EMBO/soda-nel huggingface dataset:
Effector-dependent activation and oligomerization of plant NRC class helper NLRs by sensor NLR immune receptors Rpi-amr3 and Rpi-amr1 (2022)
Figure 3

Figure 3. Rpi-amr3 and Rpi-amr1 are not present in the oligomerized NRC2 protein complex.

A. Experimental design for 2D-PAGE (blue native-PAGE/SDS-PAGE). Agro-infiltrated nrc2/3/4 knockout N. benthamiana plants were collected at 3dpi for protein extraction. Protein extracts were loaded on blue native-PAGE (1D) to separate high molecular weight (HMW) protein complexes (hypothesized as a pentamer) from low molecular weight (LMW) protein complexes. Subsequently, blue native-PAGE gels were loaded on SDS-PAGE (2D) for separation of protein complexes into individual proteins.

B. NRC2EEE and Rpi-amr3 migration in the absence of effector. N. benthamiana nrc2/3/4 knockout plants were transiently infiltrated with Rpi-amr3-HF, NRC2EEE-Myc and GUS-V5 followed by 2D-PAGE. NRC2EEE-Myc proteins (*, red) and Rpi-amr3-HF proteins (*, blue) is indicated.

C. NRC2EEE and Rpi-amr3 migration in the presence of effector. N. benthamiana nrc2/3/4 knockout plants were transiently infiltrated with Rpi-amr3-HF, NRC2EEE-Myc and AVRamr3-V5 followed by 2D-PAGE. NRC2EEE-Myc proteins (*, red) and Rpi-amr3-HF protein (*, blue) is indicated.

D. Re-localization of NRC2EEE-Myc upon effector-dependent activation of Rpi-amr1. Lysates of nrc2/3/4 knockout plants transiently expressing Rpi-amr1-Flag, NRC2EEE-Myc with AVRamr3-V5 or AVRamr1-V5 were fractionated into total (T), soluble (S), and pellet (P) fractions.

E. Re-localization of NRC2EEE-Myc upon effector-dependent activation of Rpi-amr3. Lysates of nrc2/3/4 knockout plants transiently expressing Rpi-amr3-HF, NRC2EEE-Myc with AVRamr3-V5 or AVRamr1-V5 were fractionated into total (T), soluble (S), and pellet (P) fractions.

Data information: Protein lysates boiled in SDS were loaded on the same gel as control of protein size (SDS input) for panels B, and C. Molecular weight markers for blue native-PAGE are labelled on top for panels B and C, and SDS-PAGE markers are labelled on the right. Ponceau S staining of rubisco large subunit serves as control for panels B and C. MPK6, which localizes mainly to cytosol, and H+-ATPase, which localize mainly to membrane fractions serve as fractionation control in panels D and E. Experiments were repeated with 3 biological replicates with similar results.
Effector-dependent activation and oligomerization of plant NRC class helper NLRs by sensor NLR immune receptors Rpi-amr3 and Rpi-amr1 (2022)
Figure 4

Figure 4. P-loop of Rpi-amr3 and NRC2EEE is required for NRC2EEE oligomerization.

A. P-loop of Rpi-amr3 is required for AVRamr3-dependent HR in N. benthamiana. Representative leaf phenotype of HR (hypersensitive response) in wild-type N. benthamiana is shown. The number of leaves tested and occurrences of HR are indicated in parentheses.

B. P-loop of NRC2 is required for AVRamr3-dependent HR in N. benthamiana nrc2/3/4 knockout plants. Representative leaf phenotype of HR is shown. The number of leaves tested and occurrences of HR are indicated in parentheses.

C. NRC2EEE-Myc requires functional P-loop of both Rpi-amr3 and NRC2 for oligomerization. Protein lysates from N. benthamiana nrc2/3/4 knockout plants expressing wild-type or P-loop mutants of Rpi-amr3 or NRC2EEE were loaded for blue native-PAGE. Membranes were immunoblotted with anti-Myc.

Data information: At least 3 biological replicates with multiple technical replicates were tested for panels A, and B. SDS-boiled samples of the protein lysates were loaded onto SDS-PAGE for control in panel C. Ponceau S staining serves as loading control. Molecular weight markers are shown on the right. Experiments were done with at least three biological replicates with similar results for panel C.
Effector-dependent activation and oligomerization of plant NRC class helper NLRs by sensor NLR immune receptors Rpi-amr3 and Rpi-amr1 (2022)
Figure 5

Figure 5. NRC4AAA-Myc is not oligomerized by activation of Rpi-amr1.

A. Cartoon depicting genetic requirement of Rpi-amr1 and Rpi-amr3-dependent activation of NRCs. Arrows are shown to illustrate which NRC helper NLR supports each sensor NLR.

B. Protein lysates expressing NRC4AAA-Myc with Rpi-amr3-HF or Rpi-amr1-Flag in the presence and absence of cognate effector were loaded on blue native-PAGE. Proteins were transiently expressed in N. benthamiana nrc2/3/4 knockout plants. Non-specific bands are indicated with *.

Data information: At least 3 biological replicates with technical replicates were tested. SDS-boiled samples of the protein lysates were loaded onto SDS-PAGE for control. Ponceau S staining serves as loading control. Molecular weight markers are shown on the right. Experiments were done with more than 3 biological replicates with similar results for panel B.
Effector-dependent activation and oligomerization of plant NRC class helper NLRs by sensor NLR immune receptors Rpi-amr3 and Rpi-amr1 (2022)
Figure 6

Figure 6. Recognized AVRamr3 alleles from different Phytophthora species can trigger oligomerization of NRC2EEE.

A. Cartoon depicting different alleles of AVRamr3 from different Phytophthora species, P. infestans, P. capsici, and P. parasitica. Response of the corresponding alleles by Rpi-amr3, and thus occurrence of HR is indicated as + (recognition) or - (no recognition).

B. NRC2EEE-Myc oligomerizes in response-dependent manner. Protein lysates from N. benthamiana nrc2/3/4 knockout transiently expressing NRC2EEE-Myc, Rpi-amr3-HF and AVRamr3 alleles from P. infestans, P. capsici, and P. parasitica were loaded on blue native-PAGE and blotted for NRC2EEE (anti-Myc).

C. Recognition is correlated with interaction and protein complex formation of AVRamr3 with Rpi-amr3. Protein extracts from Fig 5B were immunoprecipitated with anti-Flag antibody, separated on blue native-PAGE, and AVRamr3-GFP proteins of P. infestans, P. capsici, and P. parasitica were visualized.

Data information: SDS-boiled input and IP eluates were loaded onto SDS-PAGE as control and blotted for Rpi-amr3 (anti-Flag), and AVRamr3 alleles (anti-GFP). Ponceau S staining serves as loading control. Molecular markers are indicated on the right. Experiments were done with more than 3 biological replicates with similar results.
The protein expression profile of ACE2 in human tissues (2020)
Figure 1

Figure 1. ACE2 expression in human tissues based on transcriptomics. (A) Overview of the tissues and organs analyzed based on transcriptomics by the three independent consortia Human Protein Atlas (HPA), FANTOM and GTEx. In total, 16 organ systems (with several tissues comprising an organ system) were used to create a consensus normalized expression (defined as the unit NX) based on the expression levels of all three datasets. (B) ACE2 gene expression summary in human tissues in 61 different tissues and cells in NX. Cut-off for what is regarded as expressed was set to 1.0 NX.
The protein expression profile of ACE2 in human tissues (2020)
Figure 4

Figure 4. Cell type-specific localization of ACE2 in human tissues based on immunohistochemistry. Representative images of 20 tissue types and histological structures stained on consecutive sections with immunohistochemistry using two antibodies targeting human ACE2 protein (brown), and counterstained with hematoxylin (blue). Most intense antibody staining was observed in microvilli of the intestinal tract and renal proximal tubules, in membranes of gallbladder epithelium, epididymis epithelium, testicular Sertoli cells and Leydig cells, a subset of glandular cells in seminal vesicle and cytoplasm of cardiomyocytes, with HPA000288 also staining the cardiac muscle fibers, while MAB933 only showed staining in a few cells. Distinct ACE2 staining for both antibodies was also present in cornea and conjunctiva of the eye, interlobular pancreatic ducts, as well as in placental villi, both in cytotrophoblasts, syncytiotrophoblasts, and also in extravillous trophoblasts, while placenta decidua was negative. ACE2 staining could be observed at the base of ciliated fallopian tube epithelium, however only for one of the antibodies. Note that ACE2 protein expression was less prominent in the crypts of the mucosal intestinal layer. ACE2 also stained positive in endothelial cells and pericytes in several tissues, see Fallopian, thyroid, parathyroid, adrenal gland, pancreas and heart. Scale bar = 50µm. Scale bar in dashed squares = 10 µm. (Brunner = Brunner's gland, EVT = extravillous trophoblasts, endo=endothelial cells).
The protein expression profile of ACE2 in human tissues (2020)
Figure 5

Figure 5. Cell type-specific localization of ACE2 in human respiratory system based on immunohistochemistry. Representative images of human respiratory tissues, all stained on consecutive sections, with immunohistochemistry using two antibodies targeting human ACE2 protein (brown), and counterstained with hematoxylin (blue). Respiratory tissues were composed of different structures in nasal mucosa, bronchus, smaller bronchioles and lung tissue. ACE2 staining could be observed at the base of ciliated cells in both nasal mucosa and bronchial epithelium (arrowheads). Rare ACE2 staining was present in a few alveolar cells (arrows). No staining was observed in nasal squamous epithelium, bronchioles, or submucosal glands in either tissue. Gender and age are shown for all individuals. Red and green colored squares mark the positions in the TMA cores shown as magnifications. Scale bar = 50µm. Scale bar for images in dashed squares = 10 µm. (re=respiratory; sq=squamous).
The protein expression profile of ACE2 in human tissues (2020)
Figure 6

Figure 6. ACE2 expression in human tissues based on mass spectrometry and Western blot. (A) ACE2 protein abundance in different human tissues based on various studies processed by PaxDB, with expression levels presented as parts per million (ppm). The "Integrated" dataset corresponds to PaxDB estimation of average expression. (B) ACE2 protein abundance in different human tissues from ProteomicsDB, with the median expression presented in log10(iBAQ). (C) Western blot of ACE2 using five different human tissue lysates. For lung, two different lysates were used: one male (left) and one female (right).
Role of endopeptidases in peptidoglycan synthesis mediated by alternative cross-linking enzymes in Escherichia coli (2021)
Figure 4

Figure 4. MepM is essential for β-lactam resistance beyond the transition from 4→3 to 3→3 PG cross-linking. The mepM gene was expressed under the control of TIS1 or TIS2 (translation initiation) and of PrhaBAD (the L-rhamnose-inducible promoter of vector pHV30). Growth of BW25113(ycbB, relA') ΔmepM harboring the pHV30 derivatives was tested on BHI agar supplemented with ceftriaxone 8 µg/ml, IPTG 40 µM (induction of ycbB) and L-arabinose 1% (induction of relA'). (A) For TIS2, growth around paper disks containing 1 or 2 mg of L-rhamnose indicated that induction of the expression of mepM was required for ceftriaxone resistance. In contrast, a higher level of translation from TIS1 was sufficient for ceftriaxone resistance in the absence of the inducer. (B) The experiment was repeated with bacteria pre-exposed to L-rhamnose that were harvested at the vicinity of the disk containing 2 mg of L-rhamnose. Expression of ceftriaxone resistance remained dependent on the presence of L-rhamnose indicating that the requirement for MepM is not transient. The diameter of the growth zones is larger in panel (B) than in panel (A) as expected from the fact that bacteria in the inoculum used in (B) had been grown in the presence of the inducer and already contained MepM. In (A) growth is only possible after diffusion of L-rhamnose in the medium prior to the action of ceftriaxone. At a distance from the disk, diffusion was not sufficiently rapid to observe resistance.
Melanopsin elevates locomotor activity during the wake state of the diurnal zebrafish (2022)
Figure 1

Figure 1. melanopsin expression in the brain and knockout strategy.

(A_B) In situs on adult brain sections reveal that (A) opn4.1 and (B) opn4xb are coexpressed in many brain domains including the epiphysis cerebri (indicated with E) or pineal. A horizontal line through a representation of the brain above the sagittal section indicates the location within the brain. Scale bar: 1.0 mm. The brain domains in which all five melanopsins are expressed are presented in Figure EV1 and Table EV1.

(C) The TALEN genome editing technique was applied for site-directed mutagenesis. A 5 bp deletion was introduced proximal to the start codon of opn4.1, resulting in a frame shift and STOP codon. The grey box indicates the coding sequence, the red line indicates the location of the premature STOP codon and the grey triangle dispays the wild-type sequence aligned with the mutated sequence.

(D) An 8 bp deletion was introduced in the second exon (grey box) of the opn4xb gene resulting in a premature STOP codon.

(E) Model of the seven transmembrane G-protein coupled photoreceptor shows the light absorbing molecule, the chromophore retinal (yellow), bound to helix 7 (blue). The red crosses indicate where the Melanopsins in the mutants are truncated: the mutated Opn4.1 has lost all transmembrane helices and the mutated Opn4xb ends after the second transmembrane helix. From the structure-function relationship of the photoreceptor one can deduce that neither of the mutant Melanopsins is functional.
Paladin is a phosphoinositide phosphatase regulating endosomal VEGFR2 signalling and angiogenesis (2020)
Figure 1

Figure 1. Paladin is a lipid phosphatase.

(a) Lipid phosphatase activity of Paladin, wild-type and phosphatase-dead C/S mutant, towards PI(4,5)P2 and PI(3,4,5)P3 substrates. Positive control; wild-type phosphatase and tensin homolog (PTEN); negative control; lipid phosphatase-dead C124S PTEN. Mean±SEM, Paired t-test, n=3 biological replicates.

(b) Representative confocal image of HDMEC stained for Paladin (green), Vascular Endothelial Cadherin (VEC, red) and nuclei (DAPI, blue), scale bar: 10 μm. White boxes in the upper image are showed at higher magnification below, scale bar: 3 µm.

(c) HDMEC stained for VEGFR2 (green), Paladin (red) and nuclei (DAPI, blue) and with or without stimulation of 50 ng/ml VEGF-A for 0, 2 or 10 min. Scale bar: 10 µm.

(d) Quantification of VEGFR2-Paladin co-staining before and after VEGF-A stimulation as shown in (C). Mean±SEM, One-way ANOVA. n=3 biological replicates.

(e) HDMEC analysed using Proximity Ligand Assay (PLA) for Paladin and VEGFR2. Green dots indicate complex formation, VE-cadherin (red) and nuclei (DAPI, blue). Cells stimulated with 50 ng/ml VEGF-A for 0, 2 or 10 min. Scale bar: 10 µm.

(f) Quantification of (e), the number of PLA Paldin-VEGFR2 complexes per cell at 0, 2 and 10 minutes after VEGF-A stimulation (top). Quantification of (e), average distance for the PLA complexes to the nearest VE-cadherin positive junction at 0, 2 and 10 minutes after VEGF-A stimulation (bottom). Mean±SEM, One-way ANOVA. n=3 biological replicates.

(g) HDMEC stained for Paladin (green) and EEA1 (red), colocalization in yellow, and nuclei (DAPI, blue) after VEGF-A stimulation for 0, 2 or 10 minutes, a representative image from a single confocal plane is shown, scale bar: 10 µm. White boxes in the upper image are showed at higher magnification below, scale bar 3 µm. See Appendix Figure S1 for siRNA PALD1 knock-down controls.

(h) Quantification of (g), EEA1/Paladin double positive particles per field of view at 0, 2 and 10 minutes after VEGF-A stimulation. Mean±SEM, One-way ANOVA. n= 3 biological replicates.

Data information: *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.
Paladin is a phosphoinositide phosphatase regulating endosomal VEGFR2 signalling and angiogenesis (2020)
Figure 2

Figure 2. Paladin regulates VEGFR2 internalization and endosomal trafficking.

(a) Cell surface VEGFR2 levels detected by cell surface biotinylation, using thiol-cleavable sulfo-NHS-SS-biotin, of HDMEC transfected with PALD1 siRNA (#1 and #2) or non-targeting control ("c") siRNA, followed by VEGF-A stimulation (50 ng/ml) for indicated time periods. Total lysates (input) and streptavidin (SA) pull down, immunoblotted for VEGFR2, Paladin, and actin. 'No biotin ctrl', cells not treated with sulfo-NHS-SS-biotin.

(b) Quantification of data in (a). VEGFR2 surface levels (data pooled for the indicated time points) normalized to total VEGFR2 levels and compared between control and siRNA treated HDMEC. n=4 for each time point, biological replicates, Mean±SEM.

(c) Internalized pool of VEGFR2 after VEGF-A treatment (50 ng/ml) of non-transfected HDMEC ("NT") or HDMEC transfected with PALD1 siRNA (#1 and #2) or non-targeting control siRNA ("c"). Cell surface biotinylation was performed prior to VEGF-A stimulation and at indicated time points, remaining cell surface biotin was stripped and the internalized pool of VEGFR2 was collected by SA pull down. Immunoblotting of the total lysate (input) and SA pull down fraction for VEGFR2, Paladin and actin.

(d) Quantification of data in (c). Data were normalized to total VEGFR2 levels in the lysate after subtraction of signals in biotinylated and stripped samples. Mean±SEM, unpaired t-test for indicated time points, normalized to cntrl siRNA sample. n=3 for each time point, biological replicates.

(e) Analysis of EEA1 and VEGFR2 vesicles following PALD1 knockdown. Representative images of VEGFR2 (green)/EEA1 (red) double-positive (yellow) vesicles in negative control siRNA, and PALD1 KD#2 siRNA-silenced HDMEC at 0, 2 and 10 min of VEGF-A stimulation (50 ng/ml). DAPI in blue, scale bar: 10 µm. Inset shows only EEA1 channel, scale bar: 10 µm.

(f) Quantification of (e), number of EEA1 positive (top) or VEGFR2-EEA1 double-positive vesicles (bottom) per field of view. Mean±SEM, Two-way ANOVA, n=3 biological replicates.

(g) HDMEC stained for PI(4,5)P2 (cyan), VE-cadherin (red) and Paladin (yellow in inset) following treatment using negative control or PALD1 (KD#2) siRNA. VEGF-A stimulation for 0, 2 or 10 minutes (50 ng/ml). DAPI in blue. Scale bar: 10 µm

(h) Quantification of (g), Total PI(4,5)P2 signal (top) or intracellular PI(4,5)P2 not overlapping with VE-cadherin (bottom). Mean±SEM, two-way ANOVA, n=3 biological replicates.

Data information: *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.
Paladin is a phosphoinositide phosphatase regulating endosomal VEGFR2 signalling and angiogenesis (2020)
Figure 3

Figure 3. Paladin regulates VEGFR2 signalling.

(a-d) Signalling downstream of VEGF-A/VEGFR2 assessed in HDMEC, untreated (NT, non-transfected), transfected with non-targeting siRNA (c/cntrl), or with two different PALD1-targeting siRNAs (KD#1 and KD#2), and treated with VEGF-A for 0-10 and 0-20 min, respectively. Immunoblotting of cell lysates for phosphorylated (p) VEGFR2 (pY1175), total VEGFR2, phosphorylated Erk1/2 (pT202 and pY204), and total Erk. Actin served as loading control (a,c). PALD1 knockdown was verified by blotting for Paladin. Quantification of pVEGFR2 (normalized to total VEGFR2) (b) and pErk1/2 (normalized to total Erk1/2) (d). Mean±SEM, two-way ANOVA, n=3 biological replicates.

(e-f) Immunoblotting on total heart lysates from adult Pald1+/+ and Pald1-/- mice, tail-vein injected with VEGF-A (0.25µg/g body weight) or PBS for the indicated time points, for Paladin, phosphorylated and total levels of VEGFR2, Erk1/2 and β2-microglobulin (β 2-MG, loading control). (f) Quantification of pVEGFR pY1173 normalized to total VEGFR2 (n=3), total VEGFR2 levels normalized to total loading control (n=4), pT202/pY204 Erk1/2 normalized to Erk1/2 (n=5). Mean±SEM, multiple t-test with Holm-Sidak correction (total VEGFR2), two-way ANOVA (others).

Data information: *p < 0.05, **p < 0.01.
Paladin is a phosphoinositide phosphatase regulating endosomal VEGFR2 signalling and angiogenesis (2020)
Figure 4

Figure 4. Retinal vascular phenotype in Pald1-/- mouse.

(a, b) Delayed vascular outgrowth and hyperdense vascular front in isolectinB4-stained P5 retina from Pald1-/- mouse compared with Pald1+/+ (a). Orange arrow indicates radial expansion of the vascular plexus in the Pald1+/+ retina. Scale bar: 1 mm. Quantification of radial expansion (b) as shown in (a) normalized to wild type litter mates. Mean±SEM, unpaired t-test. n=14 litters, 18 wild type and 23 knock-out pups.

(c, d) Increased filopodia number in Pald1-/- mouse retina at vascular front, visualized by isolectinB4 staining (c). Scale bar: 50 μm. Quantification of filopodia per 100 µm at the vascular front (d). Mean±SEM, unpaired t-test. n=3 litters, 7 wild type and 5 knock-out pups.

(e, f) P5 retina vascular front (isolectinB4, green) and endothelial nuclei (Erg, red) (e). Scale bar: 100 μm. Magenta stippled square area quantified in (f). Vascular density was determined in the capillary bed (left) (11 wild type, 12 knock-out) and in area around veins (right) (10 wild type, 9 knock-out, biological replicates). Mean±SEM, unpaired t-test.

(g, h) pT202/pY204 Erk1/2 immunostaining (black) in Pald1+/+ and Pald1-/- P5 pups. IsolectinB4 (white) visualizes the entire vasculature. Scale bar: 100 μm. Quantification of pT202/pY204 Erk1/2 area (h) as in the black stippled square in (g) (400 µm from the retina rim), normalized to isolectinB4 area. Mean±SEM, unpaired t-test. n=5 litters, 14 wild type and 10 knock-out retinas per genotype.

(i) Quantitative real-time PCR analysis of P4-P5 retinas from Pald1+/+ and Pald1-/- pups. Ccnd1 transcript levels, normalized to Gapdh. Mean±SEM, unpaired t-test. n=10 Pald1+/+ and 14 Pald1-/- pups.

(j) Cyclin D1 (black) and isolectinB4 (white) staining of P5 retinas from Pald1+/+ and Pald1-/-pups. Scale bar: 100 µm.

(k) Quantification of number of Cyclin D1 positive nuclei normalized for isolectinB4 area in retinas from Pald1+/+ and Pald1-/ P5 pups. Mean±SEM, unpaired t-test. n=7 Pald1+/+ and 6 Pald1-/- pups.

(l) Quantification of relative radial expansion in vehicle (n=2 litters, 5 Pald1+/+ and 4 Pald1-/- pups) and MEK inhibitor (U0126)-treated pups (n=4 litters, 7 Pald1+/+ and 5 Pald1-/- pups). MEK inhibitor/vehicle was administered twice at 12h interval at P4 and eyes collected at P5. Each dot is one mouse. Mean±SEM, one-way ANOVA, n=4-7.

(m) Quantification of the tip cell number in vehicle- (n=3 litters, 5 Pald1+/+ and 5 Pald1-/- pups) and MEK inhibitor (U0126)-treated pups (n=3 litters, 5 Pald1+/+ and 4 Pald1-/- pups). MEK inhibitor/vehicle administered twice at P5 at 2-h intervals, and eyes collected 2 h after the second injection. Each dot is one mouse. Mean±SEM, one-way ANOVA.

Data information: *p < 0.05, **p < 0.01, ***p < 0.001.