EMBO/soda-nel huggingface dataset:
Antagonism of interferon signaling by fibroblast growth factors promotes viral replication (2020)
Figure 5

Fig 5: FGF7 promotes HSV-1 replication in human keratinocytes

(A-C) Serum-starved HaCaT cells were infected with HSV-1 (MOI=0.5) in the presence or absence of FGF7 (10 ng/ml) or IFN-α (1000 U/ml). Viral load was determined 24 h post infection (hpi) by qPCR for the HSV-1 Glyc-B gene relative to the human β-actin gene (ACTB) (A) or by HSV-1 Glyc-D immunofluorescence (red) and DAPI counterstaining (blue) (B). (C) Transmission microscopy images of HaCaT cells 48 h post infection (hpi).

(D, E) Serum-starved HaCaT cells (D) or human primary keratinocytes (HPK, from two donors) (E) were infected with HSV-1 (MOI=0.5) in the presence or absence of FGF7 at the indicated concentrations. Virus load was determined by qPCR for HSV-1 Glyc-B relative to ACTB 24 hpi.

(F) Serum-starved HaCaT cells were infected with HSV-1 (MOI=0.5) in the presence or absence of FGF7, FGF10 or FGF2 (10 ng/ml). DNA samples were analyzed by qPCR for Glyc-D DNA relative to ACTB and protein lysates were analyzed by Western blot for Glyc-D and vinculin 10 hpi.

(G) Epidermal sheets from tail skin, infected ex vivo with HSV-1 (MOI = 2) +/- FGF7 (10 ng/ml), were stained 48 hpi for Glyc-D (red) using DAPI as counterstain (blue). The infected area (red staining) was measured using Image J software. Data are expressed as A.U. = arbitrary unit. Background staining of hairs was subtracted manually.

(H) Serum-starved HaCaT cells were infected with HSV-1 in the presence or absence of FGF7 (10 ng/ml) or IFN-α (1000 U/ml). Viral load was determined 24 hpi by qPCR for the HSV-1 Glyc-B gene relative to ACTB or by Western blot for the viral protein Glyc-D and GAPDH.

(I) Serum-starved HaCaT cells were infected with HSV-1 (MOI = 0.5). Four hours hpi, infected cells were washed and incubated in fresh serum-free medium containing FGF7 (10 ng/ml). Viral load was measured 8 h later by qPCR for Glyc-B relative to ACTB.

(J) Serum-starved HaCaT keratinocytes were treated for 6 h with FGF7 (10 ng/ml). RNA samples were analyzed by qRT-PCR for NECT1 (encoding nectin-1) relative to RPLP0.

Data information: Scatter plots show mean +/- S.E.M. In (A), and (D-I) mean viral DNA levels or stained area in HSV-1-infected, but non-treated cells were set to 1. In (J) mean expression levels in CTRL cell cultures were set to 1. A: N=16-24 from four independent experiments. B, C: Representative pictures are shown. D: N=6 from one experiment. E: N=6 from two experiments and two donors. F: N=4 from two experiments. The Western blot is a representative of two experiments. G: N=16-24 measured areas from 8 mice from 4 experiments. H (scatter plot): N=6 from two experiments. The Western blot is a representative of two experiments. I: N=8 mice from two experiments. J: N=6 from 2 experiments. *P≤0.05, **P≤0.01, ****P≤0.0001 (Mann-Whitney U test). Exact P-values are shown in Dataset EV2. Magnification bars: 200 μm (B) or 80 μm (C) and 800 µm (G).
Antagonism of interferon signaling by fibroblast growth factors promotes viral replication (2020)
Figure 6

Fig 6: FGF7 suppresses virus-induced ISG expression and stimulates replication of different viruses in keratinocytes

(A-E) Serum-starved HaCaT cells were infected with HSV-1 (MOI=0.5) +/- FGF7 (10 ng/ml), added at the indicated time points (A). 12 h after infection, RNA samples were analyzed by qRT-PCR for RSAD2, IRF7 and IFNL2 relative to RPLP0 (B), DNA samples were analyzed by qPCR for Glyc-B DNA relative to ACTB (C), and protein lysates were analyzed by Western blot for Glyc-D, total and phosphorylated STAT1 (Y701), STAT2 and IRF3, for IRF1, IRF7, IRF9 and RSAD2 and for GAPDH or vinculin (loading controls) (E). Quantification of the Glyc-D, RSAD2, IRF1, IRF7, IRF9 and STAT2 band intensity relative to GAPDH is shown on the right hand side. (D) shows mean Glyc-B DNA and RSAD2, IRF7 and IFNL2 mRNA levels (based on results shown in Fig. 6B and C) in virus-infected cells treated for different periods with FGF7.

(F) HaCaT cells were infected with LCMV in the presence or absence of FGF7 (10 ng/ml) or IFN-α (1000 U/ml) and analyzed for LCMV nucleoprotein (NP) by flow cytometry 24 hpi. Arbitrary units are shown.

(G, H) HaCaT cells were infected with ZIKV strains 976 (Uganda isolate) (MOI = 0.1) or PF13/251013-18 (French Polynesia isolate) (MOI = 20) and treated 2 hpi with FGF7 (20 ng/ml) or vehicle (CTRL). Immunofluorescence staining for the ZIKV envelope protein and quantification of the percentage of cells expressing the ZIKV envelope protein (G) and qRT-PCR analysis of RNA samples for ZIKV RNA (H) 48 hpi is shown.

(I, J) HaCaT cells were infected with the ZIKV PF13/251013-18 (MOI = 20) in the presence or absence of FGF7 (20 ng/ml). RNA samples were analyzed by qRT-PCR for OAS1 and MxA relative to RPLP0 72 hpi.

Data information: Scatter plots show mean +/- S.E.M. In (B-H and J) results obtained with virus-infected, but non-treated cells were set to 1; in (I) mean expression levels in non-infected cell cultures were set to 1. B: N=4-5 from 2 experiments. C: N=4 from 2 experiments. E: Representative blots of two experiments. F: N=5-6 from two experiments. G, H: N=4 from one experiment. I, J: N=4-8 from two experiments. Magnification bar in (G): 40 μm. *P≤0.05, **P≤0.01, ***P≤0.001 (Mann-Whitney U test). Exact P-values are provided in Dataset EV2.
Antagonism of interferon signaling by fibroblast growth factors promotes viral replication (2020)
Figure 7

Fig 7: Inhibition of FGFR signaling blocks HSV-1 replication

(A) Serum-starved HaCaT cells were infected with HSV-1 (MOI = 0.5) in the presence or absence of FGF7 (10 ng/ml) and the FGFR kinase inhibitors AZD4547 (1 μM) or BGJ398 (3.5 μM). DNA and protein lysates were analyzed by qPCR for Glyc-B relative to ACTB or by Western blot for Glyc-D and GAPDH, respectively, 16 hpi.

(B, C) HaCaT cells were cultured in DMEM/10% FCS (B) or DMEM/5% FCS (C) and infected with HSV-1 (MOI=0.5) in the presence or absence of AZD45347 (1 μM) and/or BGJ398 (3.5 μM). Viral load was determined by qPCR for Glyc-D relative to ACTB (B) or by measurement of viral titers (plaque forming units (PFU)) 14 hpi (C).

(D, E) Adult C57BL/6 wild-type (D) or K5-R1/R2 mice and respective controls (E) were infected subcutaneously with HSV-1 (MOI = 10). RNA from infected skin was analyzed by qRT-PCR for Fgf7 relative to Rps29 (D) and DNA was analyzed by qPCR for ICP0, normalized to the host gene Tbx15 48 hpi (E).

Data information: Scatter plots show mean +/- S.E.M. In (A, B) mean viral DNA levels in HSV-1-infected, but non-treated cells were set to 1. In (C) absolute PFU/ml cell lysate is shown. In (D) mean expression in non-infected mice (vehicle-injected) were set to 1, and in (E) mean ICP0 DNA levels in Ctrl mice were set to 1. A: N=5-6 from two experiments. B: N=12-13 from 4 experiments. C: N=6 from two experiments. D: N=9-10 infected mouse skin spots from two experiments. F: N=20-24 infected mouse skin spots from two experiments. *P≤0.05, **P≤0.01, ****P<0.0001 (Mann-Whitney U test). Exact P-values are provided in Dataset EV2.
Mycobacterium tuberculosis Rv2626c-derived peptide as a therapeutic agent for sepsis (2020)
Figure 1

Figure 1. Effects of rRv2626c on inflammatory response in macrophages.

(A) Bacterially purified 6xHis-rRv2626c and analyzed by Coomassie blue staining (left) or immuno blotting (IB) with αHis (right). (B) BMDMs were incubated with rRv2626c for the indicated times and then cell viability measured with MTT assay. (C) Fluorescence confocal images showing in BMDMs treated with Rv2626c (2.5 μg/ml) for 30 min, fixed, immunostained with antibodies for His (Alexa 488) and DAPI. Scale bar, 10 μm. (D and E) BMDMs from WT mice were treated with LPS (100 ng/ml) or rRv2626c for indicated times (D). BMDMs from WT, TLR2-/-, TLR4-/-, MyD88-/-, TRIF-/-, IRAK1-/-, TRAF6-/-, TBK1-/- mice were treated with 2.5 μg/ml rRv2626c or rVector for 18 h (E). Culture supernatants were harvested, and the levels of TNF-α, IL-6, IL-12p40, and IL-10 were measured by ELISA. (F) BMDMs were treated with 2.5 μg/ml rRv2626c for 1 h, harvested and then subjected to IB for αIκBα and His. αActin was used as a loading control. Data shown are representative of three independent experiments with similar results (A, C, E). Data shown are the means ± SD of five experiments (B, D, E). Statistical significance was determined by Student's t-test with Bonferroni adjustment (***P < 0.001) compared with rVector.
Mycobacterium tuberculosis Rv2626c-derived peptide as a therapeutic agent for sepsis (2020)
Figure 2

Figure 2. TLR-mediated inflammatory responses in macrophages are negatively regulated by rRv2626c.

BMDMs were pretreated with 2.5 μg/ml rRv2626c or rVector for 1 h, and stimulated with 100 ng/ml LPS (A and C) or Pam2CSK4 (B) for indicated times. (A and B) Culture supernatants were harvested, and the levels of TNF-α, IL-6, IL-12p40, and IL-10 were measured by ELISA. (C) Cells were harvested and then subjected to IB for the phosphorylated forms of AKT, MAPK (ERK, p38, JNK), IκBα, total forms of IκBα. αActin was used as a loading control (left). The densitometric values were calculated from the ratios of protein levels relative to actin (right). Data shown are the means ± SD of three experiments (A and B). Data shown are representative of five independent experiments with similar results (C). Statistical significance was determined by Student's t-test with Bonferroni adjustment (*, P < 0.05; **, P < 0.01; ***, P < 0.001) compared with rVector.
Mycobacterium tuberculosis Rv2626c-derived peptide as a therapeutic agent for sepsis (2020)
Figure 3

Figure 3. TRAF6 interacts with Rv2626c.

(A) Identification of TLR2, IRAK1, RIP1, TRAF6, MAPK11 and TOLLIP as endogenous binding partners of rRv2626c. THP-1 cell lysates were incubated with His tagged-rRv2626c or rVector and then IP with His-agarose beads. Binding partners were confirmed by silver staining and mass spectrometric analysis. The red-colored letters indicate the peptides identified from mass spectrometry analysis. (B) 293T cells were co-transfected with Flag-TRAF6 and Myc-Rv2626c and IP with αFlag or αMyc. WCLs were used for IB with αFlag, αMyc or αActin. (C) THP-1 and BMDMs were stimulated with rRv2626c (2.5 μg/ml) for the indicated times, followed by IP with αHis (up) or αTRAF6 (down) and IB with αTRAF6, αHis, and αActin. (D) Immunostaining of BMDMs treated with 2.5 μg/ml rRv2626c for 30 min. and then immunolabeled with antibody to His-rRv2626c (Alexa Fluor 488) or TRAF6 (Alexa Fluor 568). DAPI (blue) stained nuclei. Scale bar, 10 μm. The data are representative of five independent experiments with similar results (A-D).
Mycobacterium tuberculosis Rv2626c-derived peptide as a therapeutic agent for sepsis (2020)
Figure 4

Figure 4. Rv2626c associates with N-terminal region of TRAF6 through CBS2 domain and attenuates E3 ubiquitin ligase activity of TRAF6.

(A) Schematic diagram of the structures of Rv2626c (up). 293T cells were co-transfected with Flag-TRAF6 together with GST-vector or GST-Rv2626c and its mutant constructs, subjected to GST pulldown, followed by IB with αFlag. WCLs were used for IB with αGST, αFlag or αActin (down). (B) At 12 hr post-transfection with mammalian Myc-Rv2626c constructs together with Flag-TRAF6 and 293T cells treated with several Tat-CBS2 peptide for 12 h (10 µM) for 6h, subjected to IP with αMyc, followed by IB with αFlag. WCLs were used for IB with αMyc, αFlag or αActin. (C) Schematic diagram of the structures of TRAF6 (up). 293T cells were co-transfected with Myc-Rv2626c together with GST-vector or GST-TRAF6 and its mutant constructs, subjected to GST pulldown, followed by IB with αMyc. WCLs were used for IB with αGST, αMyc or αActin (down). (D) 293T cells were co-transfected with Flag-TRAF6, Myc-Rv2626c as indicated doses and HA-ubiquitin (left), HA-K48-linked ubiquitin (middle), HA-K63-linked ubiquitin (right)

and then IP with αFlag, followed by IB with αHA. WCLs were used for IB with αFlag, αMyc, or αActin. (E) BMDMs were pretreated with 2.5 μg/ml rRv2626c for 1 h, and stimulated with 100 ng/ml LPS for 30 min, followed by IP with αTRAF6, IB with ubiquitin, K48-linked ubiquitin, or K63-linked ubiquitin. WCLs were used for IB with αHis, αTRAF6, or αActin. The data are representative of five independent experiments with similar results (A-E).
Mycobacterium tuberculosis Rv2626c-derived peptide as a therapeutic agent for sepsis (2020)
Figure 5

Figure 5. Design and expression of Tuftsin-conjugated Rv2626c peptide-based protein.

(A) Schematic in design of rRv2626c and its mutants. (B) Bacterially purified 6xHis-rRv2626c and analyzed by Coomassie blue staining (left) or immuno blotting (IB) with αHis (right). (C) BMDMs were incubated with rRv2626c-CA, rRv2626c-DN, or rVehicle for the indicated times and then cell viability measured with MTT assay. (D) BMDMs were pretreated with rRv2626c-WT, rRv2626c-CA, or rRv2626c-DN for 1 h, and stimulated with 100 ng/ml LPS for 18 h. Culture supernatants were harvested, and the levels of TNF-α, IL-6, IL-12p40, and IL-10 were measured by ELISA. (E) Myc-Rv2626c-expressing Raw264.7 or THP-1 cells were pretreated with rRv2626c-WT (2.5 μg/ml), rRv2626c-CA (10 ng/ml), rRv2626c-DN (2.5 μg/ml), for 1 h, and stimulated with 100 ng/ml LPS for 30 min, followed by IP with αMyc or αTRAF6, IB with αMyc and ubiquitin. WCLs were used for IB with αMyc, αTRAF6, αHis or αActin. The data are representative of five independent experiments with similar results (B and E). Data shown are the means ± SD of three experiments (C and D). SC, solvent control (PBS).
Mycobacterium tuberculosis Rv2626c-derived peptide as a therapeutic agent for sepsis (2020)
Figure 6

Figure 6. The rRv2626c-CA protects mice from cecal ligation and puncture (CLP)-induced polymicrobial sepsis.

(A-C) Schematic of the CLP model treated with rRv2626c-CA or its mutants (upper). The survival of mice was monitored for 10 days; mortality was measured for n=25 mice per group (lower). Statistical differences compared with the rVector-treated mice are indicated (log-rank test). The data are representative of two independent experiments with similar results. (D) Serum cytokine levels and (E) representative hematoxylin and eosin (H&E) staining of the lung, liver and spleen (left) from 10 mice per group. Histopathology scores were obtained from H&E stained as described in Methods (right) were determined at 20 h in CLP mice were treated with rRv2626c-CA or its mutants. Scale bar, 100 μm. (F) Splenocytes were used for IP with αHis or αTRAF6, followed by IB with αHis or αUb. WCLs were used for IB with αTRAF6, αHis or αActin. (G) In vivo imaging using IVIS spectrum-chromatography CT system. Intraperitoneal administration of rRv2626c-CA/Cy5.5 was carried out at intervals of 0, 6, 12, and 18 h in a CLP-induced sepsis model. Pharmacokinetics and biodistribution were observed in spleen, liver, lung, and kidney tissues. The data are representative of three independent experiments with similar results. (H) The percentage of F4/80+ macrophages cells and rRv2626c-CA-His were found in the spleen, liver and lung using Fluorescence-activated cell sorting analysis in the background of CLP-induced sepsis. The data are representative of five independent experiments with similar results (E-H). Statistical significance was determined by Student's t-test with Bonferroni adjustment (***P < 0.001) compared with CLP+rVehicle.
Mycobacterium tuberculosis Rv2626c-derived peptide as a therapeutic agent for sepsis (2020)
Figure 7

Figure 7. rRv2626c-CA shows bactericidal effects.

(A) The bacterial burden was evaluated 20 h after treatment of CLP mice with rRv2626c-CA or its mutants (n=10 mice per group). (B) The percentage of F4/80+ macrophages cells and rRv2626c-CA-His or its mutants were found in the spleen or lung using Fluorescence-activated cell sorting analysis in the background of CLP-induced sepsis. (C) CD86, iNOS, CD163 and Arg1 mRNA levels were determined by real-time PCR. (D) Expression of SR-A, FcR, TLR2, TLR4, NRP1 or CXCR2 were measured by flow cytometry analysis. The data are representative of five independent experiments with similar results (D). Statistical significance was determined by Student's t-test with Bonferroni adjustment (**P < 0.01; ***P < 0.001) compared with CLP+rVehicle.
Anti-tumor effects of PIM/PI3K/mTOR triple kinase inhibitor IBL-302 in neuroblastoma (2019)
Figure 3

Fig. 3. PIM/PI3K/mTOR inhibition decreases N-Myc levels and increases cellular differentiation and apoptosis A-G. Neuroblastoma PDX and SK-N-BE(2)c cells treated with IBL inhibitors at indicated concentrations for 48 hours. A-B. pAkt (at Ser473 (A) and Thr308 (B) sites) levels in LU-NB-3 and SK-N-BE(2)c cells determined by western blotting. Total Akt levels were used as loading control. C. p-p70S6K and p-p85S6K levels in LU-NB-3 cells determined by western blotting. Actin, p70S6K, and p85S6K levels were used as loading controls. D. Brightfield photomicrographs of LU-NB-3 and SK-N-BE(2)c cells treated with 0.36μM IBL-202 or 0.05 μM IBL-301. Scale bars represent 100 μm (LU-NB-3) or 200 μm (SK-N-BE(2)c). Arrows indicate neurite outgrowths and asterisks indicate where inserts are magnified. IBL-301 treated cells were stained for Tuj1. DAPI was used to visualize nuclei. E. Quantification of neurite outgrowth presented as number of neurites/cell in LU-NB-3 PDX and SK-N-BE(2) cells treated with IBL-301. For LU-NB-3 PDX cells, representative areas (n=2) were used and n=344 and n=240 cells/condition for CTRL and IBL-301, respectively, were counted. For SK-N-BE(2)c cells, representative areas (n=2 and n=3 for CTRL and IBL-301 respectively) were used and n=141 and n=130 cells/condition for CTRL and IBL-301, respectively, were counted. Statistical significance was determined by two-sided student's t-tes. P=0.003 for LU-NB-3 and p=0.08 for SK-N-BE(2)c. F. Gap43 protein levels in LU-NB-3 and SK-N-BE(2)c cells determined by western blotting. SDHA levels were used as loading control. G. N-Myc levels in LU-NB-2 and LU-NB-3 cells determined by western blotting. SDHA levels were used as loading control.
Anti-tumor effects of PIM/PI3K/mTOR triple kinase inhibitor IBL-302 in neuroblastoma (2019)
Figure 4

Fig. 4. Treatment with multikinase inhibitors results in cell death. A. Viability IBL-202 or IBL-301 treated cells determined by CellTiterGlo. B-C. Cell cycle distribution of LU-NB-3 cells determined by flow cytometry. Bars show mean values from two independent experiments. D. Cleaved caspase 3 (Cl Casp3) levels in LU-NB-3 cells determined by western blotting. SDHA levels were used as loading control. E. Flow cytometry analyses of Annexin V and PI stainings following treatment with 360 nM IBL-202 or 50 nM IBL-301. F. Quantification of live and dead cells from the AnnexinV/PI stainings. Dead cells=PI positive, Live cells=PI negative. Dot plots from two independent experiments and error bars represent SEM.
Anti-tumor effects of PIM/PI3K/mTOR triple kinase inhibitor IBL-302 in neuroblastoma (2019)
Figure 5

Fig. 5. IBL-302 reduces neuroblastoma growth in vivo. A-G. Neuroblastoma cells were treated with indicated concentrations of IBL-302 for 48 hours (pAkt and N-Myc expression, photomicrographs and flow cytometry) or 72 hours (cell viability, (F)). A. Expression of pAkt(Ser473) in LU-NB-3 and SK-N-BE(2)c cells. Total Akt and actin were used as loading controls. B. Expression of pAkt(Thr308) in LU-NB-3 and SK-N-BE(2)c cells. Total Akt and actin were used as loading controls. C. Brightfield photomicrographs of LU-NB-3 and SK-N-BE(2)c cells treated with 50 nM IBL-302. Scale bars represent 100 μm (LU-NB-3) or 200 μm (SK-N-BE(2)c). D. Quantification of neurite outgrowth presented as number of neurites/cell in LU-NB-3 PDX and SK-N-BE(2)c cells treated with IBL-302. For LU-NB-3 PDX cells, representative areas (n=4) were used and n=460 and n=216 cells/condition for CTRL and IBL-302, respectively, were counted. For SK-N-BE(2)c cells, representative areas (n=5 and n=7 for CTRL and IBL-302 respectively) were used and n=124 and n=260 cells/condition for CTRL and IBL-301, respectively, were counted. Statistical significance was determined by two-sided student's t-test. p=0.009 for LU-NB-3 and p=0.0003 for SK-N-BE(2)c. E. N-Myc protein expression determined by western blotting. SDHA was used as loading control. F. Cell viability determined by CellTiterGlo. G. Flow cytometry analyses of Annexin V and PI stainings following treatment with 50nM IBL-302 H. Quantification of live and dead cells. Dead cells=PI positive, Live cells=PI negative. Dot plots from two independent experiments and error bars represent SEM. I. Neuroblastoma SK-N-BE(2)c carrying mice (n=5 in each group) were treated with vehicle (CTRL) or 40 mg/kg IBL-302 for up to 35 days and tumor growth was followed over time. Asterisks indicate each occasion a mouse within that particular group was sacrificed. J. Kaplan-Meier survival curves comparing mice treated with vehicle (CTRL) or IBL-302. Log-rank test was used to determine statistical significance. p=0.0577.
Anti-tumor effects of PIM/PI3K/mTOR triple kinase inhibitor IBL-302 in neuroblastoma (2019)
Figure 6

Fig. 6. PIM/PI3K/mTOR inhibition enhances the effects of chemotherapy. A-B. LU-NB-3 PDX, SK-N-BE(2)c and SK-N-SH cells were treated with indicated concentrations of cisplatin and/or 0.36 μM IBL-202, 0.05 μM IBL-301, or 0.05 μM IBL-302 for 48 hours. Cell viability determined by CellTiterGlo. Graphs show mean values and SEM from three independent experiments. Theoretical additive curves were evaluated by calculating the combination index (CI) based on the Bliss Independence model. C-D. Cell cycle distribution in LU-NB-3 PDX cells after treatment with 320 nM cisplatin, 50 nM IBL-302 or the combination of these, determined by flow cytometry. Bars show mean values from two independent experiments. E. Tumor size in individual mice from (F.) presented as each group separately. F. Neuroblastoma PDX carrying mice (n=5 in each group) were treated with vehicle (CTRL), low-dose (20mg/kg) IBL-302, low-dose (1mg/kg) cisplatin, or the combination of low-dose IBL-302 and low-dose cisplatin for up to 70 days. Tumor size was measured over time. Asterisks indicate each occasion a mouse within that particular group was sacrificed. G. Kaplan-Meier survival curves comparing mice treated with vehicle (CTRL), IBL-302, cisplatin, or the combination. Log-rank test was used to determine statistical significance. p=0.0014. H. Immunohistochemical staining of N-Myc expression in tumors from (F). Scale bars represent 100 μm. Roman numbers in each box indicate which mouse the tumor derives from (see individual graphs in (E.))
A small peptide inhibits siRNA amplification in plants by mediating autophagic degradation of SGS3/RDR6-bodies (2021)
Figure 1

Figure 1. Characterization of an ATG8 interacting small peptide.

Work flow to identify virus-induced small peptides in Arabidopsis.

B. GUS activity of VISP1Pro::VISP1-GUS transgenic Arabidopsis plants infected by mock buffer or CMV-2blm at 7 dpi. Scale bar = 5 mm.

C. Amino acid sequence alignment of AtVISP1 and its homologues from Arabidopsis lyrata (Al), Brassica rapa (Br), Brassica napus (Bn), Brassica oleracea (Bo), Brassica cretica (Bc), Arabis alpine (Aa), and Arabis nemorensis (An). The conserved ubiquitin-interacting motif (UIM) and Arginine/lysine-rich motif (ARM) are indicated by lines.

D. BiFC analysis of interactions between VISP1 or VISP1mUIM and ATG8a or ATG8f in N. benthamiana leaves. Images were taken at 60 hpi. Scale bar = 20 μm.

E. Co-IP experiments examining protein interactions between ATG8a with VISP1 or VISP1mUIM. N. benthamiana leaves were agroinfiltrated and treated with 2 mM 3-MA at 48 hpi, and then for IP with anti-Flag beads 12 h later.

F. GST pull-down analysis of the interactions between ATG8a with VISP1 or VISP1mUIM.