EMBO/soda-nel huggingface dataset:
The Arabidopsis CERK1-associated kinase PBL27 connects chitin perception to MAPK activation (2016)
Figure 6

Figure 6. The phosphorylation sites of MAPKKK5 are required for chitin-induced MAPK activation.

A Chitin-induced MAPK activation in a mapkkk5 mutant (Line 1) expressing MAPKKK56xA. MAPK activity was detected using immunoblots with α-pMAPK.

B Chitin-induced callose deposition in the MAPKKK56xA plant (Line 1). Seedlings were analyzed at 18 hours after treatment with 10 μM chitin. Data are means ±SD from three independent biological replicates, where each biological replicate consists of two technical replicates. The asterisks indicate statistically significant differences from the WT controls by Student’s t-test (P < 0.05).

C Detection of MAPKKK5K375M -GFP and MAPKKK5K375M, 6xA-GFP transiently expressed in Arabidopsis protoplasts.

D Visualization of interaction between PBL27 and MAPKKK5K375M, 6xA by BiFC analysis in Arabidopsis protoplasts. Venus fluorescence indicates interaction between PBL27 and MAPKKK5K375M, 6xA. Scale bar=10µm.
The Arabidopsis CERK1-associated kinase PBL27 connects chitin perception to MAPK activation (2016)
Figure 7

Figure 7. PBL27 Phosphorylates MAPKKK5-KD:C in CERK1-Dependenet Manner.

A PBL27 does not interact with MAPKKK5-KD:C in a yeast two-hybrid assay.

B Addition of CERK1 to an in vitro kinase assay enhances phosphorylation of MAPKKK5-KDK375M:C by PBL27. Asterisks indicate artificial bands derived from CERK1:IC.

C CERK1 does not phosphorylate the C-terminal domain of MAPKKK5 in vitro. The in vitro kinase assay was performed with recombinant proteins of GST-PBL27, MAPKKK5-C and the intracellular kinase domain of CERK1 (GST-CERK1:IC). The protein loading control was shown by staining with Coomassie Brilliant Blue. The in vitro phosphorylation reaction was carried out using [32P]γ-ATP, and the phosphorylated proteins were detected by autoradiography. Asterisks indicate artificial bands.

D The alanine substitution mutations at the residues conserved in the activation loop of reduces the phosphorylation of PBL27 by CERK1:IC. The in vitro kinase assay was carried out with recombinant proteins of GST-PBL27K112E , GST-PBL27K112E, 3xA and CERK1-IC.

E The kinase activity of GST-PBL273xA was analyzed by the in vitro kinase assay using [32P]γ-ATP.
The Arabidopsis CERK1-associated kinase PBL27 connects chitin perception to MAPK activation (2016)
Figure 8

Figure 8. MAPKKK5 Phosphorylates the activation loops of MKK4 and MKK5.

A Visualization of interaction between MAPKKK5K375M and MKK1 / MKK2 / MKK4 / MKK5 by BiFC analysis in Arabidopsis protoplasts. Venus fluorescence indicates interaction. Scale bar=10µm.

B MAPKKK5-KD phosphorylates MKK4 and MKK5 in vitro. The in vitro phosphorylation reaction was carried out using [32P]γ-ATP, and the phosphorylated proteins were detected by autoradiography.

C MAPKKK5-KD does not phosphorylate a T224A/S230A mutant of MKK4 or a T215A/S221A mutant of MKK5. The above experiments were performed three times with similar results.
The mTORC1-eIF4F axis controls paused pluripotency (2021)
Figure 1

Figure 1. mTORC1 inhibition induces paused pluripotency in mESCs in 2iL.

Schematic of the mTORC1 and mTORC2 complexes and inhibitors.

Images of mESCs treated with control (Ctrl, dimethyl sulfoxide (DMSO)), INK (INK128, 200 nM) for three days and released for three days in 2iL. Scale bar, 400 µm.

Relative proliferation of mESCs as in (B). Data are the mean values ± SD with the indicated significance (P value was calculated using two-tailed unpaired Student's t test, ***P < 0.001, n.s., not significant, also hereafter in similar experiments). n = 3 biological replicates.

Western blotting for mTORC1 and mTORC2 activity in mESCs transduced with Rptor and Rictor shRNAs.

Images of mESCs transduced with Rptor and Rictor shRNAs in 2iL. Scale bar, 400 µm.

Relative proliferation of mESCs as in (E) (mean values ± SD, P value was calculated using two-tailed unpaired Student's t test, *P < 0.05, also hereafter in similar experiments). n = 3 biological replicates.

Western blotting for mTORC1 and mTORC2 activity in Rptor WT (AAV-empty) and KO (AAV-Cre) mESCs. Samples were collected after AAV transduction for four days.

Images of Rptor WT and KO mESCs in 2iL. Scale bar, 400 µm.

Western blotting for RPTOR and the indicated pluripotency proteins in mESCs as in (H) (left), and quantification of pluripotency proteins (right) (mean values ± SD). n = 3 biological replicates.

Images of mESCs transduced with Rptor siRNA on different days in 2iL. siNC, transduced with a negative control; day 0 was the day of siRNA transfection, also hereafter in similar experiments. Scale bar, 400 µm.

Relative proliferation of mESCs as in (J) (mean values ± SD). n = 3 biological replicates.

Images of EBs on different days after INK128 release for three days. Scale bar, 400 µm.

Relative number of beating EBs on D12 as in (L) (mean values ± SD). n = 3 biological replicates.
The mTORC1-eIF4F axis controls paused pluripotency (2021)
Figure 2

Figure 2. mTOR or mTORC1 inhibition does not suppress global transcription.

Numbers of DEGs from INK128 (200 nM) treatment or Rptor KO mESCs in 2iL. FC > 1.5, FPKM mean > 1, and P < 0.05.

Hierarchical RNA-seq clustering of DEGs as in (A).

RNA-seq heatmaps of the indicated pluripotency gene sets in control and INK128 (200 nM) treated mESCs in 2iL.

RNA-seq heatmaps of indicated pluripotency gene sets in Rptor WT and KO mESCs in 2iL.

GO analysis of RNA-seq data of INK128-induced paused state mESCs as in (A).

Cell cycle analysis of INK128 (200 nM) treated mESCs in 2iL (mean values ± SD). n = 3 biological replicates.
The mTORC1-eIF4F axis controls paused pluripotency (2021)
Figure 3

Figure 3. Translation initiation complex eIF4F controls mESC self-renewal in 2iL.

Schematic of how mTORC1 regulated translation through eIF4F complex.

Images of mESCs transduced with Eif4a1, Eif4e and Eif4g1 shRNAs in 2iL. Scale bar, 400 µm.

Relative proliferation of mESCs as in (B) (mean values ± SD). n = 3 biological replicates.

Western blotting for the indicated shRNAs and pluripotency proteins as in (B) (left) and quantification (right) (mean values ± SD). n =3 biological replicates.

Images of mESCs transfected with Eif4e and Eif4g1 siRNAs on different days in 2iL. siNC was same with Figure 1J, also hereafter in similar experiments. Scale bar, 400 µm.

Relative proliferation of mESCs as in (E) (mean values ± SD). n = 3 biological replicates.

Images of EBs on different days. Scale bar, 400 µm.

Relative number of beating EBs on D12 as in (G) (mean values ± SD). n = 3 biological replicates.
The mTORC1-eIF4F axis controls paused pluripotency (2021)
Figure 4

Figure 4. 4EBP1/2 and LARP1 mediate an INK128-induced paused state in 2iL.

Knockdown efficiency of Eif4ebp1 and Eif4ebp2 shRNAs in 2iL (mean values ± SD). n = 3 biological replicates.

Images of mESCs transduced with Eif4ebp1 and Eif4ebp2 shRNAs in 2iL, alone or in combination, in control or INK128 (200 nM) treated for three days. Scale bar, 400 µm.

Relative proliferation of mESCs as in (B) (mean values ± SD, P value was calculated using two-tailed unpaired Student's t test, **P < 0.01, also hereafter in similar experiments). n = 3 biological replicates.

Knockdown efficiency of Larp1 shRNAs in 2iL (mean values ± SD). n = 3 biological replicates.

Images of mESCs transduced with Larp1 shRNAs in 2iL, in control or INK128 (200 nM) treated for three days. Scale bar, 400 µm.

Relative proliferation of mESCs as in (E) (mean values ± SD). n = 3 biological replicates.
The mTORC1-eIF4F axis controls paused pluripotency (2021)
Figure 6

Figure 6. Mitochondrial translation inhibition induces paused pluripotency in mESCs in 2iL.

OCR analysis of INK128 (200 nM) treated mESCs in 2iL.

ROS measurement in mESCs treated with INK128 or transduced with Rptor shRNAs in 2iL (mean values ± SD). n = 3 biological replicates.

Mitochondrial mass (MitoTracker green) measurement in mESCs treated with INK128 or transduced with Rptor shRNAs in 2iL (mean values ± SD). n = 3 biological replicates.

qPCR for mtDNA copy number in mESCs treated with INK128 or transduced with Rptor shRNAs in 2iL (mean values ± SD). n = 3 biological replicates.

ROS, mitochondrial mass (MitoTracker green), mtDNA copy number measurements in mESCs treated with CAP in 2iL (mean values ± SD). n = 3 biological replicates.

Images of mESCs treated with Ctrl (DMSO) or CAP (Chloramphenicol, 1 mM) for three days and released for three days in 2iL. Scale bar, 400 µm.

Relative proliferation of mESCs as in (F) (mean values ± SD). n = 3 biological replicates.

Western blotting for the indicated pluripotency proteins in mESCs as in (F) (left), and quantification (right) (mean values ± SD). n = 3 biological replicates.

Images of EBs on different days after CAP release for three days. Scale bar, 400 µm.

Relative number of beating on D12 as in (I) (mean values ± SD). n = 3 biological replicates.
The mTORC1-eIF4F axis controls paused pluripotency (2021)
Figure 7

Figure 7. Translation initiation complex eIF4F regulates both self-renewal and pluripotency in SL.

Images of mESCs transduced with Rptor or Rictor shRNAs in SL. Scale bar, 400 µm.

Relative proliferation of mESCs as in (A) (mean values ± SD). n = 3 biological replicates.

Western blotting for RPTOR, RICTOR and the indicated pluripotency proteins as in (A) (left) and quantification (right) (mean values ± SD). n = 3 biological replicates.

Images of mESCs transduced with Eif4a1, Eif4e and Eif4g1 shRNAs in SL. Scale bar, 400 µm.

Relative proliferation of mESCs as in (D) (mean values ± SD). n = 3 biological replicates.

Western blotting for EIF4A1, EIF4E, EIF4G1 and the indicated pluripotency proteins as in (D) (left) and quantification (right) (mean values ± SD). n = 3 biological replicates.

Western blotting for EIF4E, EIF4G1 and the indicated pluripotency proteins in cells with the indicated KD in the indicated culture conditions.
A bacterial effector counteracts host autophagy by promoting degradation of an autophagy component (2022)
Figure 2

Figure 2: Xanthomonas T3E XopL is suppressing autophagy

(A) RLUC-ATG8a or RLUC-NBR1 constructs were coexpressed with internal control FLUC in N. benthamiana. XopL or GFP constructs were co-infiltrated. RLUC and FLUC signals were simultaneously measured in leaf extracts at 48 h post- infiltration using the dual-luciferase system. Values represent the ratio of RLUC-ATG8a and FLUC activities to the mean of control (n=4). Middle horizontal bars of boxplots represent the median, the bottom and top represent the 25th and 75th percentiles, whiskers extend to at most 1.5 times the interquartile range. Statistical significance (P < 0.01) was shown by Student's t-test. The experiment was repeated more than 3 times by with similar results.

(B) Immunoblot analysis of NBR1 and ATG8 protein levels in N. benthamiana plants transiently expressing GFP-XopL or GFP control at 2dpi verified with an anti-GFP antibody (same blot was split for visualization purpose). Ponceau Staining (PS) served as a loading control. The experiment was repeated at least three times with similar results.

(C) Immunoblot analysis of ATG8 protein levels in N. benthamiana plants transiently expressing XopL or GUS control at 2dpi after ConA or DMSO treatment. Expression of GFP-XopL was verified with an anti-GFP antibody, while expression of GUS-HA was confirmed with an anti-HA antibody. Ponceau Staining (PS) served as a loading control. The experiment was repeated twice with similar results.

(D) GFP-ATG8g-labeled autophagosomes were quantified from plants infected with Xcv or Xcv ΔxopL at 2 dpi in the presence or absence of ConA. Puncta were calculated from z-stacks (15) of n=12 individuals using ImageJ. Middle horizontal bars of boxplots represent the median, the bottom and top represent the 25th and 75th percentiles, whiskers extend to at most 1.5 times the interquartile range. Statistical significance (** P < 0.01, *** P < 0.001) was determined by one way ANOVA. The experiment was repeated twice with similar results.

(E) Immunoblot analysis of NBR1 and ATG8 protein levels in Xcv ΔxopQ, Xcv ΔxopQ ΔxopL or mock infected N. benthamiana plants at 2dpi. Ponceau Staining (PS) served as a loading control. The experiment was repeated twice with similar results.

(F) RLUC-ATG8a or RLUC-NBR1 constructs were coexpressed with internal control FLUC in N. benthamiana. Xcv ΔxopQ and Xcv ΔxopQ ΔxopL were co-infiltrated with Agrobacteria containing the respective constructs. RLUC and FLUC activities were simultaneously measured in leaf extracts at 48 h post- infiltration using the dual-luciferase system. Values represent the ratio of RLUC-ATG8a and FLUC activities (n=4). Middle horizontal bars of boxplots represent the median, the bottom and top represent the 25th and 75th percentiles, whiskers extend to at most 1.5 times the interquartile range. Statistical significance comparing Xcv ΔxopQ and Xcv ΔxopQ ΔxopL values (***P < 0.001) was revealed by Student's t-test. The experiment was repeated 3 times with similar results.
A bacterial effector counteracts host autophagy by promoting degradation of an autophagy component (2022)
Figure 4

Figure 4. XopL mediates the proteasome degradation of SH3P2 via its E3 ligase activity

(A) SH3P2-GFP was transiently coexpressed together with GUS-HA and GFP-XopL in N. benthamiana using agroinfiltration. At 42 hpi, 200 μM MG132 was infiltrated into A. tumefaciens-inoculated leaves, and leaf material was collected 48 hpi. Expression of SH3P2-GFP (lower band) and GFP-XopL (upper band) was detected using an anti-GFP antibody. GUS-HA expression was confirmed with an anti-HA antibody. Ponceau S staining serves as a loading control. The experiment was repeated three times with similar results.

(B) In vitro ubiquitination assay reveals ubiquitination of SH3P2 by XopL. GST-XopL, and MBP-SH3P2 were tested using the Arabidopsis His-AtUBA1 and His-AtUBC8. Lanes 2 to 4 are negative controls. Proteins were separated by SDS-PAGE and detected by immunoblotting using the indicated antibodies. Asterisks indicate bands that are not ubiquitinated an present in controls. The experiment was repeated twice with similar results.

(C) SH3P2-GFP was transiently coexpressed together with GFP, GFP-XopL and GFP-XopL ΔE3 in N. benthamiana using agroinfiltration. GFP protein levels were detected with an anti-GFP antibody. Ponceau S staining serves as a loading control. The experiment was repeated three times with similar results.

(D) Immunoblot analysis of ATG8 protein levels in N. benthamiana plants transiently expressing GFP-XopL, GFP-XopL ΔE3 or GFP control at 2dpi. Expression of binary constructs was verified with an anti-GFP antibody. GFP blot was split for visualization purpose. Ponceau Staining (PS) served as a loading control. The experiment was repeated twice with similar results.

(E) RLUC-ATG8a constructs were coexpressed with internal control FLUC in N. benthamiana. GFP-XopL, GFP-XopL ΔE3 or GFP control were co-infiltrated together with RLUC/FLUC mixture. Renilla and Firefly luciferase activities were simultaneously measured in leaf extracts at 48 hpi using the dual-luciferase system. Values represent the ratio of RLUC-ATG8a and FLUC activities (n=4). Middle horizontal bars of boxplots represent the median, the bottom and top represent the 25th and 75th percentiles, whiskers extend to at most 1.5 times the interquartile range. Statistical significance (* P < 0.5, ** P < 0.01) was revealed by Student's t-test. The experiment was repeated 3 times.
Recessive mutations in EPG5 cause Vici syndrome, a multisystem disorder with defective autophagy (2012)
figf2

Transverse sections from muscle biopsies from subject 3.1 (top) and a normal control (bottom) were stained with monoclonal antibody against NBR1 or polyclonal antibody to MURF2 and counterstained with antibody to the M-band titin, titin M8. Accumulation of NBR1 in puncta (autophagosomes, arrowheads) and of MURF2, as well as fiber inhomogeneity with marked fiber atrophy, characterizes Vici syndrome muscle. Numerous fibers of very small cross-sectional area (arrows) with high content of MURF2 and NBR1 puncta are frequently seen. Scale bar, 10 μm.
Recessive mutations in EPG5 cause Vici syndrome, a multisystem disorder with defective autophagy (2012)
figf3

Accumulation of autophagy adaptors NBR1 and SQSTM1 and the phagophore membrane component LC3 is induced in fibroblasts from two controls and a subject with Vici syndrome (subject 4.1) after 12 h of treatment with rapamycin or dual treatment with rapamycin and bafilomycin.
Recessive mutations in EPG5 cause Vici syndrome, a multisystem disorder with defective autophagy (2012)
figf4

In control fibroblasts subjected to 6 h of bafilomycin treatment, lysosomal structures were detected by staining with monoclonal antibody to LAMP1. Numerous LC3-positive autophagosomes are found engulfed by the LAMP1-positive vesicular structures (arrowheads). In contrast, fibroblasts from individuals with Vici syndrome consistently show smaller LC3-positive puncta that only sporadically colocalize with LAMP1, with many isolated LC3-positive puncta (arrows). Note that LC3 signal in Vici syndrome cells occurs mostly at the rim of LAMP1-positive structures, not centrally. Scale bar, 5 μm.
Adherens junction remodelling during mitotic rounding of pseudostratified epithelial cells (2020)
Figure 1

Figure 1. Epithelial cells round up and down-regulate adherens junctions at mitosis.

(A) Dynamic regulation of fluorescently tagged Beta-catenin/Armadillo (Arm-GFP) during mitosis in the growing fly wing epithelium. Notice down-regulation of Arm-GFP as cells round up for mitosis (arrow). Scale bar ~2µm. n>10 independent biological replicates.

(B) Dynamic regulation of fluorescently tagged E-cadherin (E-cad-GFP) during mitosis in the growing fly wing epithelium. Notice down-regulation of E-cad-GFP as cells round up for mitosis (arrow). Scale bar ~2µm. n>10 independent biological replicates.

(C) E-cad-GFP remains downregulated in cells arrested in mitosis with Colchicine. Scale bar ~2µm. n>10 independent biological replicates.

(D) Quantification of the degree of down-regulation of Arm-GFP and E-cad-GFP at the junction between mitotic cells and their interphase neighbours (n > 10 independent samples per cell cycle stage). Mean +/- 1 S.D. shown.

(E) Two adjacent mitotic cells show no detectable Arm-GFP at the junction between them. Scale bar ~2µm.

(F-I) Electron microscopy cross-sections of wing epithelial cells at interphase, prophase, prometaphase and telophase. Notice adherens junctions (dark staining, highlighted in insets) are downregulated as cells round up for mitosis. Scale bars ~1µm (low mag.) and ~10nm (high mag.). n>10 independent biological replicates. Asterisks indicate a single mitotic cell. Green arrowheads indicate adherens junctions.
Adherens junction remodelling during mitotic rounding of pseudostratified epithelial cells (2020)
Figure 2

Figure 2. Rho-kinase activates acto-myosin contractility to induce downregulation of adherens junctions at mitosis.

(A) Phosphorylated Myosin-II Regulatory Light Chain (p-MLC; purple) detected by antibody staining reveals high levels around the cortex of rounded mitotic cells in wild-type wing epithelial cells in the anterior (A) compartment, but not in Rho-kinase (Rok) RNAi expressing cells of the posterior (P) compartment (GFP-positive; green). Scale bar ~10µm. n>10 independent biological replicates.

(B) High-magnification view of single mitotic cell stained for p-MLC (purple) and E-cadherin (green), with metaphase plate chromosomes stained by DAPI (blue). Scale bar ~1µm. n>10 independent biological replicates.

(C) High-magnification view of single UAS.Rok-RNAi expressing mitotic cell stained for p-MLC (purple) and E-cadherin (green), with metaphase plate chromosomes stained by DAPI (blue). Note loss of cortical p-MLC staining (purple). Scale bar ~1µm. n>10 independent biological replicates.

(D) Expression of constitutively active Rho-kinase (UAS.Rok-CA) in the posterior compartment of the wing epithelium is sufficient to elevate p-MLC and reduce E-cadherin immunostaining. Zoom (right) shows high p-MLC and low E-cad in both a mitotic cell and its neighbours. Scale bar ~10µm. n>10 independent biological replicates.

(E) MyoII RLC-GFP (MyoII-GFP) accumulates at the cortex as cells round up for mitosis and later appears at the cleavage furrow during cytokinesis (basal section, diagrammed left). Treatment with the Rho-kinase inhibitor Y-27632 inhibits mitotic accumulation of MyoII-GFP. Quantification shown right (n = 8 independent samples per genotype). Scale bar ~1µm. Mean +/- 1 S.D. shown.

(F) E-cad-GFP is downregulated as cells round up for mitosis (apical section, diagrammed left). Treatment with the Rho-kinase inhibitor Y-27632 inhibits mitotic downregulation of E-cad-GFP. Extended treatment with Y-27632 leads to a general defect in all interphase and mitotic cells in which apical surface areas enlarge and E-cad-GFP is generally lost. Arrow indicates mitotic cell cortex at maximal rounding. Quantification shown right (n = 6 independent samples per genotype; mean +/- 1 S.D. shown). Scale bar ~1µm.
Adherens junction remodelling during mitotic rounding of pseudostratified epithelial cells (2020)
Figure 3

Figure 3. Loss of RhoGEF ECT2/Pbl prevents mitotic rounding and causes cell extrusion and apoptosis

(A) Nuclear localised Pbl-GFP becomes cytoplasmic in mitotic cells, co-incident with Rok-GFP relocalisation to the plasma membrane. Scale bar ~1µm. n > 5 independent biological replicates.

(B) Phosphorylated Myosin-II Regulatory Light Chain (p-MLC; purple) detected by antibody staining reveals high levels around the cortex of rounded mitotic cells in wild-type wing epithelial cells, but not those expressing UAS.pbl-RNAi. Phospho-Histone H3 (pH3) staining (green) marks mitotic cell chromosomes. Quantification shown right (n > 10 independent samples per genotype; mean +/- 1 S.D. shown). Scale bar ~1µm.

(C) A stripe of expression of ptc.Gal4 UAS.pbl-RNAi in the wing disc leads to both enlarged cells and many basally extruded cells that undergo apoptosis, marked by pyknotic nuclei and loss of aPKC/Dlg staining. Scale bars ~20µm. n > 6 independent biological replicates.
Adherens junction remodelling during mitotic rounding of pseudostratified epithelial cells (2020)
Figure 4

Figure 4. Loss of RacGAP1/MgcRacGAP/CYK4/Tum prevents mitotic rounding and causes cell extrusion and apoptosis

(A) Nuclear localised Tum-GFP becomes cytoplasmic in mitotic cells, including a distinct plasma membrane localisation (arrows). Note also localisation to the cytokinetic ring (asterisk) and remnant mid-bodies/ring canals in interphase cells. Quantification shown on the right (n > 7 independent samples per genotype; mean +/- 1 S.D. shown). Scale bars ~2µm.

(B) A stripe of expression of ptc.Gal4 UAS.tum-RNAi in the wing disc leads to basally extruded cells that undergo apoptosis, marked by Dcp1 immunostaining, similar to UAS.pbl-RNAi. Quantification shown on the right (n > 10 independent samples per genotype; mean +/- 1 S.D. shown). Scale bars ~20µm.

(C) Phosphorylated Myosin-II Regulatory Light Chain (p-MLC; red) detected by antibody staining reveals high levels around the cortex of rounded mitotic cells in wild-type wing epithelial cells, but not those cells homozygous mutant for tum347, marked by expression of GFP in single-celled clones (MARCM system). pH3 staining (blue) marks mitotic cell chromosomes. Arrow indicates a GFP-positive single cell tum347 mutant clone. Quantification shown on the right (n > 3 independent samples per genotype; mean +/- 1 S.D. shown). Scale bars ~20µm.