EMBO/soda-nel huggingface dataset:
A small peptide inhibits siRNA amplification in plants by mediating autophagic degradation of SGS3/RDR6-bodies (2021)
Figure 2

Figure 2. Overexpression of VISP1 induced autophagy and autophagosome formation.

Confocal microscopy of YFP-ATG8e-labeled autophagic bodies in root cells of Col-0/YFP-ATG8e and VISP1OE/YFP-ATG8e plants. 7-day-old seedlings were inoculated in liquid ½MS medium with 1 µM ConA for 12 h in dark. Scale bar = 20 μm.

Puncta numbers of YFP-ATG8e-labeled autophagic bodies per 10 cells in panel A. Data points represent means of three biological repeats. Error bars indicate SD. ***p < 0.001 (Student's t-test).

Western blotting analysis of the vacuolar delivery of the free YFP protein in the plants as shown in panel A. YFP-ATG8e/Col-0 plants were treated with N starvation as a positive control of autophagy induction. Total proteins were analyzed with GFP antibodies. Actin served as loading controls.

Western blotting analysis of accumulation of ATG8a and ATG8a-PE in leaves of 4-week-old Col-0, VISP1OE, VISP1mUIM/OE and VISP1mARM/OE plants.

Western blotting detecting accumulation of VISP1-Flag in 7-day-old Col-0 and VISP1OE seedlings. Seedlings were treated with 3-MA (10 mM) or buffer for 18 h at dark prior to protein detection.

Representative TEM images of autophagic structures in inoculated leaves of Col-0 and VISP1OE plants treated with mock buffer or CMV-2blm infection at 4 dpi. The leaves was treated with 1 µM ConA at 4 dpi for 12 h in dark before TEM sampling. Magenta arrowheads indicate autophagic bodies inside the vacuoles. Scale bar = 2 μm. Cp, chloroplast; CW, cell wall; V, vacuole.

Autophagic structure numbers per 10 cells in panel F. Error bars indicate SD. Letters indicate significant differences (ANOVA, P < 0.05).
A small peptide inhibits siRNA amplification in plants by mediating autophagic degradation of SGS3/RDR6-bodies (2021)
Figure 3

Figure 3. Overexpression of VISP1 promotes CMV infection by inhibiting vsiRNA amplification.

Development and pathogenic responses of Col-0, VISP1OE1, VISP1OE2, rdr6-15, and sgs3-1 plants to mock buffer or CMV-2blm infection at 21 dpi. Scale bar = 2 cm.

Long-term symptoms of Col-0, VISP1OE1, VISP1OE2, rdr6-15, and sgs3-1 plants inoculated with CMV-2blm at 50 dpi. Scale bar = 2 cm.

Western blotting showing accumulation of CMV CP and VISP1-Flag in systemically infected leaves at 14 dpi. Relative accumulation (RA) of CMV CP was calculated from band densities. Rubisco complex large subunit (RbcL) served as loading controls.

Northern blotting analyzing accumulation of genomic RNAs (gRNAs) in systemically leaves of Col-0, OE1 (VISP1OE1), OE2 (VISP1OE2), rdr6-15, and sgs3-1 plants infected by CMV-2blm at 14 dpi. Methylene blue-stained rRNA were used as loading controls.

Northern blotting analyzing accumulation of RNA3-derived vsiRNAs in the same samples as shown in panel D.

Relative accumulation of gRNA3, RNA3-derived vsiRNA, and vsiRNAs/gRNA3 ratios calculated from hybridization signal intensities of northern blotting in D, E. Values in Col-0 plants were set as 1. Data points represent means of three biological repeats. Error bars indicate SD. Letters indicate significant differences (ANOVA, P < 0.05).
A small peptide inhibits siRNA amplification in plants by mediating autophagic degradation of SGS3/RDR6-bodies (2021)
Figure 4

Figure 4. VISP1 knockout enhances antiviral RNA silencing.

Development and symptom induction of Col-0, visp1-1, visp1-2, rdr6-15, and sgs3-1 plants to mock treatment or CMV-2blm infection at 21 dpi. Scale bar = 2 cm.

Long-term systemic symptoms of Col-0, visp1-1, visp1-2, rdr6-15, and sgs3-1 plants inoculated with CMV-2blm at 50 dpi. Scale bar = 2 cm.

Western blotting showing accumulation of CMV CP in systemically infected leaves at 14 dpi. Bottom values represent relative accumulation (RA) of CMV CP. RbcL served as loading controls.

Northern blotting detecting accumulation of genomic RNAs and RNA3-derived vsiRNAs in systemically infected leaves at 14 dpi. Relative accumulation (RA) of gRNA3 was calculated from band densities.

Northern blotting analyzing accumulation of RNA3-derived vsiRNAs in the same samples as shown in panel D. Bottom values represent relative accumulation (RA) of RNA3-derived vsiRNAs.

Relative accumulation of vsiRNAs/gRNA3 ratios calculated from hybridization signal intensities of northern blotting in panel D. Data points above columns are means of three biological repeats. Error bars indicate SD. Letters indicate significant differences (ANOVA, P < 0.05).

Accumulation of the CMV CP protein in inoculated leaves of Col-0, atg5-1, atg7-3, rdr6-15, and sgs3-1 plants with CMV-2blm at 4 dpi.
A small peptide inhibits siRNA amplification in plants by mediating autophagic degradation of SGS3/RDR6-bodies (2021)
Figure 5

Figure 5. VISP1 interacts with SGS3 in vivo and in vitro.

BiFC analyses of interactions between VISP1 and SGS3 or RDR6 in N. benthamiana leaves at 60 hpi. RbcL served as a negative control. Scale bar = 20 μm.

Schematic representation of the Arabidopsis SGS3 protein and deletion mutants. Deleted regions are indicated by dashed lines. N: N terminus including Zinc finger (ZF), XS: Rice protein X and SGS3, CC: Coiled-coil domain.

Schematic representation of VISP1 substitution mutants in the UIM and ARM regions. UIM: ubiquitin-interacting motif, ARM: Arginine/lysine-rich motif.

BiFC assays analyzing interactions between VISP1 and SGS3 or its deletion mutants in N. benthamiana leaves. Scale bar = 20 μm.

BiFC assays analyzing interactions between SGS3 and VISP1, VISP1mUIM or VISP1mARM in N. benthamiana leaves. Scale bar = 20 μm.

Co-immunoprecipitation analyses of the interactions between SGS3, SGS3ΔCC, or SGS3CC and VISP1 or VISP1mARM. N. benthamiana leaves were agroinfiltrated with constructs as indicated and treated with 2 mM 3-MA at 48 hpi, and collected for IP with anti-Flag beads 12 h later.

GST pull-down analysis of the in vitro interactions of SGS3CC with VISP1, VISP1mARM or VISP1mUIM. GST- VISP1, GST- VISP1mARM, GST- VISP1mUIM or GST were incubated with SGS3CC-His for IP with anti-GST beads, and detected by Western blotting with GST or His antibodies.
A small peptide inhibits siRNA amplification in plants by mediating autophagic degradation of SGS3/RDR6-bodies (2021)
Figure 6

Figure 6. VISP1 mediates selective autophagic degradation of SGS3/RDR6 bodies.

Confocal analysis of co-localization of VISP1-GFP and RFP-SGS3 with the CFP-NbATG8f-labelled autophagic bodies in N. benthamiana leaves. The infiltrated leaves were treated with 100 µM E64d or DMSO at 48 hpi and examined at 60 hpi. Arrow heads indicate autophagic bodies. Scale bar = 10 μm.

Confocal analysis of co-localization of CFP-NbATG8f-labelled autophagic bodies with the VISP1-YN and SGS3-YC bodies in N. benthamiana leaves. The infiltrated leaves were treated with 100 µM E64d or DMSO at 48 hpi and examined for imaging at 60 hpi. Scale bar = 20 μm.

Effect of VISP1-Flag and the autophagy inhibitor 3-MA on accumulation of SGS3-Flag and SGS3ΔCC-Flag in N. benthamiana leaves. The co-infiltrated leaves were treated with 3-MA (10 mM) or buffer at 48 hpi and were harvested for western blotting 12 h later. Accumulation of AtSGS3 transcript was analyzed by RT-PCR.

Effect of VISP1mARM-Flag on accumulation of SGS3-Flag.

Effect of VISP1mUIM-Flag on accumulation of SGS3-Flag.

Confocal analysis of effect of VISP1-Flag VISP1mARM-Flag, and VISP1mUIM-Flag on RDR6-YN and SGS3-YC formed bodies in N. benthamiana leaves at 60 hpi. EV: empty vector. Scale bar = 20 μm.

Numbers of RDR6-YN and SGS3-YC formed bodies per 10 cells in E were used for quantification. Data points represent means of three biological repeats. Error bars indicate SD. Letters indicate significant differences (ANOVA, P < 0.05).

Western blotting showing accumulation of proteins as indicated in the samples of E.

Western blotting detecting accumulation of the endogenous SGS3 protein in leaves of 2-week-old Col-0, VISP1OE, visp1, and sgs3-1 Arabidopsis plants with anti-SGS3 antibodies.

Western blotting detecting accumulation of the endogenous SGS3 protein in leaves of 2-week-old Col-0, VISP1OE, VISP1mUIM/OE, and VISP1mARM/OE Arabidopsis plants.

Western blotting detecting accumulation of the endogenous SGS3 protein in 2-week-old VISP1-overexpressing Col-0, atg5-1, and atg7-1 mutants.

Data information: EV: empty vector. (B-D)Relative accumulation (RA) represent means ± SD from band densities in three biological repeats. (C-E, H) RbcL served as loading controls. (I-K) Actin served as a loading control.
A small peptide inhibits siRNA amplification in plants by mediating autophagic degradation of SGS3/RDR6-bodies (2021)
Figure 7

Figure 7. VISP1 negatively affects tasiRNA biogenesis.

Developmental phenotypes of rosette leaves of 5-week-old Col-0, visp1-1, visp1-2, VISP1OE1, VISP1OE2, and sgs3-1 seedlings. Arrowheads indicate the 8th and 9th rosette leaves from the top leaves. Scale bar = 2 cm.

The length/width ratios of the 8th and 9th rosette leaves of the seedlings shown in A. Every dot represents data from the length/width ratio of one leave (n = 20). Letters indicate significant differences (ANOVA, P < 0.05).

Northern blotting detecting accumulation of TAS1-TasiR255, TAS2-TasiR1511, TAS3-TasiR5′D8, miR173, and miR390 in leaves of Arabidopsis plants shown in panel A. Values represent relative accumulation (RA) of siRNAs and values in Col-0 plants were set as 1. rRNA served as loading controls.

RT-qPCR analyzing accumulation of TAS-tasiRNA targets in rosette leaves of 4-week-old Arabidopsis plants. Values in Col-0 plants were set as 1. Data points represent means from three biological repeats. Error bars indicate SD. Letters indicate significant differences (ANOVA, P < 0.05). PPR, pentatricopeptide repeat (PPR); ARF3, auxin response factor 3.

Model of VISP1-mediate autophagic degradation of SGS3/RDR6-bodies in Arabidopsis. SGS3/RDR6-bodies mediate dsRNA synthesis for siRNA amplification. Virus infection induce overexpression of VISP1 and selective autophagy. VISP1, a cargo receptor, interacts with SGS3 via the VISP1 Arginine/lysine-rich motif (ARM), and recruits SGS3/RDR6 bodies to ATG8-PE docking site through the VISP1 ubiquitin-interacting motif (UIM). This then promotes selective autophagic degradation of SGS3/RDR6-bodies and negatively regulates amplification of vsiRNAs and tasiRNA.
Golgi-associated microtubules are fast cargo tracks and required for persistent cell migration (2019)
Figure 1

Figure 1 - Classification of GaMTs and non-GaMTs based on super-resolution imaging.

A. Cartoon description of the segmentation of a polarized cell into 4 quadrants. The red dots indicate the center of the nucleus and the cell leading edge membrane, and the black arrow indicates the cell front-rear direction. The red dashed line box indicates the STORM imaging region.

B. Left: representative conventional image of interphase MT network in the 1st quadrant of a Human Retinal Pigment Epithelium (HRPE) cell. Grey: α-tubulin; Yellow: TGN-46; Yellow dashed line: Golgi boundary. Right: the classified MT subgroups. Red: GaMTs; Green: non-GaMTs. Scale bar: 5 μm.

C. Box-whisker plot presents the proportion of GaMT and non-GaMTs. (data were pooled from 3 independent experiments and n= 36 cells). The ends of whiskers set as the 10% and 90% of the entire population. ***p < 0.001, two-tailed Mann-Whitney.

D. Six representative images of MT networks extracted from MT STORM images of HRPE cells under combinative KDs. Cells were rotated to orient their leading edges locating in the 1st quadrant. Upper: control cell; CLASPs KD cell; GCC185 KD cell. Lower: CLASPs + GCC185 KD cell; CAMSAP2 KD cell; AKAP450 KD cell. Red: GaMT; Green: non-GaMT. Scale bar: 5 μm.

E. Box-whisker plot presents the ratio of GaMT/non-GaMT under combinative KDs. (1 representative of 3 independent experiments and n= 12 cells). The ends of the whiskers are set at 10% and 90% of the entire population. *p < 0.05, **p < 0.01, ***p < 0.001, ns, no significant difference, unpaired t-test.

F. Time series show EB1 movement on individual GaMTs. Red: GaMT; Green: non-GaMT; White: EB1 tracks. Scale bar: 1 μm.

G. Time series show EB1 movement on individual non-GaMTs. Red line: GaMT; Green line: non-GaMT; White line: EB1 tracks. Scale bar: 0.5 μm.

H. EB1 velocity distributions of GaMTs and non-GaMTs. Red curve and green curve represent the Gaussian fittings (data were pooled from 3 independent experiments and n= 92 tracks). ***p <0.001, unpaired t-test.

See Supplementary Table 1 for the source data.
Golgi-associated microtubules are fast cargo tracks and required for persistent cell migration (2019)
Figure 2

Figure 2 - Cargo velocity on GaMTs and non-GaMTs.

A. Representative trajectories of E-cad cargos tracked over 120 s in an HRPE cell. Colors represent different average velocity ranges. Red: > 400 nm/s; Purple: 200~400 nm/s; Green: 100~200 nm/s; Yellow: 0~100 nm/s. Scale bar: 20 μm.

B. Total number of cargos within different velocity ranges in the 4 quadrants, respectively (Data were pooled from 3 independent experiments and n= 45 cells).

C. Proportions of cargos with different velocity in the 4 quadrants. Red: the 1st quadrant; Dark grey: the 2nd quadrant; Light grey: the 3rd quadrant; White: the 4th quadrant (Data were pooled from 3 independent experiments and n= 45 cells).

D. Flow chart shows the method of combining STORM imaging with single particle tracking in live cells. Red lines indicate the cargo trajectories. Lower right, MT networks (Grey) were extracted and present in purple.

E. Representative time series show one fast cargo moving on a GaMT and a slow cargo moving on a non-GaMT. Upper, conventional imaging; lower, STORM imaging. Red line: GaMT; Green line: non-GaMT; White circle: cargo position. Scale bar: 2 μm.

F. Marginal distributions show the velocity on GaMT (red dots) or non-GaMTs (green dots). Red curve and green curve, Gaussian fitting curves of velocity distribution on GaMT or non-GaMT, respectively. (Data were pooled from 3 independent experiments and n= 69 MTs). Peak of red curve (431.14 ± 9.7 nm/s) and peak of purple curve (188.96 ± 22.03 nm/s). ***p < 0.001, unpaired t-test.

G. Upper panel: time series show an example of several fast cargos present on GaMT. Lower panel: time series show one slow cargo on non-GaMT switching onto a GaMT and becoming faster. Red line: GaMT; Green line: non-GaMT; White circle: cargo position. White line, cargo trajectory. Scale bar: 2 μm.

H. Box-whisker plot presents the averaged acetylation level of fast or slow MT tracks. (Data were pooled from 2 independent experiments and n= 63 MTs). The ends of the whiskers are set at 10% and 90% of the entire population, ns, no significant difference, unpaired t-test.

I. Marginal distributions of the mean velocity of cargos on segments of Ac-MTs (orange dots) and non-Ac-MTs (purple dots). Orange curve and purple curve represent the Gaussian fittings (Data were pooled from 2 independent experiments and n= 63 MTs). ns, no significant difference, unpaired t-test.

See Table S1 for the source data.
Golgi-associated microtubules are fast cargo tracks and required for persistent cell migration (2019)
Figure 3

Figure 3 - non-GaMT harbors more MT repair sites where cargos frequently pause and reverse.

A. Five representative images of MT repair sites in HRPE cells under combinative KDs. Cells were rotated to orient their leading edges locating in the 1st quadrant. From left to right, control cell, CLASPs&GCC185 KD cell, CAMSAP2 KD cell, AKAP450 KD cell and Centrinone-B treated cell. Grey: α-tubulin; red: GTP-tubulin. Scale bar: 20 μm.

B. Box-whisker plot presents intensity analysis of MT repair sites in HRPE cells under combinative KDs. (1 representative of 3 independent experiments and n= 20 cells). ***p < 0.001, unpaired t-test.

C. Conventional image (left) and STORM image (right) of MTs and MT repair sites. Grey: α-tubulin; yellow: GTP-tubulin; green: Golgi. Scale bar: 5 μm.

D. MT repair sites on GaMTs and non-GaMTs extracted from C. Red: GaMTs; Green: non-GaMTs; yellow: GTP-tubulin.

E. MT repair sites on GaMTs and non-GaMTs separately presented in detail. White arrows indicates GTP-tubulin labeling sites. Scale bar: 5 μm.

F. Box-whisker plot presents the ratio of MT repair sites on GaMTs and non-GaMTs separately. (1 representative of 3 independent experiments and n= 8 cells). ***p < 0.001, unpaired t-test.

G. Representative time series show one fast cargo moving on a GaMT and slowing down at the MT repaire site. Red: GaMT; Green: non-GaMT; Yellow: GTP-tubulin; White circle: cargo position. Scale bar: 2 μm.

H. Representative time series show one cargo moving fast between two MT repair sites and slowing down at the MTrepair sites. Red: GaMT; Green: non-GaMT; Yellow: GTP-tubulin; White circle: cargo position. Scale bar: 2 μm.

l-J. Pause (I) and reverse (J) events of cargos on MT repair segment and non-MTs repair segment, respectively. (Data were pooled from 2 independent experiments and n= 22 cells). ***p <0.001, ns, no significant difference, unpaired t-test.

See Table S1 for the source data.

Data information: The ends of the whiskers are set at 10% and 90% of the entire population.
Golgi-associated microtubules are fast cargo tracks and required for persistent cell migration (2019)
Figure 5

Figure 5 - GaMTs are required for persistent cell migration but not for cell to change directions.

A. Random migration of HRPE cells during 24 h. Upper: control cell; Middle: CAMSAP2 KD cell; Lower: CLASPs&GCC185 KD cell. Red arrow and white dashed lines indicate the migration direction. Different migration trajectories are distinguished with different colors. Scale bar: 50 μm.

B-D. Rose-plot presents the representative directionality of an HRPE cell. (B) control cell; (C) CAMSAP2 KD cell; (D) CLASPs&GCC185 KD cell.

E. Box-whisker plot presents the migration persistence of control cell, CAMSAP2 KD cell and CLASPs&GCC185 KD cell. (1 representative of 3 independent experiments and n= 34 cells). The ends of the whiskers are set at 10% and 90% of the entire population, ***p < 0.001, unpaired t-test.

F. Controlled cell migration of 3 representative cells in PA-Rac1 assay. White spot: 440 nm laser spot; Green area: the original position of the cell; Red area: the current position of the cell; Yellow area: the overlapped area. Scale bar: 10 μm.

G. Cartoon presents a controlled migrating HRPE cell with Golgi, cell, nucleus. Black arrow with red dot: laser direction; Black arrow with orange dot: cell body direction; Black arrow with blue dot: nuclear direction; Black arrow and green dot: Golgi direction. H-J. Angle change curve of cell, nucleus and Golgi. Red-dashed line: laser angle; Blue line: nucleus angle; Orange line: cell angle; Green line: Golgi angle. H: control cell; I: CAMSAP2 KD cell; J: CLASPs&GCC185 KD cell.

K. Box-whisker plot shows the total time required for the nucleus, Golgi and cell body reaching the given laser angle. Blue: time required for nucleus; Green: time required for Golgi; Orange: time required for cell body. (1 representative of 3 independent experiments and n= 14 cells). The ends of the whiskers are set at 10% and 90% of the entire population, ***p < 0.001, ns, no significant difference, unpaired t-test.

See Table S1 for the source data.
Golgi-associated microtubules are fast cargo tracks and required for persistent cell migration (2019)
Figure 6

Figure 6 - GaMTs efficiently deliver post-Golgi cargos to the cell leading edge.

A Projections of 120 frame movies present leading edge delivery of two different types of cargos. Left: Ecad cargo; Right: Rab5 cargo. Yellow line mark the cell boundary. Scale bar: 20 μm.

B-D Examples of cargo delivery towards the leading edge, time series present at 40 s, 80 s and 120 s, respectively. B: control cell; C: CAMSAP2 KD cell; D: CLASPs&GCC185 KD cell. Red arrow heads indicate cargo current position. Size of cropped regions: 30 μm x 5 μm.

E Box-whisker plot presents the Ecad cargo number on GaMTs and non-GaMTs. (Data were pooled from 3 independent experiments and n= 36 cells). The ends of the whiskers are set at 10% and 90% of the entire population, **p < 0.01, unpaired t-test. F Box-whisker plot presents the number of Ecad cargos finally reached the leading edge membrane during 120 s. (1 representative of 3 independent experiments and n= 24 cells). The ends of the whiskers are set at 10% and 90% of the entire population, **p < 0.01, two-tailed Mann-Whitney.

G Box-whisker plot shows the number of Rab5 cargos that finally reached the leading edge membrane during 120 s. (1 representative of 3 independent experiments and n= 23 cells). The ends of the whiskers are set at 10% and 90% of the entire population, ns, no significant difference, two-tailed Mann-Whitney.

See Table S1 for the source data.
miR-10b expression in breast cancer stem cells supports self-renewal through negative PTEN regulation and sustained AKT activation (2016)
Figure 2

Figure 2. Self-renewal ability is controlled by miR-10b expression in MCF-7 cells.

(A) Upper panel, left: MCF-7/CD44+ cells were transfected with anti-miR-10b or a scrambled control, and qRT-PCR was performed. Upper panel, right: An aliquot of these cells was subjected to mammosphere-forming assays. The graph shows the mean ± SEM of the number of formed mammospheres per 4000 seeded cells in 3 independent experiments. Lower panel, left: Expression of miR-10b relative to RNU6B in MCF-7 cells stably transfected with a plasmid containing the miR-10b gene versus an empty vector. Results are the mean ± SEM values from 3 independent experiments. Lower panel, right: miR-10b-overexpressing MCF-7 cells analyzed by serial mammosphere assays. The mean ± SEM of the number of formed mammospheres per 4000 seeded cells in 3 independent experiments was plotted on each bar.

(B) Upper panel: Mammosphere formation was performed by seeding 20,000 MDA-MB-231/EpCAM+ cells, and after 3 weeks, the cells were disaggregated, counted, and reseeded. Stable miR-10b-overexpressing cells from 3 independent clones were tested in 3 passages, and cell number was plotted as the mean ± SEM.

(C) In vivo limiting dilution assays. miR-10-OE or control cells were subcutaneously injected in the backs of mice at 3 doses (1,000, 10,000 and 50,000 cells for MCF-7 and 5000, 10 000 and 20 000 for SKBR-3). Mice were sacrificed after eight weeks, and tumor burden was analyzed using the ELDA software. Plots are depicted in Fig EV2. The tables show the number of mice with tumors versus the total number of mice injected. Below the tables, the estimated frequency (freq) of stem cells for each group and p is shown. Stem cell frequency for MCF-7 cells was 1 in 34252 (range 94888-12364) in miR-10b-OE cells versus 1 in 238965 (Range 1817801-31414) in miR-scr cells. Stem cell frequency for SKBR3 cells was 1 in 5013 (Range 11135-2257) in miR-10b-OE cells versus 1 in 18778 (Range 45397-7767) in miR-scr cells.

(D) Stemness and EMT marker mRNA levels following miR-10b modulation. miR-10b, OCT4/3, SNAIL and Vimentin expression was quantified in breast cancer cell lines using transient transfection of miR-10b versus a miR-Scr vector (left) or anti-miR-10b versus anti-miR-ctl (right). Log-scale plots show the mean and SEM for the relative expression of each gene from triplicate independent transient transfections, each performed three times. A Mann-Whitney U test was used to compare expression between groups. SOX2, OCT4 and Vimentin were significantly modulated by up- or down-regulation of miR-10b (p=0.0043, p=0.0054 and p=0.000908, respectively). SEM, standard error of the mean.