EMBO/soda-nel huggingface dataset:
Selective autophagy degrades DICER and AGO2 and regulates miRNA activity (2012)
figf3

(a) Western blot analysis of GEMIN4 in fractions from a discontinuous gradient of Histodenz (15%, 20%, 24%, 26%) described to enrich autophagosomes (light AV) and autophagolysosomes (heavy AV). HEK293T cells were treated with CQ (20 μM, 16 h). Fractions enriched in multivesicular body (MVB) and lysosomal markers are indicated. (b) Western blot of GEMIN4 or loading control (TUBA) in HeLa cells treated with control siRNA or siRNA targeting ATG5, ATG6 or ATG7. (c) Western blot analysis of GEMIN4 or control (TUBA) in HeLa cells cultured in DMEM 10% FBS (Serum), Hank’s buffered salt solution (Starve) for 2 h, or treated for 16 h with control (DMSO), BAF (200 nM), RAP (20 nM), RAP+BAF, or PP242 (100 nM). (d) Top, western blot of GEMIN4 immunoprecipitates (IP) from HeLa cells. Bottom, western blot analysis of GEMIN4 in immunoprecipitates of anti-NDP52, -p62 or control antibody from HeLa cells. (e) Western blot of DICER immunoprecipitates from HeLa cells. (f) Western blot of AGO2 immunoprecipitates from HeLa cells. (g) Western blot analysis of GEMIN4, compared with loading controls (DCP1A, CLN3) in HeLa cells treated with control siRNA or siRNA targeting GEMIN3 and GEMIN4. (h) Western blot analysis of HeLa cells treated with indicated combinations of control siRNA or siRNA targeting GEMIN3, GEMIN4 and ATG5. Western blot analysis of CLN3 and DCP1A are used to demonstrate equal loading of wells. (i) Western blot analysis with anti-UB monoclonal antibody (mono- and polyubiquitylated proteins, FK2 clone) in Flag immunoprecipitates performed under stringent denaturing conditions. Flag–AGO2 was induced for 24 h with tetracycline in HEK293T cells with Tet-inducible Flag–AGO2 stably integrated that had been treated 24 h before with the indicated siRNAs. (j) Western blot analysis of HeLa cells treated with control siRNA or siRNA targeting ATG5 for 24 h. TUBA,
α-tubulin loading control. (k) Top, RT–qPCR analysis of miR-16 or let-7a levels in AGO2 immunoprecipitates from DMSO- and BAF-treated HeLa cells. Error bars represent s.e.m. Bottom, a representative western blot of AGO2 immunoprecipitates used in one replicate of these experiments. Uncropped images of blots are shown in Supplementary Fig. S3.
Selective autophagy degrades DICER and AGO2 and regulates miRNA activity (2012)
figf4

(a) Northern blot of miR-16 and let-7a in HeLa cells treated with siRNA for 84 h. Levels of U6, ethidium-bromide-stained total RNA on northern membrane (loading) and 28S and 18S ribosomal RNA on 1% agarose gel are shown as controls for equal loading. (b–d) RT–qPCR analysis of let-7a and miR-16 (b), pre-let-7a and pre-miR-16 (c) and let-7a* and miR-16* (d) in HeLa cells depleted of NDP52 or ATG5 with siRNA (84 h).
n = 3 independent experiments; error bars represent s.e.m. (e) Quantification of fluorescence signal in AGO2 immunoprecipitates from HeLa cell extracts incubated with duplexes of let-7a–let-7a* labelled on the 3′ end of either let-7 or let-7a* with FITC. Cells were treated 16 h before with DMSO (control), BAF (200 nm) or geldanamycin (10 μM). Representative results of one experiment are shown. (f) Western blot analysis of AGO2 immunoprecipitates (IP) used in e. (g) Normalized results of four independent experiments (n = 4) identical to e. Error bars represent s.e.m. Uncropped images of blots are shown in Supplementary Fig. S3.
Selective autophagy degrades DICER and AGO2 and regulates miRNA activity (2012)
figf5

(a) Renilla luciferase expression controlled by an exogenous siRNA–siRNA* duplex targeting two partially complementary sites in HeLa cells depleted of ATG5 or ATG7 with siRNA (black and grey bars correspond to two independent siRNAs). Results are normalized to firefly luciferase and Renilla luciferase expression in cells treated with a nonspecific siRNA–siRNA* duplex.
n = 2 independent experiments. (b) Renilla luciferase expression controlled by let-7 in HeLa cells depleted of NDP52 or ATG5 with siRNA (black and grey bars are independent siRNAs). Results are normalized to firefly luciferase and a version of the Renilla luciferase construct with mutated let-7 binding sites.
n = 3 independent experiments. The asterisks denote mutations in the let-7 target sites of the reporter. (c) Western blot analysis of endogenous miRNA-repressed proteins (RAS, HMGA2 and CDK6 are repressed by let-7; CDK6 is also repressed by miR-16 (bottom panel)) or control (TUBA,
α-tubulin) in HeLa cells treated with control siRNA or either of two independent siRNAs targeting ATG7. Cells were treated in parallel with nonspecific antagomir or let-7 targeting antagomir. (d) Renilla luciferase expression controlled by the DICER 3′UTR in HeLa cells treated with control antagomir or anti-let-7 antagomir. (e) Western blot analysis of DICER in HeLa cells treated with control siRNA or either of two independent siRNAs targeting ATG7. Cells were also treated with nonspecific antagomir or let-7 targeting antagomir. (f) Renilla luciferase expression controlled by the DICER 3′UTR in HeLa cells treated with either of two independent siRNAs targeting ATG5 or a control siRNA. All error bars are s.e.m. Uncropped images of blots are shown in Supplementary Fig. S3.
Peroxisomal fission is modulated by the mitochondrial Rho-GTPases, Miro1 and Miro2 (2019)
Figure 1

Figure 1: The first GTPase domain and transmembrane domain of Miro1 control its peroxisomal localisation.

A) Representative zooms of WT MEFs transfected with GFP-tagged Miro1 and Miro2 (GFPMiro1 and GFPMiro2). Control is GFP fused to the first 70 amino acids of Tom70. Tom20 and catalase stain mitochondria and peroxisomes, respectively. Merge is of Miro (green) and catalase (magenta) with colocalisation shown as white. White arrowheads highlight an area where an isolated peroxisome is situated.

B) Quantification of the extent of GFP signal on peroxisomes.

C) Pulldown of overexpressed Pex19 in Cos7 cells with a Pex19 antibody shows interaction with full-length Miro1, but not Miro1 lacking the transmembrane domain.

D) Schematic of Miro1 truncation mutants used in E-G. TM denotes the transmembrane domain.

E) Representative images of Miro1 truncation constructs expressed in DKO MEFs. Mitochondria and peroxisomes are stained with Tom20 and catalase, respectively. White arrowheads highlight an area where an isolated peroxisome is situated.F) Quantification of the extent of peroxisomal localisation of the Miro1 truncation constructs.

G) Co-immunoprecipation of Miro1 truncation constructs and mycPex19 following transfection into Cos7 cells. GFP-tagged Miro1 truncation constructs were pulled down with GFP-Trap agarose beads and Pex19 was probed with myc antibody.

Data information: B) and F) One-way ANOVA with Newman-Keuls post hoc test was used for all comparisons (n=18 cells per condition over 3 independent experiments). *, ** and *** denote p < 0.05, p < 0.01 and p < 0.001 in comparison to Control, respectively, and ## and ### denote p < 0.01 and p < 0.001 in comparison to GFPMiro1, respectively. Data are represented as mean ± SEM. Scale bar is 5 μm.
Peroxisomal fission is modulated by the mitochondrial Rho-GTPases, Miro1 and Miro2 (2019)
Figure 2

Figure 2: Loss of Miro1 or Miro2 does not affect long-range peroxisomal trafficking.

A) Six consecutive frames of live trafficking of peroxisomes (pxDsRed signal at 1.5 seconds per frame). Yellow arrows show the trajectory of a fast-moving peroxisome in both WT and DKO MEFs. Scale bar is 5 μm.

B) Blind scoring of the number of long-range peroxisomal trafficking events in WT, Miro1KO and Miro2KO and DKO MEFs (n=42 cells over six independent experiments. Two different MEFs lines were used for each genotype).

C) and D) Quantification of individual track length and velocity, respectively (n=24 cells over three independent experiments).

E) Representative blots of Miro1, Miro2 and actin from whole cell lysates of wild-type and Miro1-floxed ERT Cre-recombinase MEFs treated with and without 4-OH tamoxifen for 48 hours. Lysates were taken one day after the end of treatment.

F) Quantification of long-range peroxisomal trafficking events by blind scoring of pxDsRed signal from a two-minute movie (n=18 cells per condition over three independent experiments).

G) Representative blot of Miro1 and Miro2 following knockdown of either Miro1 or Pex14 in HeLa cells.

H) Quantification of long-range peroxisomal trafficking events (visualised with pxGFP) in HeLa cells transfected with scrambled, Miro1 and Pex14 siRNA, by blind scoring of a five-minute movie (n=18 cells over three independent experiments).

Data information: For B) a Kruskall-Wallis with a Dunn's correction posthoc test was used to test for significance. Analysis for C), D) and H) is a one-way ANOVA with Newman-Keuls post-hoc test. ** denotes p < 0.01. Statistical significance in F) was calculated by two-way ANOVA. For B), C), D), F) and H), no statistical difference between conditions was observed, unless stated. Data are represented as mean ± SEM.
Peroxisomal fission is modulated by the mitochondrial Rho-GTPases, Miro1 and Miro2 (2019)
Figure 3

Figure 3: Loss of Miro does not impact basal peroxisomal distribution.

A) Representative images of WT, Miro1KO, Miro2KO and DKO MEFs seeded onto Y-shaped fibronectin micropatterns stained for mitochondria (Tom20 in red) and peroxisomes (Catalase in white). Scale bar represents 10 μm.

B) Normalised cumulative distribution of mitochondria in WT, Miro1KO, Miro2KO and DKO MEFs.

C) Distance at which 95% of mitochondria are distributed (Mito95) for all four genotypes of MEF.

D) Normalised cumulative distribution curves comparing the peroxisomal distribution from the centre of the cell to the periphery of WT, Miro1KO, Miro2KO and DKO MEFs.

E) Bar graph comparing the average distance at which 95% of the peroxisomal signal is distributed (Perox95).

Data information: A-E is n = 60 cells over three independent experiments. For C) and E) a one-way ANOVA with Newman-Keuls post-hoc test was used to test for statistical significance. *** represents p < 0.001. Data are represented as mean ± SEM.
Peroxisomal fission is modulated by the mitochondrial Rho-GTPases, Miro1 and Miro2 (2019)
Figure 4

Figure 4: Loss of Miro1/2 reduces short-range peroxisomal displacements.

A) Snapshots and tracks of pxDsRed signal in WT, Miro1KO, Miro2KO and DKO MEFs following live imaging at 1.5 seconds per frame for two minutes.

B) Quantification of median net displacement of individual peroxisomes (pxDsRed signal) for WT, Miro1KO, Miro2KO and DKO MEFs (n=42 cells over six independent experiments. Two different MEFs lines were used for each genotype).

C) Still images every 30 seconds of ER and peroxisomes (ER-DsRed pseudo-coloured to green and pxGFP pseudo-coloured to magenta, respectively) in WT and DKO MEFs taken from two-minute movies by live-cell spinning-disk microscopy. Arrows track individual peroxisomes associated with an ER tubule. Scale bar is 5 μm.

D) Quantification of the relative ER displacement over a two-minute movie using ER-DsRed signal in WT and DKO MEFs (n=20 cells per condition over three independent experiments).

E) Median net displacement of peroxisomes in WT and DKO MEFs. n=20 cells per condition over three independent experiments.

F) Median net displacement of peroxisomes in DKO MEFs with and without myc-tagged variant 4 of Miro1 (mycv4) (n=12 cells per condition over three independent experiments).

Data information: For multiple comparisons in B) a one-way ANOVA with Newman-Keuls post-hoc test was used to test for statistical significance. For D-F Student's t-test was used to calculate statistical significance. * and *** represent p<0.05 and p<0.001, respectively. Data are represented as mean ± SEM.
Peroxisomal fission is modulated by the mitochondrial Rho-GTPases, Miro1 and Miro2 (2019)
Figure 5

Figure 5: Miro regulates peroxisomal size, morphology and number.

A) Representative images of catalase staining (peroxisomes) in WT, Miro1KO, Miro2KO and DKO MEFs.

B) Bar graph comparing average peroxisomal area of WT Miro1KO, Miro2KO and DKO MEFs ( n = 60 cells per condition over three independent experiments).

C) Comparison of total peroxisomal number between WT, Miro1KO, Miro2KO and DKO MEFs. n = 60 cells per condition over three independent experiments.

D) Representative images of Control (GFP-tagged 1-70 of Tom70), GFPMiro1 and GFPMiro2 in WT MEFs stained with catalase (peroxisomes).

E) Representative images of Pex14 (peroxisomes) in DKO MEFs expressing either GFP (control) or GFP-tagged variant 4 of Miro1 (GFPv4).

F) Bar graph comparing average peroxisomal area of Control, GFPMiro1 and GFPMiro2 overexpressing WT MEFs (n=60 cells per condition over three independent experiments).

G) Comparison of average area of individual peroxisomes between DKO MEFs expressing either GFP (control) or GFP-tagged variant 4 of Miro1 (GFPv4) (n=30 cells per condition over three independent experiments).

Data information: Statistical test used in G) was a two-tailed Student's t test. Calculation of statistical significance in B), C) and F) was a one-way ANOVA with Newman-Keuls post-hoc. *, ** and *** denote p < 0.05, p < 0.01 and p < 0.001, respectively. Data are represented as mean ± SEM. Scale bar is 10 μm. Scale bar in zooms is 5 μm.
Peroxisomal fission is modulated by the mitochondrial Rho-GTPases, Miro1 and Miro2 (2019)
Figure 6

Figure 6: Miro negatively regulates the recruitment of Drp1 to peroxisomes and mitochondria.

A) Representative images of proximity-ligation assay (PLA) of Fis1 and Drp1 in WT and DKO MEFs. Scale bar is 10 μm. Red dots indicate interaction of Fis1 and Drp1. Blue is DAPI.

B) Quantification of fluorescent dots from PLA of Fis1 and Drp1 in WT and DKO MEFs. Dotted line indicates the sum of the Fis1 and Drp1 single antibody controls (n=3 experiments).

C) Representative images of a Fis1-Drp1 PLA in DKO MEFs expressing either GFP, GFPv1 (GFP-tagged variant 1 of Miro1) or GFPv4 (GFP-tagged variant 4 of Miro1) n=30 cells per condition over three independent experiments). Scale bar is 10 μm. Red dots indicate interaction of Fis1 and Drp1. Blue is DAPI.

D) Quantification of PLA fluorescent dots per cells between GFP, GFPv1 or GFPv4 transfected DKO MEFs (n=30 cells over three independent experiments).

E) Western blot of Drp1, Fis1 and actin from WT and DKO whole cell lysates.

F) Quantification of normalised band intensity of Drp1 and Fis1 in WT and DKO MEFs (n=3).

G) Zooms of endogenous Drp1, mitochondria (Mitotracker) and peroxisomes (PMP70) in WT and DKO MEFs. Scale bar is 5 μm.

H) Integrated density of Drp1 signal on Mitotracker-positive and PMP70-negative structures in WT and DKO MEFs.

I) Integrated density of Drp1 signal on PMP70-positive and Mitotracker-negative structures in WT and DKO MEFs.

Data information: G-I n=42 cells per condition over three independent experiments. For B), F), H) and I), statistical significance was quantified by a two-tailed Student's t test. In D) a one-way ANOVA with a Newman-Keuls post-hoc test was used to calculate statistical significance. *, ** and *** denote p < 0.05, p < 0.01 and p < 0.001, respectively. Data are represented as mean ± SEM.

Expanded view Figure legends
Karyopherin Kap114p-mediated trans-repression controls ribosomal gene expression under saline stress (2020)
Figure 1

Figure 1. Biochemical and structural characterization of yTBP and Kap114p interaction

A. Pull-down assays of yTBP (61-240) with Kap95p, Kap114p and Kap121p. GST-fused yTBP (61-240) was incubated with recombinant Kap95p, Kap114p and Kap121p. Unbound (S) and bound (B) samples were analyzed by SDS-PAGE and stained with Coomassie blue.

B. Band intensities of Kap-βs from the SDS-PAGE gels in (A) were quantified and normalized by GST-yTBP intensity. Quantitative plots show percent Kap-βs binding on yTBP.

C-E. ITC titration curves (upper) and binding isotherms (lower) of (C) Kap114p, (D) Kap95p, and (E) Kap121p with yTBP (61-240). Salt concentration and Kd values are indicated.

F. Cartoon representation of the Kap114p crystal structure. The A and B helices composed of each HEAT repeat are shown as gray cylinders. The HEAT8 hairpin, HEAT19 loop and additional HEAT18/19 loop are highlighted in blue, red and green, respectively. A 45o rotated view is shown on the right. Overall dimensions of the Kap114p structure are indicated.

G. Superimposition of Kap114p (gray) with cargo-bound Cse1p (light orange; PDB code: 1WA5). The HEAT8 hairpin and HEAT19 loop of Kap114p are colored blue and light green, respectively.

H. Schematic representation of the Kap114p domain configuration using the same color code as shown in F.

Data information: In (B-E), data represent mean ± standard deviation from three independent experiments. Differences were assessed statistically by two-tailed Student's t test; *P<0.05; ***P<0.001.
Karyopherin Kap114p-mediated trans-repression controls ribosomal gene expression under saline stress (2020)
Figure 2

Figure 2. Ran and yTBP binding by Kap114p

A, B. Purified recombinant Kap114p was mixed with RanQ69L or yTBP and analyzed by SEC. SEC (Superdex 200) elution profiles of the (A) Kap114pRanQ69L and (B) Kap114pyTBP complexes. Peak fractions were analyzed by SDS-PAGE and stained with Coomassie blue. The void volume (Vo) of the peak fraction and absorbance (a.u.) at 280 nm for each complex is indicated.

C-E. SV-AUC analysis of (C) yTBP, (D) Kap114p and (E) the Kap114pyTBP complex. The experimental data were analyzed using the Sedfit program and are presented above molecular weights calculated using standards (in brackets).

F. SE-AUC analysis of the Kap114pyTBP complex. The average molecular mass determined by the Sedfit program was ~133.5  kDa. The lower panel shows the residual difference (standard deviation) between experimental and fitted values.

G-N. Plots of the distance distribution function P(r) for the (G) Kap114pRanQ69L and (K) Kap114pyTBP complexes derived from experimental SAXS data using the GNOM program. (H, L) Rg-based dimensionless Kratky plot of the Kap114pRanQ69L (H) and Kap114pyTBP (L) complexes. Intersection of the two dotted gray lines indicates features of a compact protein. The ab initio envelopes determined by GASBOR for the (I) Kap114pRanQ69L and (M) Kap114pyTBP complexes. Models of the (J) Kap114pRanQ69L and (N) Kap114pyTBP complexes derived from SAXS profiles using FoXSDock and docked into the ab initio envelopes obtained from GASBOR.
Karyopherin Kap114p-mediated trans-repression controls ribosomal gene expression under saline stress (2020)
Figure 3

Figure 3. The HEAT8 hairpin of Kap114p is cruical for Ran binding

A. Schematic representation of the domain structures of the two deletion mutants, Kap114p (Δ347-371) and Kap114p (Δ899-956).

B. Pull-down assays of yTBP with Kap114p, Kap114p (Δ347-371) and Kap114p (Δ899-956). GST-fused yTBP (61-240) was incubated with recombinant Kap114p, Kap114p (Δ347-371) and Kap114p (Δ899-956). Unbound (S) and bound (B) samples were analyzed by SDS-PAGE and stained with Coomassie blue.

C. Band intensities of Kap-βs from the SDS-PAGE gels in (B) were quantified and normalized by GST-yTBP intensity. Quantitative plots show percent Kap-βs binding on yTBP.

D, E. ITC titration curves (upper) and binding isotherms (lower) of (D) Kap114p (Δ347-371) and (E) Kap114p (Δ899-956) with yTBP (61-240). Salt concentration and Kd values are indicated.

F. Relative fluorescence GFP intensities in the nucleus and cytoplasm of wild type, KAP114 knockout strains, and KAP114 knockout strains rescued by KAP114 deletion mutants.

G. His-tagged RanQ69L was incubated with recombinant Kap114p, Kap114p (Δ347-371) and Kap114p (Δ899-956). Unbound (S) and bound (B) samples were analyzed by SDS-PAGE and stained with Coomassie blue.

H. Band intensities of Kap-βs from the SDS-PAGE gels in (G) were quantified and normalized by His-Ran intensity. Quantitative plots show percent Kap-βs binding on His-Ran.

I. GST pull-down assays of yTBP (61-240) with Kap114p or Kap114p (Δ347-371) in the presence or absence of RanQ69L. GST-fused yTBP (61-240) was incubated with recombinant Kap114p and His-tagged RanQ69L at a molar ratio of 1:4:8 Kap114p:yTBP:RanQ69L. Unbound (S) and bound (B) samples were analyzed by SDS-PAGE and stained with Coomassie blue.

J. Analysis of Kap114p dissociation from yTBP in the presence of RanQ69L. Band intensities of Kap114p and Kap114p (Δ347-371) from the SDS-PAGE gels were used to determine the average fraction of bound protein.

Data information: In (C-E, H and J), data represent mean ± standard deviation from three independent experiments. In (F), data represent mean ± standard deviation from four independent experiments (40 yeast cells in total for each strain; 10 cells for one experiment). Differences were assessed statistically by two-tailed Student's t test; n.s.: not significant; **P < 0.01; ***P < 0.001.
Karyopherin Kap114p-mediated trans-repression controls ribosomal gene expression under saline stress (2020)
Figure 4

Figure 4. Kap114p modulates interaction between yTBP and DNA/TFIIA

A. Protein sequence alignment of the HEAT8 hairpin and TAF1-TAND1, and the HEAT19 loop and TAF1-TAND2 (numbers represent amino acid positions). α-helices were assigned based on crystal structures and are depicted as blue cylinders. The Phe57 anchor residue of TAND2 is indicated 24[]. Black color highlights amino acid identity and gray shading indicates amino acids with similar properties.

B. EMSA analysis of yTBP binding to DNA. We incubated 5 nM FAM-labeled oligonucleotide (TGTATGTATATAAAAC) with indicated concentrations of purified yTBP (0.125 to 8 μM) and then analyzed binding by 6% non-denaturing PAGE. Band intensities of free DNA from the gels (Top panel) were quantified and plotted (Bottom panel).

C. Indicated concentrations (0.25 to 16 μM) of purified recombinant Kap114p were incubated with yTBP (4 μM) and FAM-labeled DNA (5 nM), followed by native PAGE analysis. Kap114p (16 μM) was incubated with DNA without yTBP as a control. Band intensities of free DNA from the gels (Top panel) were quantified and plotted (Bottom panel).

D. Super-shift EMSA analysis of TFIIA binding to yTBPDNA complex. We incubated 5 nM FAM-labeled DNA and yTBP (0.5 μM) with indicated concentrations of purified TFIIA (0.08 to 5 μM) and then analyzed binding by 6% non-denaturing PAGE. Band intensities of free DNA from the gels (Top panel) were quantified and plotted (Bottom panel).

E. Indicated concentrations (0.06 to 4 μM) of purified Kap114p were added to the reaction containing 0.5 μM of TFIIA, yTBP and FAM-labeled DNA (TGTATGTATATAAAAC), before conducting native PAGE analysis. TFIIA and Kap114p (4 μM) were incubated with DNA without yTBP as a control. Band intensities of free DNA from the gels (Top panel) were quantified and plotted (Bottom panel). Electrophoretic bands containing protein-DNA complex and free DNA are indicated.

Data information: In (B-E), data represent mean ± standard deviation from three independent experiments.