EMBO/soda-nel huggingface dataset:
POLO REGULATES SPINDLY TO PREVENT PREMATURE STABILIZATION OF KINETOCHORE-MICROTUBULE ATTACHMENTS (2019)
Figure 10

Figure 10. Polo activity fine-tunes the RZZ-Spindly-dynein module to ensure the accuracy of KT-MT attachments.

(A) Representative immunofluorescence images of ZW10 streaming from KTs in Drosophila S2 cells in metaphase expressing either SpindlyWT-, SpindlyS499A- or SpindlyS499D-EGFP. ZW10 streaming is also shown in S2 cells expressing SpindlyWT- or SpindlyS499A-EGFP in a dynein-depleted background. Insets display magnifications of the outlined regions, which highlight streaming robustness.

(B) Graph represents the percentage of cells in metaphase showing different levels of ZW10 streaming (n≥35 cells for each condition, n=2 independent experiments).

(C) Graph represents the percentage of ZW10 levels at aligned KTs normalized to ZW10 levels at the spindle region. All values were normalized to the mean fluorescence intensity quantified in SpindlyWT-EGFP expressing cells, which was set to 100% (n≥470 KTs from at least 35 cells for each condition, n=2 independent experiments).

(D) Representative immunofluorescence images of calcium-stable KT-MT attachments in metaphase S2 cells expressing SpindlyWT-, SpindlyS499A-, SpindlyS499D- or SpindlyS499D-EGFP in a ZW10-depleted background. Insets display magnifications of the outlined regions which highlight different attachment configurations (E - end on; L - lateral; M - merotelic). Cartoon depicts the attachment configuration of the respective KT pair. Asterisk highlights an aligned KT pair in which a sister KT appears to be laterally attached to the end of a MT fiber. Plotted profiles show the overlap between CENP-C and tubulin signals for the highlighted KT. CENP-C was used as a KT reference.

(E) Graph represents the percentage of metaphase cells showing only end-on attachments or at least 1 KT with lateral or merotelic attachment, as shown in D (n≥44 cells for each condition, n≥2 independent experiments).

(F) Proposed model for Polo-mediated regulation of RZZ-Spindly and its impact on KT-MT attachment. High levels of active Polo during early mitosis render Spindly phosphorylated on Ser499, thus weakening its interaction with the RZZ complex at KTs. On laterally attached KTs, the presence of minus end-directed motor dynein allows the removal of phosphorylated Spindly while the RZZ complex is retained at high levels to inhibit premature formation of stable end-on attachments and therefore avoid merotely. As protein phosphatases are recruited to KTs, Polo activation status declines and Spindly is eventually dephosphorylated. Spindly is now able to bind Zwilch and the Spindly-RZZ complex is stripped from KTs through dynein, which enables the stable conversion from lateral to end-on attachments and SAC silencing.

Data information: Statistical analysis was calculated using a Kruskal-Wallis test for multiple comparisons. p values: ****, <0.0001. Data are shown as mean ± SD. Scale bar: 5 μm.

EXPANDED VIEW FIGURE LEGENDS
Atg1 kinase regulates autophagosome-vacuole fusion by controlling SNARE bundling (2020)
Figure 2

Figure 2: Ykt6 phosphorylation inhibits autophagy flux.

A) ykt6ts pho8∆60 cells containing an empty plasmid, GFP-Ykt6 or 3HA-Ykt6 on a centromeric plasmid and pho8∆60 cells containing an empty plasmid were grown at permissive temperature (23 ºC) to late exponential phase in selective medium. Cells were shifted to the restrictive temperature (37 °C) for 1 hour (light grey bars) and subsequently starved for 4 hours in SD-N medium at restrictive temperature (black bars). Pho8Δ60 alkaline phosphatase activity was measured in three independent biological experiments and the mean was plotted normalized to starved Pho8Δ60 alkaline phosphatase activity. Error bars represent the standard deviation.

B) Trichloroacetic acid extracts were prepared from ykt6ts cells containing an empty plasmid, 3HA-Ykt6 wild type, 3HA-tagged Ykt6 mutant variants or atg1∆ and wild type cells as controls. Cells were grown at the permissive temperature (23 ºC) to late exponential phase in selective medium. Cells were shifted to the restrictive temperature (37 °C) for 1 hour preincubation to inactivate the endogenous Ykt6 temperature sensitive protein, and harvested (0) or further incubated at 37 ºC for an additional 4 hours (4). Samples were analyzed by anti-Ape1 and anti-HA Western blotting. One representative experiment out of three biological replicates is shown. Wild type or atg1∆ cells served as controls. Note that the temperature shift results in enhanced Ape1 processing.

C) ykt6ts pho8∆60 cells containing an empty plasmid, 3HA-Ykt6 or 3HA-tagged Ykt6 mutant variants as indicated were analyzed as in A).

D) ykt6ts pho8∆60 cells containing an empty plasmid, 3HA-Ykt6, or ADH1 promoter driven 3HA-Ykt6 wild type or 3HA-tagged Ykt6 mutant variants as indicated were grown and Pho8∆60 activity was monitored as described in A).

E) Ykt6 levels were analyzed from the strains shown in D) and corresponding cell extracts were analyzed by anti-HA and anti-Pgk1 Western blotting.
Atg1 kinase regulates autophagosome-vacuole fusion by controlling SNARE bundling (2020)
Figure 3

Figure 3: Ykt6 phosphorylation affects early and late steps in autophagy.

A) ykt6ts cells containing centromeric plasmids as indicated were grown at permissive temperature (23 ºC), shifted to the restrictive temperature at 37 ºC for 1 hour before starvation for 1 hour at 37 ºC. Cells were lysed and the total extract (T) separated into a cytoplasmic (S) and a 100'000x g membrane pellet (P) fraction. Fractions were analyzed by anti-HA, anti-Tom70 and anti-Pgk1 Western blotting. One representative experiment out of three biological replicates is shown.

B) vam3∆ or ykt6ts vam3∆ cells containing the indicated centromeric plasmids were grown at permissive temperature (23 ºC), shifted to the restrictive temperature at 37 °C for 1 hour followed by 1 hour starvation in SD-N at 37 ºC. After cell lysis, samples were subjected to proteinase K (ProtK) and Triton X-100 (TX100) treatment as indicated and analyzed by anti-Ape1 Western blotting. Ape1*: proteinase K-resistant fragment of Ape1. One representative experiment out of three biological replicates is shown.

C) Trichloroacetic acid extracts were prepared from ykt6ts cells containing an empty plasmid, 3HA-Ykt6 wild type, or 3HA-tagged Ykt6 mutant variants. Cells were grown at the permissive temperature (23 ºC) to late exponential phase in selective medium, shifted to the restrictive temperature (37 °C) for 1 h preincubation and continued to grow at the restrictive temperature for another 4 hours before harvesting. Samples were analyzed by anti-CPY and anti-Pgk1 Western blotting. One representative experiment out of three biological replicates is shown.

D) Exponentially growing ykt6ts cells at permissive temperature (23 ºC) containing centromeric plasmids as indicated were shifted to the restrictive temperature (37 ºC) for 1 hour before starvation for 4 hours in SD-N. Trichloroacetic acid extracts were prepared. Lipidation of Atg8 was analyzed on a 15 % SDS-PAGE containing 6 M urea by anti-Atg8 and anti-Pgk1 Western blotting. One representative experiment out of three biological replicates is shown.
Atg1 kinase regulates autophagosome-vacuole fusion by controlling SNARE bundling (2020)
Figure 4

Figure 4: Ykt6 phosphorylation prevents autophagosome-lysosome fusion by inhibiting SNARE bundling.

A and B) GFP-Atg8 atg1∆, GFP-Atg8 atg1∆ vam3∆, GFP-Atg8 wild type, GFP-Atg8 vam3∆, GFP-Atg8 ykt6ts or GFP-Atg8 ykt6ts vam3∆ cell containing centromeric plasmids as indicated were grown at permissive temperature (23 ºC), shifted to the restrictive temperature at 37 ºC for 2 hours before starvation for 1 hour at 37 ºC in SD-N. Representative microscopy images are shown in A). GFP-Atg8 puncta per cell were quantified from 3 biological replicates (n≥100 cells) and the mean was plotted in B). Error bars are SD. Scale bar: 5 µm.

C and D) Autophagosomes were prepared from GFP-Atg8 vam3∆ pep4∆ or GFP-Atg8 ykt6ts vam3∆ pep4∆ cells containing centromeric plasmids as indicated. Cells were starved for 16 hours at the permissive temperature (23 °C) to allow formation of mature, closed autophagosomes and shifted for 1 hour to the restrictive temperature before harvesting. Vacuoles were isolated from Vph1-4xmCherry atg15∆ pep4∆ cells grown under rich conditions at 30 °C. Fusion reactions were incubated at the restrictive temperature (30 °C) for 2 hours. Representative images are shown in C). Scale bar: 1 µm. The graph in D) shows the mean from at least three independent biological experiments. Error bars represent the standard deviation.

E) The indicated strains were grown to late exponential phase at permissive temperature (23 ºC), shifted to the restrictive temperature at 37 °C for 1 hour followed by starvation in SD-N for 1 hour at 37 ºC. 3HA-Ykt6 and its mutant variants expressed from a centromeric plasmid were immunoprecipitated (IP) and analyzed by anti-HA Western blotting. Co-precipitating proteins were analyzed by anti-Vam3 and anti-Vti1 Western blotting. One representative experiment out of three biological replicates is shown.
Foxd3 controls heterochromatin-mediated repression of repeat elements and 2-cell state transcription (2021)
Figure 1

Figure 1: FOXD3 binds to MERVL and MSR elements.

(A) Transcription factor binding sites with Transfac IDs predicted by PROMO in repeat element consensus sequences.

(B) Electrophoretic mobility shift assay (EMSA) with increasing concentration (0, 15nM, 30nM, 60nM, 1.25μM and 2.5μM) of GST-FOXD3 and fixed concentration (50nM) of 5'-Cy5 labelled dsDNA oligonucleotides (35 bp each) from Sox15 promoter (positive control), MSR, MERVL-LTR, MERVL-int, L1MdA and Hprt promoter (negative control). The experiment was repeated 3 times.

(C): Metadata analysis plots of FOXD3 ChIP-Seq (blue) and input (green) over MSR, MERVL-int, MT2_Mm and L1MdA consensus sequences including 1 kb flanking the start (RS) and end (RE) of each repeat. Y axes indicate normalized reads per genome coverage (RPGC). Predicted FOXD3 binding sites are marked in yellow and enrichment of FOXD3 over input is indicated in red. FOXD3 enrichment is seen in MSR, MERVL-int, MERVL-LTR but not in L1MdA.

(D) ChIP-qPCR enrichment of FOXD3 in mESCs using primers specific for MSR, MERVL-LTR, MERVL-int (including and excluding the FOXD3 cognate motif), L1MdA promoter sequences. Sox15 promoter and Actb promoter primers are used as positive and negative control respectively. Data is represented as percentage of input and the average of 3 biological replicates is plotted. Error bar indicates standard error of the mean (SEM). Asterisks indicate statistically significant differences compared to no antibody control levels (*: p< 0.05, paired t-test).
Foxd3 controls heterochromatin-mediated repression of repeat elements and 2-cell state transcription (2021)
Figure 2

Figure 2: MSR, MERVL and 2CLC specific genes are upregulated in Foxd3 KO cells:

(A) MA plot showing analysis of up and down regulated genes and repeats in total RNA-Seq preparations of control and Foxd3 KO cells. Significantly dysregulated genes are shown in blue and repeats are shown in yellow. The most highly upregulated repeat is marked in red. Data from 2 biological replicates is plotted.

(B) Validation of select upregulated repeats in Foxd3 KO cells by RT-qPCR. The data is plotted as average fold change relative to control, after normalization to Gapdh. Error bars indicate SEM (n=3 biological replicates). Asterisks indicate statistically significant differences when compared to control (**: p<0.005, *: p<0.05, paired t-test).

(C) Immunofluorescence analysis of the MERVL-gag protein in control and Foxd3 KO cells. Nuclei are labelled with DAPI. The percentage of counted cells (~150 cells per experiment) exhibiting immunofluorescence signal is indicated. Scale bar represents 10 μm, n= 3 biological replicates.

(D) Venn diagram representing the overlap between genes upregulated in Foxd3 KO cells (yellow) and in MERVL: Tomato+ cells (representing 2CLCs, blue) (Macfarlan et al, 2012).

(E) Immunofluorescence analysis of 2CLC markers ZSCAN4 and DPPA3 in control and Foxd3 KO cells. Nuclei are labelled with DAPI. The percentage of counted cells (~150 cells per experiment) exhibiting the immunofluorescence signal is indicated. Scale bar represents 10 μm, n=3 biological replicates.

(F) Heat map depicting the comparative gene expression profiles of the top 100 upregulated genes in Foxd3 KO cells. The columns indicate relative expression in Foxd3 KO cells, 2-cell stage embryos and MERVL: tomato+ cells.
Foxd3 controls heterochromatin-mediated repression of repeat elements and 2-cell state transcription (2021)
Figure 3

Figure 3: FOXD3 DNA binding domain is essential for regulation of MERVL and 2CLC genes

(A) Electrophoretic mobility shift assay (EMSA) with increasing concentrations (0, 30nM, 60nM, 1.25μM, 2.5μM and 5μM) of recombinant GST-FOXD3, DNA binding mutants GST-FOXD3 M1 and GST- FOXD3 M2 and fixed concentration (50nM) of 5'-Cy5 labelled dsDNA oligonucleotides (35 bp each) representing MERVL and MSR. The experiment was repeated 3 times.

(B) RT-qPCR analysis of MERVL, MSR and L1MdA in Foxd3 cKO cells, Foxd3 cKO cells over expressing full length FOXD3 or FOXD3 M1 without and with 4-OHT. Data is represented as fold change relative to respective untreated cells after normalizing to Gapdh. The average data for 3 biological replicates is plotted, error bars represent standard error mean (SEM). Asterisks indicate statistically significant differences. (**: p< 0.005, *: p<0.05 paired t-test).

(C) Immunofluorescence analysis of MERVL-gag protein in Foxd3 cKO cells, Foxd3 cKO cells over expressing full length FOXD3 or FOXD3 M1 without and with 4-OHT. Nuclei are labelled with DAPI. The percentage of counted cells (~150 cells per experiment) exhibiting immunofluorescence signal is indicated. Scale bar represents 10μm n= 3 biological replicates.

(D) RT-qPCR analysis of, 2C genes Zscan4c and Dppa3 in Foxd3 cKO cells, Foxd3 cKO cells over expressing full length FOXD3 or FOXD3 M1 without and with 4-OHT. Data is represented as fold change relative to respective untreated cells after normalizing to Gapdh. The average data for 3 biological replicates is plotted, error bars represent standard error mean (SEM). Asterisks indicate statistically significant differences. (**: p< 0.005, *: p<0.05 paired t-test).

(E) Immunofluorescence analysis of the ZSCAN4 protein in Foxd3 cKO cells, Foxd3 cKO cells over expressing full length FOXD3 or FOXD3 M1 without and with 4-OHT. Nuclei are labelled with DAPI. The percentage of counted cells (~150 cells per experiment) exhibiting fluorescence signal is indicated. Scale bar represents 10μm n=3 biological replicates.
Foxd3 controls heterochromatin-mediated repression of repeat elements and 2-cell state transcription (2021)
Figure 4

Figure 4: FOXD3 recruits SUV39H1 to repeat elements.

(A) GFP immunoprecipitation analysis using GFP-SUV39H1 and GFP-SUV39H2 expressing Suv39h dn mESCs. The top panel represents immunoblot using GFP antibody and the bottom panel represents immunoblot using FOXD3 antibody. The experiment was repeated 3 times using biological replicates.

(B) Volcano plot depicting significantly upregulated and downregulated repeats in Suv39h dn ES cells compared to wild type ES cells determined by RNA-Seq analysis. MSR (GSAT_MM), MERVL-int, L1MdA and MT2_Mm are labelled.

(C) Metadata profile showing enrichment of SUV39H1 and H2 over the MERVL consensus sequence including 1kb sequences flanking the start (RS) and end (RE) of the repeat. Y axes represent reads per genome coverage (RPGC).

(D) ChIP-qPCR analysis of endogenous SUV39H1 enrichment over MERVL, MSR and L1MdA in control and Foxd3 KO cells. Actb promoter is used as negative control. Data is represented as average percentage input from 3 biological replicates. Error bar indicates SEM (*: p<0.05 paired t-test).

(E) ChIP-qPCR analysis revealing H3K9me3 enrichment over MERVL, MSR and L1MdA in control and Foxd3 KO cells. Actb promoter is used as negative control. Data is represented as average percentage input from 3 biological replicates. Error bar indicates SEM (**: p<.005, *: p<0.05 paired t-test).

(F) Metadata analysis of H3K9me3 enrichment over MSR and MERVL consensus sequences in WT mESCs (blue) and Suv39h dn mESCs (green). Relative enrichment is plotted along the repeat start (RS) and repeat end (RE) including ± 1 kb flanking sequences. Y axis represents reads per genome coverage (RPGC).

(G) Immunofluorescence analysis of control and Foxd3i GFP-SUV39H1 expressing Suv39h dn mESCs stained with DAPI, FOXD3 and H3K9me3. The percentage of counted cells (~150 cells per experiment) exhibiting fluorescence signal is indicated. Scale bar=2μm. n= 3 biological replicates.

(H) FOXD3 represses MERVL and MSR in mouse ES cells: FOXD3 (green) binds to MERVL and MSR and recruits SUV39H1 (yellow) to the repeats. SUV39H1 facilitates repression of the repeats by establishing H3K9me3 (orange hexagons). Foxd3 depletion disrupts the recruitment of SUV39H1 and leads to a reduction in H3K9me3 over MERVL and MSR. This in addition to an increase in H3K4me3 (green hexagons) on the region results in the activation of MSR and MERVL and a concomitant increase in 2CLC specific genes, shifting the balance between mESCs (Green and purple) and 2CLCs (Brown and blue). This is accompanied by a perturbation of the classical condensed heterochromatic foci in the nucleus. Foxd3 thus helps maintain ES cells in a pluripotent state by regulating MERVL and MSR.

Expanded View Figure legends
Functional interaction between autophagy and ciliogenesis (2013)
f1

a, Percentage of ciliated cells in control (Ctrl) and IFT20− MEFs (*P = 0.030, n = 3) and wild-type (WT) and Ift88−/− KECs (**P = 0.003, 25 cells each, n = 3) after 24 h of serum removal. Arrows, cilia. b, LC3 immunoblot (left) for autophagic flux quantification (right) in Ctrl and IFT20− MEFs (top, **P = 0.0003, n = 7), and in WT and Ift88−/− KECs (bottom, *P = 0.035, n = 9). PI, protease inhibitors; S, serum. c, Autophagic flux in WT and Ift88−/−KECs by mCherry–GFP–LC3 puncta quantification. Puncta: yellow, autophagosomes; red only, autophagolysosomes; total, both together (**P = 0.008, *P = 0.025). d, LC3 immunoblot (left) for quantification of autophagosome formation (right). (*P = 0.048, n = 4). e, Electron microscopy images (left) and morphometric quantification of autophagic vacuoles (AV, right) in Ctrl and IFT20− MEFs (top, **P = 0.001, 10 fields in 2 experiments) and KECs WT and Ift88−/− (bottom, *P = 0.037, 5 fields). Arrows, autophagic vacuoles (AV, black) and endosomal compartments (yellow). f, Percentage of ciliated MEFs after induction of cilia resorption with 50 ng ml−1 platelet-derived growth factor (PDGF) treatment. (**P = 0.001, ***P = 0.0009, 121, 230 and 162 cells, n = 5). g, LC3 immunoblot (top) for autophagic flux quantification (bottom) upon PDGF treatment (*P = 0.005, n = 3). Scale bars in a, 10 &mgr;m. n.s., statistically non-significant. Mean ± s.d. in b, d and mean ± s.e.m. in other panels.
Functional interaction between autophagy and ciliogenesis (2013)
f2

a, GFP–LC3 puncta pattern (left) and quantification (right) in MEFs treated with purmorphamine (Purmo; top, *P = 0.028, n = 4), in Ptc−/− MEFs (middle, *P = 0.015, n = 4) and after myc–GLI1 overexpression (bottom, *P = 0.012, n = 3). b, Autophagic flux by mCherry–GFP–LC3 puncta quantification upon purmorphamine treatment in MEFs in the presence of serum. Yellow, autophagosomes; red only, autophagolysosomes; total, both compartments. (*P = 0.0323, 40 fields). c, LC3 immunoblot (left) for autophagic flux quantification (right) in Ctrl (**P = 0.001) and MEFs knocked down for SMO (SMO−) (**P = 0.0001) upon purmorphamine treatment (*P = 0.009, n = 4). d, Autophagic flux by mCherry–GFP–LC3 puncta quantification in WT and Ift88−/− KECs upon purmorphamine treatment (*P = 0.046, **P = 0.008, 40 fields). e, LC3 immunoblot (left) for autophagic flux quantification (right) in Ift88−/− KECs overexpressing myc–GLI1 (**P = 0.00006, n = 3). f, Autophagic flux by mCherry–GFP–LC3 puncta quantification in MEFs treated with cyclopamine upon serum removal (*P = 0.011, **P = 0.013, 25 fields). Scale bars, 10 &mgr;m. Mean ± s.d. in c, f and mean ± s.e.m. in other panels.
Functional interaction between autophagy and ciliogenesis (2013)
f3

a, Co-immunostaining (top) and 3D reconstruction (middle and bottom) for the indicated autophagy-related proteins (ATGs, green) and acetylated tubulin (red, ciliary axoneme marker) in KECs maintained in the absence of serum for 24 h. Yellow arrows, colocalization. b, Schematic representation of the ATGs associated with the axoneme (top left). Co-immunostaining for ATGs and acetylated tubulin in cilia isolated by peeling off. Individual channels and merged images are shown. c, Co-immunostaining (bottom) and 3D reconstruction (top) for ATGs (green) and gamma-tubulin (red, basal body marker) in the same cells and conditions. Arrows, colocalization (yellow), no colocalization (white). d–f, Percentage of cells with colocalizing ATGs in the basal body (BB) in WT and Ift88−/− KECs in the presence or absence of serum. d, Serum- and IFT-dependent association of ATGs with the BB (VPS34, †P = 0.04, **P = 0.002, n = 4; ATG16L, ††P = 0.0003, **P = 0.0009, 15 cells each per experiment, n = 7). e, IFT-dependent but serum-independent association of ATGs with the BB (**P = 0.001, ***P = 9.33768 × 10−6, n = 4; *P = 0.018, 15 cells each per experiment, n = 5). f, BB association of ATGs independent on serum or IFT (ATG5, n = 5; LC3, n = 8; 15 cells each per experiment). Scale bars, 10 &mgr;m. Mean ± s.e.m.
Functional interaction between autophagy and ciliogenesis (2013)
f4

a, b, Immunoblot for the indicated proteins of WT and Ift88−/− KECs subjected to co-immunoprecipitation of IFT20 (a) and ATG16L (b). Inp, 1/10 input; IP, immunoprecipitate; FT, 1/10 flow-through. c, Immunoblot for ATG16L and clathrin in MEF homogenate (Hom), and the pellets from 1 h centrifugation at 100,000g (organelles (Org)), 300,000g (300k) and 500,000g (vesicles, 500k). d, Immunoblot for IFT20 and ATG16L in the same fractions isolated from Ctrl, IFT20− and IFT88− MEFs in the presence (top) and absence (bottom) of serum. e, Immunogold electron microscopy for ATG16L (15-nm particles) and IFT20 (10-nm particles) in vesicles from MEFs isolated by the same method as in c. Right, quantification of IFT20 and ATG16L presence in the vesicles (11 fields). Mean ± s.e.m.
Functional interaction between autophagy and ciliogenesis (2013)
f5

a, b, Ciliated Atg5−/− MEFs in serum+ (*P = 0.038, 25 cells each per experiment, n = 3) (a) or serum− (*P = 0.006, nonlinear fit regression, 25 cells each per experiment, n = 3) (b). c, Scanning electron microscopy (SEM) of primary cilia. Arrows, cilia-associated vesicles; arrowheads, ciliary pocket. d, Cilia length quantification from SEM (*P = 0.027 n = 28; **P = 0.001, ††P = 0.0001, n = 23). e, f, GLI1 (*P = 0.04, n = 3) (e) and GLI2 (***P = 0.0005, **P = 0.0046, †P = 0.0132, n = 3) (f) messenger RNA expression in Atg5−/− MEFs. g, h, IFT20 immunoblot in Atg5−/− (g) and WT MEFs with lysosomal inhibitors (NL) (h). i, IFT20 immunoblot in cytosol (Cyt), homogenate (Hom) and autophagosomes (APG). j, IFT20 protein levels in Atg5−/− MEFs (*P = 0.038, n = 6). k, IFT20 immunoblot in Atg5−/− MEFs. n.s., statistically non-significant. Mean ± s.d. in d and j and mean ± s.e.m. in other panels.