EMBO/SourceData huggingface dataset:
Figure 5MITOL added polyubiquitin chains to K481 of IRE1α. Lysates of HEK293 cells transfected with each lysine mutant of IRE1α and indicated vectors were immunoprecipitated with anti-FLAG antibody, followed by immunoblotting with indicated antibodies. 481: K481R, 545: K545R, 568: K568R.IRE1α K481R interacted with MITOL. Lysates of HEK293 cells transfected with indicated vectors were immunoprecipitated with anti-FLAG antibody, followed by immunoblotting with indicated antibodies.Overexpression of IRE1α K481R induced apoptosis. MEFs were transfected with indicated vectors. After 24 hours, these cells were stained with Annexin V-FITCOverexpression of IRE1α K481R induced apoptosis. or subjected by immunoblotting with indicated antibodies (D).Enhanced RIDD activity in IRE1α K481R. MEFs were transfected with indicated vectors 24 hours prior to analysis (E, F). The RIDD activity was measured by qRT-PCR.Hyper-oligomerization of IRE1α K481R. MEFs were transfected with IRE1α-GFP or IRE1α K481R-GFP. After 24 hours, these cells were exposed with Tu for indicated periods and GFP signals were observed. The right panels show 5.5-fold magnification images of the boxed regions. Percentages of cells with IRE1α-foci were calculated from 100 cells by visual inspection in each independent experiment.Increased interaction of IRE1α K481R with BIM. Cells transfected with indicated vectors 24 hours prior to immunoprecipitation, followed by immunoblotting with indicated antibodies.
Figure 6Accumulations of MITOL and IRE1α in the MAM fraction. Each organelle fraction was isolated from MEFs by percoll density-gradient centrifugation, followed by immunoblotting with indicated antibodies. mito: pure mitochondria, micro: microsome, cyto: pure cytosol.High solubility of MITOL and IRE1α in the low dose digitonin. Raft-like structure including MAM was solubilized from the crude mitochondrial fraction of MEFs with indicated concentration of digitonin. S: supernatant, P: pellet.IRE1α regulation by MITOL occurred in the MAM-rich mitochondrial fraction. Crude mitochondrial fraction containing MAM (Mito plus MAM) and the cytosolic fraction containing ER (Cyto plus ER) were isolated from HEK293 cells transfected with indicated vector and subsequently immunoprecipitation assay was performed to detect the interaction between MITOL and IRE1αIRE1α regulation by MITOL occurred in the MAM-rich mitochondrial fraction. Crude mitochondrial fraction containing MAM (Mito plus MAM) and the cytosolic fraction containing ER (Cyto plus ER) were isolated from HEK293 cells transfected with indicated vector and subsequently immunoprecipitation assay was performed to detect the level of IRE1α ubiquitylation (D).Knockdown of PACS2 impaired the interaction of MITOL with IRE1α. HEK293 cells were transfected with either scramble RNA or siPACS2 and indicated vectors for 24 hours prior to immunoprecipitation assay.Knockdown of PACS2 inhibited IRE1α ubiquitylation by MITOL. HEK293 cells were transfected with either scramble RNA, siPACS2 and indicated vectors 24 hours prior to immunoprecipitation assay.Knockdown of Mfn2 inhibited IRE1α ubiquitylation by MITOL. HEK293 cells were transfected with either scramble RNA, siMfn2 and indicated vectors 24 hours prior to immunoprecipitation assay.
Figure 7MITOL ubiquitylated IRE1α in the spinal cord of mice. Lysates of the spinal cord in mice were immunoprecipitated with anti-IRE1α antibody, followed by immunoblotting with indicated antibodies. WT: MITOLF/F mice, MITOLnestin: MITOLF/F, Nestin-Cre mice.Loss of MITOL led to hyper-oligomerization of IRE1α in the spinal cord under ER stress. 3 month-old WT or MITOLnestin mice were treated with 1 mg/kg Tu for 24 hours. Spinal cord was solubilized with NP-40 lysis buffer and separated by sucrose-density gradient centrifugation.MITOL deletion resulted in hyper-activation of IRE1α RNase in ER stress. WT or MITOLnestin mice were injected with Tu for 24 hours and spinal cord was analyzed by qRT-PCR.Increased ER stress-induced apoptosis in the MITOL-KO spinal cord. 3 month-old WT or MITOLnestin mice were treated with Tu for 24 hours and each spinal cord was stained using TUNEL and hoechst (F) Arrowheads indicate the representative neurons.Increased ER stress-induced apoptosis in the MITOL-KO spinal cord. 3 month-old WT or MITOLnestin mice were treated with Tu for 24 hours and each spinal cord was stained using cresyl violet (G). Arrowheads indicate the representative neurons.
Figure 1The Dyf phenotype of the ts-che-3 mutant was confirmed, with animals grown at the permissive temperature (15ºC) showing the ability to uptake dye, while those raised at the restrictive temperature (25ºC) are Dyf. Scale bar, 20 µmUsing the homodimeric kinesin OSM-3 as a marker for IFT, proper localisation is seen in the ts-che-3 mutant at the permissive temperature, with the strongest signal at the basal body (bb). At the restrictive temperature, most of the OSM-3 signal is found within the axoneme of the short, bulbous cilia. Scale bar is 4 µmThe ts-che-3 mutant shows normal osmotic avoidance at permissive temperature but not at the restrictive temperature, indicating defective chemosensation. The osm-9 mutant is included as a positive control. n = 50 animals; error bars are standard error
Figure 2Three TZ markers (NPHP-4, CEP-290 and MKS-6) are shown at both the permissive (15ºC) and restrictive (25ºC) temperatures in the ts-che-3 mutant. At the permissive temperature, all three markers localise to the TZ, while at the restrictive temperature exhibit ectopic localisation (leakage) distally into the ciliary axoneme. While NPHP-4 and MKS-6 show significant accumulations, CEP-290 is grossly normal. At the permissive temperature, both IFT co-markers (CHE-13 and XBX-1) display strong localisation to the basal body (bb) and along the axoneme. At the restrictive temperature, the IFT markers show accumulation in the axoneme. Accumulations emphasized using asterisksTo determine if ciliary compartmentalisation could be restored, young ts-che-3 mutant adults were transferred to the permissive temperature after being raised at the restrictive temperature. The TZ proteins reassembled at the TZ, with loss of the ectopic accumulations, and IFT protein localisation was also restored-strongest at the basal body, similar to animals grown at the permissive temperature. (c) After 24 hours at the restrictive temperature, TZ proteins appear to begin to accumulate distally within the axoneme in ts-che-3 mutants. This is seen, in small amounts, for NPHP-4 and CEP-290 but with stronger ectopic localisation for MKS-6. The IFT proteins CHE-13 and XBX-1 also show accumulations in the axoneme after 24 hours at the restrictive temperature. Data information: For (A), (B), and (C), fluorescence quantification is shown for each marker at the indicated temperature in the heat maps on the right. Each point in the plot represents one pixel along the centre of the basal body (BB), transition zone (TZ) and middle segment (MS) regions. Dotted areas (three pixels) in the MS were used to quantify ectopic localisation (TZ markers) or accumulation (IFT markers) for statistical analyses. n = 10 cilia (4-7 animals), Kruskal-Wallis test, *P<0.05, **P<0.01. tz, transition zone; bb, basal body; scale bars are 4 µm
Figure 3In the ts-che-3 mutant grown at the permissive temperature (15ºC), both RPI-2/RP2 and TRAM-1/TRAM1 are localised to the PCMC, just proximal to the ciliary axoneme. At the restrictive temperature, TRAM-1 shows leakage into the axoneme with its strongest localisation at the PCMC. Intriguingly, RPI-2 now displays strong localisation to the axoneme, indicating that in the absence of IFT, RPI-2 may be targeted into the cilium. When ts-che-3 mutant animals grown at the restrictive temperature (25ºC) are shifted at young adult to the permissive (15ºC) for 24 hours, both TRAM-1 and RPI-2 exhibit restoration of their PCMC localisation. Consistent with other TZ markers, both MKS-2 and MKSR-1 show ectopic localisation in the axoneme when grown at the restrictive temperature compared to the permissive temperature. Also consistent with what was observed for other markers, after 24 hours at the permissive temperature, animals grown at the restrictive temperature show loss of the ectopic localisation. Fluorescence quantification is shown for each marker at the indicated temperature in the heat maps on the right. Each point in the plot represents one pixel along the centre of the basal body (BB), transition zone (TZ) and middle segment (MS) regions. Dotted areas (three pixels) in the MS were used to quantify ectopic localisation (MKS-2 and MKSR-1) or accumulation (RPI-2 and TRAM-1) for statistical analyses. n = 10 cilia (4-6 animals), Kruskal-Wallis test, *P<0.05, **P<0.01; scale bars are 4 µmLoss of CHE-3 function results in short, bulbous cilia containing electron dense accumulations and abnormal membrane-microtubule connections. Shown are transmission electron micrograph (TEM) cross sections of an amphid channel cilium in wild-type and che-3(e1124) null mutant animals. Representative images of wild-type cilia show intact middle segment (containing doublet microtubules) and transition zone (with Y-shaped links) (left top and bottom panels) regions. Representative images of che-3(e1124) cilia reveal apparently intact transition zones, with visible Y-link structures, but enlarged ciliary ends filled with electron-dense accumulations, which often appear vesicular. The bulbous structures reveal doublet microtubules associated with the membrane, and occasionally ectopic microtubule-to-membrane connections, which sometimes appear similar to transition zone Y-links in the region just distal to the seemingly "normal" TZ structure. Schematics show longitudinal (left images) and cross section (right images) representations of wild-type and che-3(e1124) cilia. tz, transition zone; pcmc, periciliary membrane compartment; scale bars are as indicated in (nm) for
Figure 4Using the IFT marker BBS-7, cilia lengths were measured for wild-type and the ts-che-3 mutant animals raised at the permissive (15ºC) and shifted to the restrictive temperature (25ºC) for three time points. After 12 hours some accumulations (indicated by asterisks in the fluorescence images) are observed within the cilia but there is not a significant reduction in cilium length. After 18 and 24 hours, significant reduction in ciliary length is observed and large accumulations of BBS-7 is seen within short bulbous cilia, similar to what is observed in ts-che-3 animals raised at the restrictive temperature. n = 30-34 cilia, one per animal; Kruskal-Wallis test, **P<0.01; error bars are mean absolute deviation; scale bars are 4 µmIFT velocities were measured for wild-type or ts-che-3 mutant animals (grown at permissive temperature) and imaged at the permissive temperature, restrictive temperature or after 3 hours at the restrictive temperature. Compared to wild-type, anterograde velocities in the ts-che-3 dynein mutant are similar, while IFT retrograde rates are highly reduced for both the middle and distal ciliary segments. Interestingly, the ts-che-3 IFT dynein mutant appears to not only have slow retrograde IFT trains, but the trains also appear much larger and less frequent. Initially upon the temperature shift (<15min) IFT rates are much faster for both wild-type and ts-che-3 dynein mutant, although retrograde remains much slower in the mutant. After 3 hours at the restrictive temperature, IFT velocities are intermediate between the rates seen at 15ºC and those observed at the initial shift to 25ºC
Figure 1A. Colony formation assay (CFA) with imatinib with representative images of colonies formed by each cell line within the panel. Error bars represent mean ±S.E.M., n=9, *P < 0.001 by two-tailed unpaired Student's t-test (H1915 P= 0.0004, H2110 P=0.0001).B. Western blot analysis after 1 hour treatment with imatinib.C. MTT assay for NSCLC cell lines and Beas-2B cells with dasatinib or DMSO. Error bars represent mean ±S.E.M., n=9, *P<0.001 by two-tailed unpaired Student's t-test (H1915 P=1.09×10-5, H2110 P=0.009 and A549 P=6×10-6).D. Western blot analysis after 1 h treatment with dasatinib.
Figure 2A -C. Drug-resistant ABL1-T315I rescues dasatinib- and imatinib-induced cytotoxicity. Western blot analysis of (A) transient and (B) tetracycline-inducible overexpression of ABL1-T315I in H1915 cells treated with dasatinib or imatinib.C. CFA with dasatinib or imatinib for H1915 cells with or without tetracycline-inducible expression of ABL1-T315I. Error bars represent mean ±S.E.M., n=9, *P<0.001 by two-tailed unpaired Student's t-test (control, DMSO vs dasatinib P=4.3×10-12 vs imatinib P=6×10-6; dasatinib, control vs tetracycline, P=9×10-7; imatinib, control vs tetracycline P=9.7×10-5).D-F. Knockdown of mutated ABL1 reduces viability of lung cancer cells. Top, western blot analysis of ABL1 knockdown in (D) Beas-2B, (E) H1915 and (F) H2110 cells upon transfection with three ABL1 siRNAs, negative control (scrambled) and CyclophilinB positive control. Bottom, MTT assay showing viability of (D) Beas-2B, (E) H1915 and (F) H2110 cells in response to ABL1 knockdown. Error bars represent mean ±S.E.M, n=9, *P<0.005, **P<0.001 by two-tailed unpaired Student's t-test (Beas-2B, scrambled vs siABL1-3 P=0.002; H1915, scrambled vs siABL1-1 P=0.004, vs siABL1-2 P=0.001, vs siABL1-3 P=0.002; H2110 scrambled vs siABL1-1 P=5×10-6, vs siABL1-2 P=2×10-5, vs siABL1-3 P=4.2×10-6).
Figure 4A and C. Root mean square deviation (RMSD) analysis of the movement of (A) αC-helix and (C) DFG motif in the active and inactive conformation of ABL1 kinase with R351W mutation (orange) compared to the wild type (WT) kinase (black). B and D. Time-lapse images show movement within the αC-helix (B) and DFG motif (D) of the inactive structure of the WT (black outline) and R351W (orange outline) constructs.E. left, western blot analysis of transient overexpression of wild type ABL1 (WT) and ABL1-R351W, compared to empty vector control (EV) in HEK293T cells. Right, quantification of p-CRKL signal based on densitometry analysis of blots from 6 independent experiments. Error bars represent ±S.D., n=6, *P=0.0004 by two-tailed unpaired Student's t-test.F. Overexpression of mutant ABL1 constructs with mutations identified in selected primary lung tumors (Ding et al, 2008) in H157 cells. Values indicate quantified band intensity for p-CRKL, relative to α-tubulin and HA-tagged ABL1.
Figure 2siRNA-mediated knockdown of PHPT1 in HeLa cells (24 h) with reference to GAPDH loading control. NT - non-targeting siRNA control.Representative SAX profile of trypsin digested HeLa lysate (Abs280 nm).Representative SAX profile of trypsin digested HeLa lysate (Abs280 nm). Base peak chromatograms are shown for select SAX fractions following high-resolution LC-MS/MS using an Orbitrap Fusion mass spectrometer: (D) fraction 3 (green); (E) fraction 6 (red); (F) fraction 10 (grey). Peptides were fragmented by HCD, with neutral loss of 98 amu from the precursor ions triggering EThCD (Ferries et al, 2017). Tandem mass spectra were separated according to fragmentation strategy in Proteome Discoverer prior to searching with Mascot. The ptmRS node was used for phosphosite localisation. Analysis was performed on three independent biological replicates for each condition (NT or PHPT1 siRNA).
Figure 1(B) RAW 264.7 cells transfected for 48 h with control (sc), MTMR6 or Jumpy siRNA, were pretreated for 30 min with 100 nM Bafilomycin A1 (BafA1), 10 μg/ml E64d and 10 μg/ml pepstatin, then incubated for 30 min in full or starvation media in the presence of BafA1, E64d and pepstatin. Cells were lysed and analysed by immunoblotting with anti-LC3 or anti‐actin. Densitometric LC3‐II/actin ratios are shown underneath the blot. Graph: LC3‐II/actin ratio for Jumpy siRNA in full medium is equal to or exceeds LC3‐II/actin ratio for control siRNA (sc) in starvation medium.(C, D) RAW 264.7 cells were transfected for 36 h with control (scramble) or Jumpy siRNA, transfected once more with corresponding siRNA and mRFP‐GFP‐LC3 DNA construct overnight and incubated for 2 h in full or starvation media. Cells were fixed and LC3 puncta analysed by confocal fluorescence microscopy. (C) Representative confocal images of RAW 264.7 cells in full or starvation media after transfection with control (scramble) or Jumpy siRNA (siJumpy). Red and yellow arrows indicate GFP−RFP+ and GFP+RFP+ puncta, respectively. (D) Quantitation of number of LC3 puncta per cell, total puncta per cell (GFP+RFP+ and GFP−RFP+ puncta) GFP+RFP+ puncta per cell and GFP−RFP+ puncta per cell. Data, mean±s.e.m. n=5 independent experiments, 30 cells per experiments. *P0.05, **P0.01, ***P0.001 (t‐test). Scale bars, 5 μm.(E, F) C2C12 myoblasts were transfected for 48 h with control (scramble) or Jumpy siRNA, followed by a second transfection overnight with corresponding siRNA and mRFP‐GFP‐LC3 DNA construct. Cells were fixed and LC3 puncta were counted by confocal fluorescence microscopy. (E) Representative confocal images of C2C12 cells in full media after transfection with control (scramble) or Jumpy siRNA (siJumpy). (F) Quantification of the number of LC3 puncta per cell. Data, mean±SEM for n=3 (independent experiments), 30 cells per experiments. *P 0.05 (t-test). Scale bars, 10 μm.(G, H) C2C12 cells were transfected for 48 h with control (sc) (lanes 1, 2, 5, 6) or Atg5 siRNA (lanes 3, 4, 6, 7) followed by a second transfection for 48 h with same siRNA and control (lanes 1, 3, 5, 7) or Jumpy siRNA (lanes 2, 4, 6, 8). Cells were incubated for 1 h with or without 100 nM Baf A1, 10 μg/ml E64d and 10 μg/ml pepstatin in full media, lysed and analysed by immunoblotting with anti‐LC3 or anti‐actin (G). (H) Densitometric LC3‐II/actin ratios for samples treated with BafA1 and protease inhibitors from G (lanes 5-8). Inset shows Atg5 knock‐down by immunoblotting.
Figure 2(A) Proteolysis of long‐lived proteins in C2C12 myoblasts. C2C12 cells were transfected with control (scramble) or Jumpy siRNA (siJumpy), labelled overnight in media containing [3H] leucine, washed, incubated for 2 h in complete media (containing cold leucine) and incubated for 4 h in full or starvation media. Leucine release was calculated from radioactivity in the tricarboxylic acid‐soluble form relative to total cell radioactivity. Results shown represent mean±s.e.m. for combined data from three independent experiments.(B) Quantitation of p62 protein levels. C2C12 cells were transfected for 48 h with control (sc) or Jumpy siRNA and p62 levels analysed by immunoblotting, quantitated by densitometry and represented as a percentage of control. Data are mean±s.e.m. (n=4 independent experiments).(C) C2C12 cells were transfected for 48 h with control (sc) or Jumpy siRNA, incubated for 4 h with or without 100 nM Bafilomycin A1 in full media, lysed, probed for endogenous p62 and actin by immunoblotting and percentage of p62 were quantitated (mean±s.e.m., n=3).(D) C2C12 cells were transfected for 48 h with control (sc) (lanes 1, 2) or Beclin siRNA (lanes 3, 4) followed by a second transfection for 48 h with same siRNA and control (lanes 1, 3) or Jumpy siRNA (lanes 2, 4). Cells were lysed, probed for p62, Beclin and actin by immunoblotting and percentage of p62 were quantitated (mean±s.e.m., n=5).(E-M) C2C12 cells were transfected for 48 h with GFP, GFP‐Jumpy (Jumpy), GFP‐MTM1 (MTM1) or GFP‐MTMR2 (MTMR2), fixed and immunostained with anti‐p62 antibody (red). p62 puncta were quantitated by confocal fluorescence microscopy. Representative confocal images of GFP (E), GFP‐Jumpy (F), GFP‐MTM1 (G), GFP‐MTMR2 (H) transfected cells, immunostained for p62 (I), (J), (K) and (L), respectively. Scale bars, 5 μm. White lines represent outline of the cells. Quantitation of p62 puncta per cell (M). Bars, SEM (n=3 independent experiments, with an average of 30 cells per experiments).(N, O) C2C12 cells were transfected for 48 h with GFP, GFP‐Jumpy (Jumpy), GFP‐MTM1 (MTM1) or GFP‐MTMR2 (MTMR2), lysed, analysed for p62, GFP and actin by immunoblotting (N) and percentage of p62 were quantitated (mean±s.e.m., n=3) (O).(P, Q) C2C12 cells were transfected for 48 h with GFP or GFP‐Jumpy (Jumpy), incubated with or without 100 nM Bafilomycin A1 (BafA1) for 4 h, lysed, analysed for p62, GFP and actin by immunoblotting (P) and percentage of p62 were quantitated (mean±s.e.m., n=4) (Q). *P0.05, ***P0.001, †ns (t‐test).
Figure 3(A) Transient association of Jumpy WT with LC3+ organelles. Time lapse sequence of C2C12 myoblasts transfected for 24 h with GFP‐Jumpy WT and Cherry‐LC3 and analysed by live confocal microscopy in a 5LIVE Zeiss microscope. EBSS was added to the cells and z‐stacks collected at 3‐min intervals for a total of 45 min. The collected images were processed to generate a maximum projection (collapsing a 3D image into an x-y projection) for each time point. Arrows indicate colocalization of GFP‐Jumpy WT with LC3. Graph: Plots of relative fluorescence intensity (GFP fluorescence pixel intensity) over time of GFP‐Jumpy WT colocalizing with Cherry‐LC3+‐positive puncta indicated by arrows.(B-I) Association of Jumpy CS mutant with autophagic organelles. C2C12 cells were transfected for 24 h with GFP‐Jumpy C330S (Jumpy CS) and tdTomato‐LC3 (LC3), fixed and immunostained with anti‐G58K (Alexa 648‐labelled secondary antibody). Jumpy CS (green), LC3 (red) and G58K (white in D, H or blue in E, I). Boxed areas (B-E) are shown at higher magnification in the corresponding panel below (F-I). Scale bars, 2 μm. White arrows indicate colocalization between Jumpy CS, LC3 but not G58K, blue arrow indicates colocalization between Jumpy CS and G58K but not LC3.
Figure 4(A-H) C2C12 cells were transfected for 24 h with GFP‐Jumpy C330S (Jumpy CS) and tdTomato‐LC3 (LC3), fixed and immunostained with anti‐Atg16 (Alexa 648‐labelled secondary antibody). Jumpy CS (green), LC3 (red) and Atg16 (white in B and F or blue in D and H). Boxed areas (A-D) are shown at higher magnification in the corresponding panel below (E-H). White arrows indicate colocalization among Jumpy CS, LC3 and Atg16, yellow arrow indicates colocalization between Jumpy CS and LC3 but not Atg16, blue arrow indicates colocalization between Jumpy CS and Atg16 but not LC3.(I, J) C2C12 cells were transfected for 24 h with GFP (I), fixed and immunostained with anti‐Atg16 (J).(K-R) C2C12 cells were transfected for 24 h with GFP‐Jumpy C330S (Jumpy CS) and tdTomato‐LC3 (LC3), fixed and immunostained with anti‐Atg12(M) antibody. Jumpy CS (green), LC3 (red) and Atg12 (white in L, P or blue in N, R). Boxed areas (K-N) are shown at higher magnification in the corresponding panel below (O-R). White arrows indicate colocalization among Jumpy CS, LC3 and Atg12, yellow arrow indicates colocalization between Jumpy CS and LC3 but not Atg12, blue arrow indicates colocalization between Jumpy CS and Atg12 but not LC3.(S-ZA) C2C12 cells were transfected for 24 h with Jumpy CS and Cherry‐LC3 (LC3), fixed and immunostained with anti‐lamp1. Jumpy CS (green), LC3 (red) and Lamp1 (white in T, X or blue in V, ZA). Boxed areas (S-V) are shown at higher magnification in the corresponding panel below (W-ZA). Yellow arrows indicate colocalization between Jumpy CS and LC3 but not Lamp1. Scale bars, 2 μm.
Figure 5(A-C) C2C12 were transfected for 48 h with Jumpy siRNA, followed by a second transfection overnight with Jumpy siRNA and GFP‐WIPI‐1, incubated for 4 h in starvation media, fixed and immunostained with anti‐Atg16 antibody (red). GFP‐WIPI1‐transfected cell (A), endogenous Atg16 immunostaining (B), colocalization of GFP‐WIPI‐1 and Atg16 is indicated in yellow in merge image (C). Boxed areas are shown at higher magnification as inset. Scale bars, 10 and 5 μm in insets.(D-I) C2C12 cells were transfected for 48 h with control (scramble) (D, E) or Jumpy siRNA (F-H), followed by a second transfection overnight with corresponding siRNA and GFP‐WIPI‐1. Cells were incubated for 4 h in full (D, G) or starvation media (E, F, H) in presence (F) or absence (E-H) of 100 nM wortmannin, fixed and analysed by confocal fluorescence microscopy. Quantitation of WIPI‐1 puncta per cell (I). Bars, s.e.m. (n=3 independent experiments, with an average of 30 cells per experiment). *P0.05, **P0.01 (t‐test).