0 and two independent transgenic lines of 35S-SNIPER1H129Y. Scale bar = 1 cm.

B. PR1 and PR2 gene expression in the indicated plants as determined by RT-PCR. Error bars represent mean ± SD (n = 3). Two independent experiments were carried out with similar results.

C. Morphology of four-week-old soil-grown plants of Col-0, sniper1-1, sniper2-1, eds1-2, SNIPER1H129Y, SNIPER1H129Y eds1-2, sniper1-1 sniper2-1 eds1-2 and sniper1-c1 sniper2-1 eds1-2. sniper1-1 is an exonic T-DNA insertional mutant. sniper1-c1 is a SNIPER1 knockout mutant containing 807bp deletion that was generated by CRISPR-Cas9 system. Scale bar = 1 cm.

D. Quantification of H.a. Noco2 sporulation in the indicated plants 7 days post inoculation (dpi) with 105 spores per ml water. Error bars represent mean ± SD (n=4). Two independent experiments were carried out with similar results.

E-H. Immunoblot of RPS4HA (E), SNC1 (F), RPS2HA (G), and RPM1myc (H) in SNIPER1H129Y background. Equal loading is shown by Ponceau S staining of a non-specific band. The numbers below represent the normalized ratio between the intensity of the protein band and the Ponceau S band ± SD (n=3). Molecular mass marker in kilo Daltons is indicated on the left. Three independent experiments were carried out with similar results.

Data information: Color squares shown on the right of panel B are for panel B. Color squares for panel D are shown below. One-way ANOVA followed by Tukey's post hoc test were performed for panel B and D. Statistical signiﬁcance is indicated by different letters (p < 0.01).
Plant E3 ligases SNIPER1 and SNIPER2 broadly regulate the homeostasis of sensor NLR immune receptors (2020)
Figure 7

Figure 7. SNIPER1 interacts with diverse NLRs through the NB domain.

A-C. Immunoprecipitation and biotinylation of SUMM2NB-FLAG (A), RPP4NB-FLAG (B), or CHS1NB-FLAG (C) by SNIPER1 H129Y-HATurboID in N. benthamiana. Two biological repeats were carried out with similar results.

D-F. Interaction of SNIPER1 H129Y-CLuc and SUMM2NB-NLuc (D), RPP4NB-NLuc (E) or CHS1NB-NLuc (F) as tested by split-luciferase complementary assay in N. benthamiana. Three biological repeats with four technical repeats each were carried out with similar results.
Plant E3 ligases SNIPER1 and SNIPER2 broadly regulate the homeostasis of sensor NLR immune receptors (2020)
Figure 8

Figure 8. In vitro ubiquitination of the NB domains by SNIPER1.

A-C. Ubiquitination of the NB domains from SUMM2 (A), RPP4 (B) and CHS1 (C). Immunoblot of bacterial lysates form E. coil strains expressing AtUBA1-S, MBP-NB-HA, AtUBC8-S, HIS-FLAG-UBQ10 and SNIPER1-myc, or strains without one of these components. Two biological repeats were carried out with similar results.

Expanded View Figure legends
Therapeutic inhibition of TRF1 impairs the growth of p53-deficient K-RasG12V-induced lung cancer by induction of telomeric DNA damage. (2015)
Figure 1

Efficient oncogenic K-RasG12V expression and Trf1 depletion in lung lesions

Genetic model. Trf1lox and K-RasLSLGV12 alleles are depicted before and after Cre-mediated excision.

In vivo imaging schedule. Eight- to ten-week-old mice were intratracheally infected with adeno-Cre, mice were analyzed every 2 weeks by computerized tomography (CT), and 22 weeks post-infection, a positron emission tomography (PET) was performed. Mice were sacrificed 24 weeks post-infection for further histological analysis.

TRF1 immunofluorescence of the lungs. Notice the absence and presence of TRF1 signal in the carcinomas and surrounding healthy tissue of Trf1Δ/Δ mice, respectively.

Analysis of Trf1 excision by PCR. Notice the completed excision in carcinomas of Trf1lox/lox lungs.

Detection of β-galactosidase activity in the lungs as a surrogate marker of oncogenic K-RasG12V expression.
Therapeutic inhibition of TRF1 impairs the growth of p53-deficient K-RasG12V-induced lung cancer by induction of telomeric DNA damage. (2015)
Figure 2

Trf1 deficiency impairs K-Ras-mediated lung cancer development

Tumor growth curve of Trf1+/+
K-Ras+/G12V
p53+/+ and Trf1Δ/Δ
K-Ras+/G12V
p53+/+ measured by computed tomography (CT).

Quantification of the number and size of Trf1+/+
K-Ras+/G12V
p53+/+ and Trf1Δ/Δ
K-Ras+/G12V
p53+/+ carcinomas at death point.

Percentage of tumors that have deleted Trf1 quantified by TRF1 immunofluorescence after mice had been sacrificed. Post-mortem analysis of Trf1 deletion in each tumor revealed that none of the Trf1lox/lox
K-Ras+/G12V
p53+/+ ones had excised Trf1.

Tumor growth curve and tumor growth slope of Trf1+/+
K-Ras+/G12V
p53−/− and Trf1Δ/Δ
K-Ras+/G12V
p53−/− measured by CT.

Tumor maximal section of Trf1+/+
K-Ras+/G12V
p53−/− and Trf1Δ/Δ
K-Ras+/G12V
p53−/− lungs measured by histological analysis before death point by CT.

Maximum 18F-FDG-glucose uptake by Trf1+/+
K-Ras+/G12V
p53−/− and Trf1Δ/Δ
K-Ras+/G12V
p53−/− tumors 22 weeks after infection by positron emission tomography (PET).

Representative PET-CT image of Trf1+/+
K-Ras+/G12V
p53−/− and Trf1Δ/Δ
K-Ras+/G12V
p53−/− lungs.

Survival curve of Trf1+/+
K-Ras+/G12V
p53−/− and Trf1Δ/Δ
K-Ras+/G12V
p53−/− mice.

Data information: Error bars represent standard error. t-test, chi-squared (B) test, or log-rank (Mantel–Cox; H) test was used to assess statistical significance. The number of mice and the number of tumors are indicated in each case.
Therapeutic inhibition of TRF1 impairs the growth of p53-deficient K-RasG12V-induced lung cancer by induction of telomeric DNA damage. (2015)
Figure 3

Trf1-deficient carcinomas present high amount of telomeric DNA damage and apoptosis

Percentage of cells showing γH2AX foci in carcinomas of the indicated genotypes (left panel). Representative images of γH2AX immunohistochemistry (right panel).

Percentage of cells showing 3 or more γH2AX and RAP1 colocalizing foci (TIFs) (left panel). Representative images of γH2AX and RAP1 double immunofluorescence (right panel). Yellow arrowheads: colocalization of γH2AX and RAP1.

Percentage of active caspase-3 (AC3)-positive cells (left panel). Representative images of AC3 immunohistochemistry (right panel).

Data information: Error bars represent standard error. The number of mice and carcinomas analyzed per genotype is indicated. t-test was used to assess statistical significance.
Therapeutic inhibition of TRF1 impairs the growth of p53-deficient K-RasG12V-induced lung cancer by induction of telomeric DNA damage. (2015)
Figure 4

Trf1 deficiency leads to G2 arrest and mitotic defects

Percentage of Ki67-positive cells in the carcinomas of the indicated genotypes (left panel). Representative images of Ki67 immunohistochemistry (right panel).

Percentage of pH3-positive cells in the carcinomas of the indicated genotypes (left panel). Representative images of pH3 immunohistochemistry (right panel). Red arrowheads: pH3-positive cells.

Percentage of giant nuclei in the carcinomas of the indicated genotype.

Percentage of anaphase bridges out of total anaphases in the carcinomas of the indicated genotypes.

Representative images of giant nuclei, multilobulated nuclei, anaphase bridges, and multipolar mitoses. Red arrowheads indicate the corresponding mitotic aberrations indicated in the image.

Data information: Error bars represent standard error. The number of mice and carcinomas analyzed per genotype is indicated. t-test was used to assess statistical significance.
Therapeutic inhibition of TRF1 impairs the growth of p53-deficient K-RasG12V-induced lung cancer by induction of telomeric DNA damage. (2015)
Figure 5

Trf1 downregulation in K-RasG12V-transformed lung cells leads to a decreased tumor growth and decreased metastatic potential in allograft and xenograft models

A–C The latency (A), volume (B), and weight (C) of subcutaneous tumors generated by control and Trf1-downregulated K-RasG12V-transformed lung cells in athymic mice.

D Representative images of the subcutaneous tumors.

E Trf1 expression levels measured by qPCR in the injected cell line and in the generated subcutaneous tumors.

F–H Number of Ki67-positive (F), number of γH2AX-positive (G), and number of active caspase-3-positive (H) cells per field in the subcutaneous tumors.

I Representative images of aberrant giant nuclei and anaphase bridges in the Trf1-downregulated subcutaneous tumors compared to the normal nuclei of control tumors.

J TRF1 immunofluorescence shows the downregulation of Trf1 in lung tumors of the mice intravenously injected with control and Trf1-downregulated cells.

K Tumor area measured in the lungs of the mice intravenously injected with control and Trf1-downregulated cells.

L Representative images of the lungs colonized by control and Trf1-downregulated cells, respectively.

M–O Number of Ki67-positive (M), number of γH2AX-positive (N), and number of active caspase-3-positive (O) cells per field in the lung tumors.

P Trf1 expression levels measured by qPCR in the A549 cell line infected either with sh-scrambled or sh-Trf1.

Q Growth of A549-derived tumors.

Data information: Error bars represent standard error. The number of mice and tumors analyzed per condition is indicated. t-test was used to assess statistical significance.
Therapeutic inhibition of TRF1 impairs the growth of p53-deficient K-RasG12V-induced lung cancer by induction of telomeric DNA damage. (2015)
Figure 6

Systemic Trf1 depletion in healthy tissues does not compromise tissue function nor organism viability

Trf1lox/loxhUBC-CreERT2 genetic mouse model.

Trf1 expression levels in the indicated tissues of wild-type and Trf1lox/lox
hUBC-CreERT2 mice subjected to a tamoxifen-containing diet for 7 weeks.

Representative images of TRF1 immunofluorescence and quantification of the percentage of TRF1-positive cells in skin and intestine sections of wild-type and Trf1lox/lox
hUBC-CreERT2 mice subjected to a tamoxifen-containing diet for 7 weeks.

Survival curve of wild-type and Trf1lox/lox
hUBC-CreERT2 mice subjected to a tamoxifen-containing diet for 7 weeks.

Quantification of the histological alterations observed in tamoxifen-treated Trf1lox/lox
hUBC-CreERT2 mice and 4 months after tamoxifen retrieval compared to their wild-type counterparts.

Quantification of blood cell populations in wild-type and Trf1lox/lox
hUBC-CreERT2 mice subjected to a tamoxifen-containing diet for 7 weeks and after 3 weeks and 4 months of tamoxifen retrieval.

Trf1 expression levels in blood, intestine, skin, and bone marrow of Trf1lox/lox
hUBC-CreERT2 mice subjected to a tamoxifen-containing diet either for 7 weeks or for 6 months and after 3 weeks tamoxifen retrieval compared to wild-type mice.

Data information: Error bars represent standard error. The number of mice analyzed per genotype is indicated in each case. t-test was used to assess statistical significance. TMX, tamoxifen.
Therapeutic inhibition of TRF1 impairs the growth of p53-deficient K-RasG12V-induced lung cancer by induction of telomeric DNA damage. (2015)
Figure 7

Efficient chemical inhibition of TRF1 telomere binding by compounds ETP-47228 and ETP-47037 in mouse lung adenocarcinoma-derived cells

Quantification of TRF1 levels by immunofluorescence in lung tumor-derived cell line treated with DMSO, with 10 μM ETP-47228 (24 h), and with 10 μM ETP-47037 (48 h). Representative images are shown to the right.

Quantification of γH2AX levels by immunofluorescence in lung tumor-derived cell line treated with DMSO, with ETP-47228 (24 h), and with ETP-47037 (48 h). Representative images are shown to the right.

Quantification of telomere-induced foci (TIFs) by double immunofluorescence with anti-RAP1 and anti-γH2AX antibodies. Representative images are shown to the right. White arrowheads: colocalization of γH2AX and RAP1.

Effect of different ETP-47228 and ETP-47037 concentrations during 24 h on proliferation in lung tumor-derived cell line relative to the growth of DMSO-treated cells.

Tumor growth quantification in allograft model ETP-47037 or with ETP-47228.

Data information: The data represent the mean values of two to three independent experiments (A–D). Error bars represent standard errors. t-test was used to assess statistical significance.
Therapeutic inhibition of TRF1 impairs the growth of p53-deficient K-RasG12V-induced lung cancer by induction of telomeric DNA damage. (2015)
Figure 8

“In vivo” treatment with ETP-47037 compound blocks the progression of lung carcinoma

Schematic representation of the ETP-47037 treatment protocol. Mice with already developed lung carcinomas were subjected to computerized tomography (CT) measurements before the start of the treatment. ETP-47037 was given at a dose of 75 mg/kg body weight by oral gavage 8 days out of the ten that the experiment lasted as indicated. At the end of the treatment, a CT was performed for quantification of tumor area and mice were sacrificed for further histological and molecular analysis. Control mice were similarly treated with vehicle.

Quantification of tumor growth relative to initial tumor size. Representative CT images are shown to the right. The white arrowhead indicates a tumor within a highly inflammatory region.

Quantification of TRF1 levels by immunofluorescence in intestine and lung tumors samples of mice treated with vehicle or with ETP-47037 for 10 days. Representative images are shown to the right (n = 4).

Number of cells showing γH2AX foci in intestine and lung tumors samples of mice treated with vehicle or with ETP-47037 for 10 days. Representative images are shown to the right (n = 4).

Data information: The data represent the mean values obtained from three mice in each group. Error bars represent standard errors. t-test was used to assess statistical significance.
Therapeutic inhibition of TRF1 impairs the growth of p53-deficient K-RasG12V-induced lung cancer by induction of telomeric DNA damage. (2015)
Figure 9

“In vivo” treatment with ETP-47037 compound does not compromise tissue viability

A–C Quantification of the number of (A) Ki67-, (B) pan-nuclear p-H3 pattern-, and (C) foci p-H3 pattern-positive cells in untreated and ETP-470037-treated lung carcinomas. The data represent the mean values obtained for three mice in each group. Error bars represent standard errors.

D Representative Ki67 and p-H3 images.

E Representative H&E images of intestine samples corresponding to untreated and ETP-47037-treated animals. High-magnification images are shown to the right indicating the presence of normal mitosis, giant multinucleated and aberrant mitotic figures.

F Representative H&E images of bone marrow and skin samples corresponding to untreated and ETP-47037-treated animals. High-magnification images are shown indicating the presence of necrosis, hemosiderosis, multinucleated cells, and giant nuclei. Bone marrow showed moderated aplasia.

G Telomere length in untreated and ETP-47037-treated lung tumor samples. Representative images are shown to the right.

Data information: t-test was used to assess statistical significance.
A Protein Palmitoylation Cascade Regulates Microtubule Cytoskeleton Integrity in Plasmodium (2020)
Figure 1

Figure 1. Parasites lacking both isp1 and isp3 genes have impaired zygote to ookinete differentiation

A In vitro differentiation to mature ookinete of wildtype (WT), Δisp1, Δisp3, and Δisp1/3 parasites. Values are means ± SD (n=4 biological replicates); two-tailed t test, *, P<0.05, **, P<0.01, ***, P<0.001, ns: not significant.

B Giemsa staining of zygotes/ookinetes from in vitro culture. Upper diagrams indicate morphological changes from zygote to ookinete. Black arrow indicates the apical. Scale bar = 5 μm.

C Time-course analysis of ookinete differentiation from in vitro culture. Values are means ± SEM (n=3 biological replicates), two-tailed t test, ***, P<0.001.

D In vivo ookinete differentiation in midgut of the infected mosquitos. Right panel indicates the parasite smear stained with Giemsa solution. Values are means ± SEM (n=3 biological replicates), two-tailed t test, **, P<0.01. Scale bar = 5 μm.

E Number of oocysts in mosquito midgut 7 days post blood feeding. n is the number of mosquitoes in each group. The horizontal line shows the mean value of each group, Mann-Whitney test, ****, P<0.0001. Right panel shows mosquito midguts stained with 0.5% mercurochrome. Scale bar = 50 μm. Three biological replicates performed.

F Formation of salivary gland sporozoites in mosquitoes 14 days post blood feeding. In each group, at least twenty blood-fed mosquitoes were counted. Values are means ± SEM (n=3 biological replicates), two-tailed t test, **, P<0.01.

G Western blot of ISP1 and ISP3 in zygote of the Δisp1/3 parasites episomally complemented with 3V5-tagged isp1 or 6HA-tagged isp3 genes from either P. yoelii or P. falciparum. P28 as loading control. The DTS (isp1::3V5;isp3::6HA) is a doubly-tagged parasite described in Figure 2B.

H IFA analysis of tagged ISP1 and ISP3 in zygotes of the complemented parasites. Nuclei are stained with Hoechst 33342. Scale bar = 5 μm.

I In vitro ookinete differentiation of the complemented parasites. Values are means ± SEM (n=3 biological replicates), two-tailed t test, **, P<0.01, ***, P<0.001.

J Proposed model for ISP1 and ISP3 during zygote to ookinete differentiation.