EMBO/soda-nel huggingface dataset:
RGI-GOLVEN signaling promotes cell surface immune receptor abundance to regulate plant immunity (2022)
Figure 3

Figure 3. RGI receptors are involved in PTI.

(A) ROS burst in WT and rgi5x after elicitation with 100 nM flg22. Shown is the kinetic of ROS production over 40 min -/+ SE, n=8 biological replicates. RLU = Relative light units. Similar results were obtained in 3 independent experiments.

(B) Ethylene production was measured in the indicated genotypes 3.5 h after elicitation with mock or 500 nM flg22. Ethylene accumulation was normalized to the average of WT responses to flg22 set as 1. Shown is the mean of n=18 biological replicates from 3 pooled experiments -/+ SD (one-way ANOVA, Tukey post-hoc test, a-b p<0.001; a-c, p<0.001; b-c, p<0.001).

(C) 5-day-old seedlings of WT and rgi5x were treated with 100 nM flg22 for 7 days before measuring fresh weight. Shown is the mean of relative fresh weight compared to the MS medium control -/+ SD, n=16 biological replicates (Students T-test, ***p<0.001). Similar results were obtained in 3 independent experiments.

(D) Mean colony-forming units (cfu) of Pto DC3000 COR- 3 days after spray infection of the indicated genotypes; n=7-8 biological replicates from 2 pooled experiments -/+ SD (one-way ANOVA, Tukey post-hoc test, a-b p<0.001; a-c p<0.001, b-c p<0.05).

(E) Ethylene production was measured in WT and rgi5x and rgi5x pRGI3::RGI3 #2 and #3 3.5 h after elicitation with 500 nM flg22. Ethylene production was normalized to the average of WT responses to flg22 set as 1. Shown is the mean of n=8 biological replicates from 2 pooled experiments -/+ SD (one-way ANOVA, Dunnett post-hoc test, a-b p<0.05).

(F) 5-day-old seedlings of the indicated genotypes were treated with 100 nM elf18 for 7 days before measuring fresh weight. Shown is the mean of relative fresh weight compared to the MS medium control -/+ SD, n=36 biological replicates from 3 pooled experiments (Students T-test, ***p<0.001).
RGI-GOLVEN signaling promotes cell surface immune receptor abundance to regulate plant immunity (2022)
Figure 4

Figure 4. RGI3 is required for GLV2 perception during PTI.

(A) Ethylene production was measured in WT and rgi5x 3.5 h after elicitation with 500 nM flg22 or co-treatment with 1 μM GLV2. Shown is the mean of n=17-24 biological replicates from 4 pooled experiments -/+ SD (one-way ANOVA, Tukey post-hoc test, a-b p<0.001; a-c, p<0.05). Ethylene production was normalized to the average of WT responses to flg22 set as 1.

(B) 5-day-old seedlings of WT and rgi5x were treated with 10 nM flg22 or co-treated with 1 μM GLV2 for 7 days before measuring fresh weight. Shown is the mean of relative fresh weight compared to the MS medium control -/+ SD, n=36-57 biological replicates pooled from 4 experiments (one-way ANOVA, Tukey post-hoc test, a-b p<0.001; a-c, p<0.01).

(C) 5-day-old seedlings of WT, rgi5x and rgi5x pRGI3::RGI3 #2 and #3 were treated with 10 nM flg22 or co-treated with 1 μM GLV2 for 7 days before measuring fresh weight. Shown is the mean of relative fresh weight compared to the MS medium control -/+ SD, n=32 biological replicates pooled from 4 experiments (Students T-test, **p<0.01).

(D) Quantitative analysis of synthetic GLV2 or GLV2-S peptide binding to RGI3ECD using microscale thermophoresis. Shown is the mean -/+ SD, n=3 technical replicates.

(E) Co-immunoprecipitation experiment upon co-expression of BAK1-HA and RGI3-GFP in N. benthamiana. IP was performed using GFP-TRAP agarose beads. Western blots for protein detection were probed with α-GFP or α-HA antibodies. Similar results were obtained in 3 independent experiments.
RGI-GOLVEN signaling promotes cell surface immune receptor abundance to regulate plant immunity (2022)
Figure 5

Figure 5. RGI3 forms a flg22 and GLV2-induced complex with FLS2.

(A) Co-immunoprecipitation experiment in 12-day-old Col-0 pFLS2::FLS2-GFP seedlings. Protein extraction was performed 10 min after mock, 1 μM flg22 or 1 μM flg22 + 1 μM GLV2 treatment. IP was performed using GFP-TRAP agarose beads. Western blots for protein detection were probed with α-GFP or α-BAK1 antibodies.

(B) Co-immunoprecipitation experiment upon co-expression of FLS2-HA with either CLV1-GFP or RGI3-GFP in N. benthamiana. IP was performed using GFP-TRAP agarose beads. Western blots for protein detection were probed with α-GFP or α-HA antibodies.

(C) Co-immunoprecipitation experiment upon co-expression of FLS2-HA, BAK1-HA and RGI3-GFP in N. benthamiana. Protein extraction was performed 30 min after mock, 1 μM flg22 or 1 μM GLV2 treatment. IP was performed using GFP-TRAP agarose beads. Western blots for protein detection were probed with α-GFP or α-HA antibodies.

Data information: Similar results were obtained in 3 independent experiments.
RGI-GOLVEN signaling promotes cell surface immune receptor abundance to regulate plant immunity (2022)
Figure 6

Figure 6. GLV2 perception by RGIs promotes maximal FLS2 abundance independent of transcription.

(A) 12-day-old WT or 35S::GLV2 8-5 seedlings were treated with mock or 1 μM GLV2 for 24h before protein extraction. Western blots were probed with α-FLS2 or α-BAK1 antibodies. CBB: Coomassie brilliant blue.

(B) Quantitative real-time PCR of FLS2 transcripts from 12-day-old seedlings treated with mock or 1 μM GLV2 for the indicated time, n=3 biological replicates. UBQ was used as a house keeping gene. Students T-test revealed no statistical difference for each of the different timepoints.

(C) Protein extraction was performed from 12-day-old seedlings of the indicated genotypes. Western blot was probed with α-FLS2 antibodies.

(D) Protein extraction was performed from 12-day-old seedlings after 1 hour treatment with mock or 50 μM MG132. Western blots were probed with α-FLS2 antibodies.

Data information: Similar results were obtained in 3 independent experiments.

Expanded View Figure legends
A dual-AAV approach restores fast exocytosis and partially rescues auditory function in deaf otoferlin knock-out mice (2018)
Figure 1

Figure 1 -Dual-AAV mediated otoferlin expression is restricted to auditory hair cells in the cochlea.

A Low magnification views of a CD1 Otof-/- organ of Corti (P23) transduced with otoferlin dual‑AAV‑TS vectors. IHCs: inner hair cells, OHCs: outer hair cells. B,C High magnification views of CD1B6F1 Otof-/- IHCs transduced with otoferlin dual‑AAV‑TS (P26) (B) and dual‑AAV‑Hyb (P26) (C) vectors. Individual eGFP and otoferlin immunostainings are depicted as color lookup tables in (A-C) with warmer colors representing higher pixel intensities. See Fig EV3 for comparison to wild-type IHCs. In (A-C), maximum intensity projections of confocal optical sections. Scale bars: 100 µm (A), 10 µm (B,C).

D Percentage of N‑ and C‑terminal otoferlin labelled IHCs in dual‑AAV‑TS (n=10 mice) and dual‑AAV‑Hyb (n=9 mice) injected CD1B6F1 Otof-/- mice (P18‑30).

E Average N-terminal and C‑terminal otoferlin immunofluorescence levels in dual‑AAV transduced Otof-/- and wild-type IHCs (P23-30). Otoferlin levels were normalized to immunofluorescence levels in non-transduced B6 wild-type IHCs for each antibody separately.

Data information: In (D), individual animals are depicted with open symbols. In (E), the number of quantified IHCs is indicated inside the bars. In (D-E), data are displayed as mean ± s.e.m. ns P>0.05; *P≤0.05; **P≤0.01;***P≤0.001, (Wilcoxon matched-pair singed rank test (D), unpaired t-test with Welch's correction (D), and Kruskal-Wallis test followed by Dunn's multiple comparison test (E)).
A dual-AAV approach restores fast exocytosis and partially rescues auditory function in deaf otoferlin knock-out mice (2018)
Figure 2

Figure 2 - Otoferlin dual-AAV injection at P6-7 partially restores synaptic function in Otof-/- IHCs.

A High magnification views of IHCs immunolabeled for otoferlin and synaptic ribbons (CtBP2) from wild‑type (B6: P27, CD1B6F1: P29), dual-AAV injected CD1B6F1 Otof-/- (dual-AAV-TS: P26, dual-AAV-Hyb: P28), and their contralateral non-injected ears. (*) Transduced cells. Maximum intensity projections of optical confocal sections. Scale bars: 5 µm.

B Synaptic ribbon numbers quantified from IHCs in apical cochlear turns of wild‑type (B6: n=48 IHCs, CD1B6F1: n=108 IHCs), transduced Otof-/- (dual‑AAV‑TS: n=59 IHCs, dual-AAV-Hyb: n=37 IHCs), and non-transduced Otof-/- IHCs from injected (-AAV injected ear, n=65 IHCs) and contralateral non-injected (-AAV non-injected ear, n=46 IHCs) ears (P25-29).

C IHC synapses labelled with CtBP2 and the postsynaptic marker Shank1a in B6 wild-type and Otof-/- P6 and P14 organs of Corti. Maximum intensity projections of optical confocal sections. Scale bars: 5 µm.

D Synapse numbers quantified from IHCs in apical cochlear turns (C) of B6 wild-type (P6: n= 53 IHCs; P14: n=73 IHCs) and B6 Otof-/- (P6: n=62 IHCs; P14: n=65 IHCs) mice at two different developmental stages (P6 and P14).

E Ca2+‑current-voltage relationship of control CD1B6F1 wild‑type (n=6 IHCs), dual-AAV-TS transduced (n=8 IHCs), and non‑transduced CD1B6F1 Otof-/- (n=10 IHCs) IHCs (P14‑18).

F Representative Ca2+‑currents (Ica) and IHC plasma membrane capacitance increments (ΔCm) of a wild‑type control, transduced, and non‑transduced Otof-/- IHC in response to a 20 ms depolarization pulse at maximum Ca2+‑current potentials (typically -14 mV).

G,H Average exocytosis level measured as ΔCm (G) and corresponding Ca2+‑current integrals (QCa2+) (H) in wild‑type (CD1B6F1: n=6 IHCs; B6: n=11 IHCs (B6 data replotted from Strenzke et al, 2016)), dual-AAV-TS transduced Otof-/- (n=8 IHCs), and non‑transduced (n=11 IHCs) Otof-/- IHCs. Transduced IHCs that showed eGFP expression, but had almost no exocytosis are depicted as dashed lines (not included into the average).

Data information: Individual animals are depicted with open symbols (B,D) or lighter color (G). In (B, D, E,G,H), data are displayed as mean ± s.e.m., ns P>0.05; **P≤0.01; ***P≤0.001 (Kruskal-Wallis test followed by by Dunn's multiple comparison test).
A dual-AAV approach restores fast exocytosis and partially rescues auditory function in deaf otoferlin knock-out mice (2018)
Figure 3

Figure 3 - Dual‑AAV mediated otoferlin gene delivery partially restores auditory function in deaf Otof-/- mice.

A Representative ABR wave traces in response to broadband click sound stimuli from otoferlin dual‑AAV‑TS and dual‑AAV‑Hyb injected CD1B6F1 Otof-/- animals. AAV2/6.eGFP (+AAV.eGFP) and dual‑AAV‑TS injected CD1B6F1 wild‑type, and non‑injected control Otof-/- littermate (-AAV) mice served as controls. SP: summating potential; ABR waves are indicated from I-V.

B Average ABRs evoked by 80 dB SPL click sound stimuli for 20 clicks/s.

C,D ABR click sound (C) and tone burst (D) thresholds in otoferlin dual‑AAV treated Otof-/- mice compared to wild‑type and non‑treated Otof-/- control animals. In D, the two best animals are depicted with open circles. Animals with thresholds exceeding the maximum loudspeaker output (arrows) of 100 dB SPL for clicks and 90 dB SPL for tone bursts were set to 110 dB SPL and 100 dB SPL, respectively. Apical and basal cochlear turns are indicated as Apex and Base, respectively.

E Summed ABR wave I-V amplitudes at different click sound intensities in otoferlin dual‑AAV injected, non‑injected Otof-/-, and wild‑type control mice.

F Summed ABR wave I‑V amplitudes of individual dual‑AAV‑TS treated CD1B6F1 Otof-/- animals (n=8 animals) (from Fig 3E) plotted against their full-length otoferlin IHC transduction rates (from Fig 1D, C-term otoferlin). r: correlation coefficient.

Data information: In (B-E), data are represented as mean ± s.e.m. In (C ,F), individual animals are depicted with open symbols. In (F), r≥0.5 positive correlation (70 dB SPL and 90 dB SPL: Pearson correlation test; 50 dB SPL: Spearman correlation test ). In (C), ns P>0.05; *P≤0.05; ***P≤0.001, (Kruskal-Wallis test followed by Dunn's multiple comparison test).

Expanded View Figures Legends
Characterization of the c10orf76-PI4KB complex and its necessity for Golgi PI4P levels and enterovirus replication (2019)
Figure 3

Figure 3. PI4KB recruits c10orf76 to the Golgi in vivo

(A) Transfections of HEK293 cells revealed that both wild-type GFP-PI4KB and RL494EA GFP-PI4KB localize to the Golgi.

(B) WT c10orf76 also localized to the Golgi, however, the PI4KB binding deficient mutant of c10orf76 (FLH409AAA) predominantly localized to the cytosol.

(C) Cartoon schematic of rapamycin-inducible mitochondria recruitment. The AKAP1-FRB-CFP construct is localized to the outer mitochondrial membrane, while the mRFP-FKBP12-PI4KB and eGFP-c10orf76 are localized in the Golgi as well as within the cytoplasm where they can form a complex. Upon addition of rapamycin, the mRFP-FKBP12-PI4KB construct is translocated to the mitochondria.

(D) Mitochondria recruitment experiment with wild-type PI4KB and c10orf76. Left: AKAP1-FRB-CFP is localized to the mitochondria before (top) and 5 minutes after rapamycin (100 nM) treatment (bottom). Middle: mRFP-FKBP12-PI4KB at high expression level is located in the cytosol before rapamycin (top) and translocates to the mitochondria after rapamycin induction (bottom). Right: eGFP-c10orf76 at a high expression level is located in the cytosol before rapamycin (top) and translocates to the mitochondria after rapamycin induction (bottom).

(E) Schematic of the rapamycin-inducible mitochondria recruitment experiment with mutant PI4KB and WT c10orf76.

(F) Mitochondria recruitment experiment with mutant PI4KB and WT c10orf76. Left: AKAP1-FRB-CFP is localized to the mitochondria before (top) and 5 minutes after (bottom) rapamycin treatment. Middle: mRFP-FKBP12-PI4KB(RL494EA) is located in the cytosol before rapamycin (top) and translocates to the mitochondria after rapamycin induction (bottom). Right: eGFP-c10orf76 is located in the cytosol before (top) and after (bottom) rapamycin induction.

Data information: Scale bars represent 10 μm.
Characterization of the c10orf76-PI4KB complex and its necessity for Golgi PI4P levels and enterovirus replication (2019)
Figure 4

Figure 4. Knockout of c10orf76 in HAP1 cells leads to decreased PI4P levels and disruption of GBF1 / active Arf1 localization despite minimal effects on Golgi morphology.

(A, B) HAP1 cells were fixed and stained with antibodies examining PI4P and PI4KB (A) and Golgi morphology markers (B).

(C) Western blot of GBF1, PI4KB and B-Actin levels in both wild-type and c10orf76 KO HAP1 cells.

(D) Markers of Arf1 activation - the coatomer proteins COPIα/γ and βCOP act as a readout for GTP-bound Arf1, while the native coatomer was detected with the CM1 antibody.

Data information: Nuclei were stained with DAPI (blue). Scale bars represent 10 μm.
Characterization of the c10orf76-PI4KB complex and its necessity for Golgi PI4P levels and enterovirus replication (2019)
Figure 5

Figure 5. The c10orf76-PI4KB complex is essential for Coxsackievirus A10 replication

(A) Viral infection assays determining viral titers by end-point titration at 0 hours and 8 hours in HAP1 wild-type or c10orf76 knockout cells. Left: Coxsackievirus A10 infection. Middle: Poliovirus-1 infection. Right: Coxsackievirus B3 infection. Titre values for c10orf76 knockout cells at 8 hours were statistically evaluated compared to c10orf76 knockout titres at 0 hours and wild-type titres at 8 hours using a one-way ANOVA. **, P<0.01; N.S., P>0.05.

(B) Viral infection assays determining virus titers by end point titration at 8 hours in HeLa wild-type and PI4KB knockout cells upon transfection of wild-type PI4KB, the complex-disrupting RL494EA PI4KB mutant or the kinase dead D674A PI4KB mutant. Left: Coxsackievirus A10 infection. Middle: Poliovirus-1 infection. Right: Coxsackievirus B3 infection. Values were statistically evaluated compared to the GalT control using a one-way ANOVA. **, P<0.01; *, P<0.05; N.S., P>0.05.

Data information: For all panels error bars represent standard error of biological replicates (n=3).
Degradation of WTAP Blocks Antiviral Responses by Reducing the m6A Levels of IRF3 and IFNAR1 mRNA (2021)
Figure 1

Figure 1. WTAP protein is decreased in virus-infected or nucleic acid analog-treated cells.

A, B Immunoblot analyses of the protein abundance of the METTL3/METTL14/WTAP heterotrimer in PBMCs (A) and BMDMs (B) upon challenge with VSV-eGFP, poly(I:C), HSV-1 or HSV60 at the indicated time points. Data are representative of 3 independent biological experiments.

C, D Immunoblot analyses of the protein abundance of the METTL3/METTL14/WTAP heterotrimer in A549 cells upon challenge with VSV-eGFP or SeV (C) and poly (I:C) or poly (dA:dT) (D) at the indicated time points. Data are representative of 3 independent biological experiments.
Degradation of WTAP Blocks Antiviral Responses by Reducing the m6A Levels of IRF3 and IFNAR1 mRNA (2021)
Figure 2

Figure 2. WTAP is degraded through the K48-linked ubiquitination-proteasome pathway upon the activation of IFN-I signaling.

A, B Real-time PCR (qRT-PCR) analyses of WTAP mRNA expression in PBMCs (A) or BMDMs (B) upon challenge with VSV-eGFP, poly(I:C), HSV-1 or HSV60 at indicated time points. n = 3 independent biological replicates, and error bars represent standard deviations. Data were compared using Student's t-test.

C qRT-PCR analyses of WTAP mRNA expression in A549 cells upon challenge with VSV-eGFP, poly(I:C), HSV-1 or poly (dA:dT) at the indicated time points. n = 3 independent biological replicates, and error bars represent standard deviations. Data were compared using Student's t-test.

D qRT-PCR analyses of WTAP mRNA expression in A549 cells stimulated with IFN-α or -β at the indicated time points. n = 3 independent biological replicates, and error bars represent standard deviations. Data were compared using Student's t-test.

E A549 cells were stimulated with poly (I:C) for 16 hrs and then treated with PR-171 (10 μM) or PS-341 (10 μM) for additional 4 hrs and 8 hrs. The cell lysates were analyzed by immunoblotting. Representative images from 3 independent biological experiments are shown (left), and the relative levels of WTAP are presented as the mean ± SD (right).

F A549 cells were stimulated with poly (I:C) for 16 hrs and then treated with CQ (50 mM) or Baf A1 (0.2 mM) for additional 4 hrs and 8 hrs. The cell lysates were analyzed by immunoblotting. LC3B served as a good marker of the autophagy process. Representative images from 3 independent biological experiments are shown (left), and the relative levels of WTAP are presented as the mean ± SD (right).

G Immunoassays of extracts of A549 cells infected with VSV-GFP for 16 hrs and then treated with PR-171 (10 μM) or DMSO for additional 4 and 8 hrs, followed by immunoprecipitation with anti-WTAP antibody and immunoblot analysis with anti-Ub antibody. Data are representative of 3 independent biological experiments.

H Coimmunoprecipitation and immunoblot analyses of extracts of 293T cells transfected with various combinations of plasmids encoding FLAG-tagged WTAP, and HA-tagged K48-linked, K63-linked or wild-type ubiquitin and treated with DMSO or PR171 (10 μM). Data are representative of 3 independent biological experiments.
Degradation of WTAP Blocks Antiviral Responses by Reducing the m6A Levels of IRF3 and IFNAR1 mRNA (2021)
Figure 3

Figure 3. WTAP positively regulates the antiviral immune responses.

A WTAP knockout efficiency in 293T cells detected by immunoblotting.

B Luciferase activity analyses in wild-type (WT), or WTAP-/- 293T cells, transfected with a luciferase reporter for IFN-β (IFN-β-luc) or ISRE (ISRE-luc) followed by treatments with intracellular poly(I:C), poly(dA:dT), or VSV-eGFP. The results are expressed relative to Renilla luciferase activity. n = 3 independent biological replicates, and error bars represent standard deviations. Data were compared using Student's t-test.

C qRT-PCR analyses to detect the mRNA abundances of IFNB1, RANTES, DDX58, and ISG15 in WT and WTAP-/- 293T cells followed by treatments with intracellular poly(I:C), poly(dA:dT), or VSV-eGFP. Data are representative of 3 independent biological experiments.

D Immunoblot analyses to detect the WTAP knockdown efficiency in A549 cells after transfection with Ctrl or WTAP-specific siRNAs for 48 hrs.

E, F qRT-PCR analyses to detect the mRNA abundance of IFNB1 (E), RANTES and ISG15 (F) in A549 cells transfected with Ctrl or WTAP-specific siRNA, followed by treatment with poly(I:C)-HMW at the indicated time points. n = 3 independent biological replicates, and error bars represent standard deviations. Data were compared using Student's t-test.

G, H ELISA analyses to detect IFN-β (G) and CCL5 (H) secretion in supernatants from E at 16 h. n = 3 independent biological replicates, and error bars represent standard deviations. Data were compared using Student's t-test.

I, J Immunoblot analyses to detect the protein expression of ISGs in shCtrl and shWTAP A549 cells that were stimulated with poly(I:C)-LMW (I) or poly(dA:dT) (J) at different time points with the indicated antibodies. Data are representative of 3 independent biological experiments.

K, L qRT-PCR analyses to detect the mRNA abundances of IFNB1 (K) and RANTES (L) in A549 cells transfected with empty vector (EV) or WTAP-HA plasmid, followed by treatment with poly(I:C)-HMW at the indicated time points. n = 3 independent biological replicates, and error bars represent standard deviations. Data were compared using Student's t-test.

M, N ELISA analyses to detect IFN-β (M) and CCL5 (N) secretion in supernatants from K. n = 3 independent biological replicates, and error bars represent standard deviations. Data were compared using Student's t-test.
Degradation of WTAP Blocks Antiviral Responses by Reducing the m6A Levels of IRF3 and IFNAR1 mRNA (2021)
Figure 4

Figure 4. WTAP regulates the antiviral responses by targeting both IFN-I and Jak-STAT signaling.

A, B Immunoblot analyses of total and phosphorylated (p-) TBK1 and IRF3 in A549 cells transfected with Ctrl or WTAP-specific siRNA, followed by stimulation with poly (I:C) (A) or poly (dA:dT) (B) at different time points. Data are representative of 3 independent biological experiments.

C Immunoblot analyses of IRF3 dimerization in A549 cells transfected with Ctrl or WTAP-specific siRNA, followed by stimulation with poly (I:C) or poly (dA:dT) for 6 hrs. Data are representative of 3 independent biological experiments.

D Immunoblot analyses to detect the protein expression of ISGs in A549 cells transfected with Ctrl or WTAP-specific siRNA, followed by stimulation with IFN-α or -β (50 U/ml) for 4 hrs. Data are representative of 3 independent biological experiments.

E, F Immunoblot analyses of total and phosphorylated (p-) STAT1 in shCtrl and shWTAP A549 cells, followed by stimulation with IFN-α (E) or -β (F) at different time points. Data are representative of 3 independent biological experiments.