EMBO/soda-nel huggingface dataset:
Inositol-Requiring Enzyme-1 Regulates Phosphoinositide Signaling Lipids and Macrophage Growth (2020)
Figure 6

Figure 6. miR-2137 regulates PI(3,4,5)P3/PIP2 ratio and growth signaling in macrophages

(A-C) BMDMs were transfected with scrambled miRNA mimic (40 nM), miR-2137 mimic (40 nM) or miR-205 mimic (40 nM) for 24 hours and cells were processed for lipidomics analysis as described in materials and methods section. The ratio of lipids from this analysis is shown for (A) PI(3,4,5)P3 to PIP2.; (B) PI(3,4,5)P3 to PI; (C) PI(3,4,5)P3 to PIP. Data from LC/MS/MS using a Waters Xevo TQ-S MS/MS in MRM mode represent mean values ± SEM of peak areas normalized to internal standards as described in Methods. (n=3 biological replicates)

(D-E) BMDMs were transfected with (D) scrambled miRNA mimic (40 nM) or miR-2137 mimic (40 nM) for 48 hours and (E) with control antagomir (200 nM) or miR-2137 antagomir for 24 hours and protein lysates were analyzed by western blotting using specific antibodies against INPPL1, pAKTS473, AKT, p70S6KT389, p70S6K, pS6S235/236, S6, p4E-BP1S65, 4E-BP1, pmTORS2448, mTOR and β-actin. Protein quantifications are shown next to the western blots (n=3 biological replicates).

Data information: All data are mean ± SEM (n=3); unpaired t-test with Welch's correction. *P≤ 0.05, **P≤ 0.01, ***P≤ 0.001.
VCP/p97 cooperates with YOD1, UBXD1 and PLAA to drive clearance of ruptured lysosomes by autophagy (2016)
Figure 1

Figure 1. p97 is a direct and essential component of the lysosomal damage response

A HeLa cells expressing LAMP1-RFP were treated with vehicle alone (control) or LLOMe for 1 h to induce lysosome damage. Cells were fixed 2 h after washout, stained with p62 and galectin-3 (Gal3) antibodies, and analyzed by confocal microscopy.

B p97 translocates to ruptured lysosomes. Cells co-expressing LAMP1-RFP and GFP-p97 were treated as in (A) and stained with the pan-ubiquitin antibody FK2 (Ub). Percentage of cells showing recruitment of p97 in three independent experiments (mean ± SD).

C Immunodetection of endogenous p97 after LLOMe treatment as in (A). Percentage of cells showing recruitment of p97 in three independent experiments (mean ± SD).

D Clearance of damaged lysosomes depends on p97. Cells were transfected with control (Ctrl) or p97 siRNA oligos, fixed at indicated time points after LLOMe washout and stained for endogenous Gal3. Quantification of the number of Gal3-positive vesicles per cell. Data represent mean ± SD of four independent experiments with ≥30 cells quantified per condition (one-sided Welch's unequal variances t-tests comparing the siCtrl with sip97).

E Cells were chased after LLOMe treatment for 12 h with vehicle alone (DMSO), bortezomib (Btz, 10 nM), NH4Cl (20 mM) or the p97 inhibitor NMS-873 (5 µM), and Gal3 vesicles quantified. Data represent mean ± SEM of three independent experiments with ≥30 cells quantified per condition (one-sided Welch's t-tests).

F p97 is essential for cell survival after lysosome rupture. Cells were transfected with control (Ctrl), p97 or SEL1L siRNA oligos and treated with indicated concentrations of LLOMe for 12 h. Cell viability was measured using the MTS assay. Data represent mean ± SD of three independent experiments.

G Tau fibrils are endocytosed and induce lysosomal damage. Tau fibrils were generated in vitro, fluorescently labelled by Dylight488 and fragmented by sonication (488-labelled tau). HeLa cells expressing mCherry-Gal3 were incubated with 300 nM tau fibrils for 24 h. Arrows indicate compartments that contain 488-labelled tau and are positive for mCherry-Gal3 indicating lysosome rupture. Percentage of cells with Gal3-positive vesicles was quantified from three independent experiments (mean ± SD).

H Accumulation of tau-induced lysosomal damage upon p97 depletion. Cells transfected with control (Ctrl) or p97 siRNA oligos were treated as in (G). Percentage of cells with Gal3-positive vesicles was quantified from three independent experiments (mean ± SD). * = p < 0.05; ** = p < 0.01; *** = p < 0.001. Scale bars, 10 µm.
VCP/p97 cooperates with YOD1, UBXD1 and PLAA to drive clearance of ruptured lysosomes by autophagy (2016)
Figure 2

Figure 2. Disease-associated mutations of p97 compromise clearance of damaged lysosomes

A Impaired clearance of damaged lysosomes in cells expressing a p97 disease-mutant. Wild-type and p97R155H/+ MEFs were LLOMe-treated for 1 h and released for indicated times. Percentage of cells with Gal3-positive vesicles was quantified from three independent experiments (mean ± SD, Student’s unpaired t-test). Scale bar, 25 µm.

B Stable U2OS cell lines were doxycycline-induced to express myc-tagged p97 wild-type or disease-mutants as indicated. Induced parental cells served as control (U2OS -). Cells were treated with LLOMe for 1h and chased for indicated time before fixation. Percentage of cells with Gal3-positive vesicles was quantified from three independent experiments. Comparable expression levels were verified (see Figure EV2A).

C Stable U2OS cell lines described in (B) were treated with LLOMe for 1 h and chased for indicated time. LC3 and GAPDH levels were analyzed by immunoblotting. Quantification is shown in Figure EV2B.

D Accumulation of damaged lysosomes in affected p97 patient skeletal muscle. Patient biopsies immunostained for Gal3 and caveolin-3 (Cav3). Quantification from 4 random 10X fields for each biopsy and means for controls and patients, respectively. Patients carry p97 mutations R155H (#1-3) or R93C for (#4). Patients #2 and #3 are cousins. Scale bar, 100 µm.
VCP/p97 cooperates with YOD1, UBXD1 and PLAA to drive clearance of ruptured lysosomes by autophagy (2016)
Figure 3

Figure 3. A subset of p97 cofactors is directly involved in the lysosomal damage response and forms a substrate-stabilized complex

A Candidate screen for p97 cofactors involved in autophagy of damaged lysosomes. HeLa cells were transfected with indicated siRNA oligos and LLOMe-treated for 1 h. Cells were chased for 12 h, fixed and stained for Gal3. See Figure EV3 for depletion efficiency.

B Quantification of the percentage of cells with more than three Gal3 vesicles per cell for each condition. This threshold was chosen so that less than 1% of untreated cells were considered positive. Data represent mean ± SEM of three independent experiments with ≥30 cells quantified per condition. * = p < 0.05; ** = p < 0.01; *** = p < 0.001 (one-sided Welch's t-tests).

C YOD1, UBXD1 and PLAA are translocated along with p97 to ruptured lysosomes. Cells expressing indicated tagged proteins were LLOMe-treated for 1 h or with vehicle alone (control), fixed 2 h after washout, and stained for endogenous LAMP1 and, in the case of PLAA, with Alexa-488 conjugated anti-HA antibodies. See Figure EV3B for quantification.

D An ATP-stabilized complex of p97 containing both UBXD1 and PLAA. Stable HEK293 cell lines were doxycycline-induced to express p97 wild-type (wt) or the ATPase-mutant E578Q (EQ) at near endogenous level. Endogenous UBXD1 was immunoprecipitated and associated proteins detected by immunoblotting with indicated antibodies. Arrowheads indicate endogenous (lower band) and induced p97 (upper band).

E Stable HEK293 cells treated as in (D) and transiently expressing GFP-tagged YOD1-wt or the YOD1-C160S catalytic mutant (CS) were processed for co-immunoprecipitation with GFP nanobodies. Asterisk, unspecific band. Note that the YOD1-CS mutant stabilized p97 binding and, in the p97-EQ background, also associated with UBXD1 and PLAA.

F Control (IgG) and UBXD1 co-immunoprecipitation with indicated associated proteins from HeLa cells expressing GFP-YOD1-CS after formaldehyde cross-linking. Ufd1 served as a negative control. Scale bars, 10 µm.
VCP/p97 cooperates with YOD1, UBXD1 and PLAA to drive clearance of ruptured lysosomes by autophagy (2016)
Figure 4

Figure 4. The ELDR components p97, UBXD1, PLAA and YOD1 selectively target K48-linked ubiquitin-conjugates on K63-decorated damaged lysosomes

A K63- and K48-ubiquitination of damaged lysosomes. Untransfected HeLa cells, or cells expressing mCherry-Gal3 (Ch-Gal3) were LLOMe-treated for 1 h. 2 h after washout, cells were stained with antibodies specific for K63 or K48 chains, and LAMP1 or p62 as indicated. Percentage of K63- or K48-ubiquitinated LAMP1 vesicles and percentage of p62-positive K63 or K48 vesicles were quantified from three independent experiments (mean ± SD, Student’s unpaired t-test for LAMP1). * = p < 0.05.

B Colocalization of K63- and K48-linked ubiquitin conjugates on individual lysosomes. Cells expressing GFP-TAB2-NZF (K63-sensor) were treated as in (A) and stained for K48 chains and LAMP1. Note that the apparent nuclear localization of the sensor is a consequence of the extraction procedure (van Wijk et al., 2012).

C Cells expressing GFP-p97-EQ were treated as in (A) and stained with antibodies as indicated.

D Quantification of the total number of K63- or K48-ubiquitinated vesicles per cell, and number of p97-EQ positive K63- or K48-ubiquitinated vesicles (mean ± SD).

E Percentage of GFP-p97-EQ vesicles positive for K63 or K48 chains was determined (mean ± SD).

F ELDR components target K48-conjugates on lysosomes. Detection of GFP-YOD1-CS or UBXD1-GFP alone, or co-transfected with PLAA-HA and GFP-p97-EQ, 2 h after LLOMe washout and co-stained with indicated antibodies.

G Scheme of ubiquitination of lysosomes after damage. K48-linked ubiquitination occurs on a subpopulation of lysosomes decorated with K63-linked chains. The ELDR components specifically target K48-linked ubiquitin-conjugates. Scale bars, 10 µm.
VCP/p97 cooperates with YOD1, UBXD1 and PLAA to drive clearance of ruptured lysosomes by autophagy (2016)
Figure 5

Figure 5. The ELDR components specifically remove K48-linked ubiquitin conjugates from damaged lysosomes

A Time course of K63- and K48-ubiquitination. Control-depleted cells were fixed at indicated time points after LLOMe washout and stained with chain-specific antibodies and LAMP1. Automated quantification of LAMP1 vesicles positive for K63 or K48 chains. Data represent mean ± SD of three independent experiments.

B Depletion of p97 or YOD1 results in increase and persistence of K48 chains on damaged lysosomes. Experiment and quantification as in (A) in cells treated with p97 or YOD1 siRNA oligos.

C The p97-EQ mutant stabilizes K48-conjugates. Lysosome damage was induced as in (A) in cells transfected with GFP-tagged p97-wt or p97-EQ. Cells were chased after washout for 12 h and stained for K48 chains.

D Quantification of K48-positive lysosomes 2 h and 12 h after damage in cells treated as in (A). Data represent mean ± SD of three independent experiments.

E The catalytically-inactive mutant of YOD1 stabilizes K48-conjugates. Cells transfected with GFP-tagged YOD1-wt or the YOD1-CS were treated and stained as in (C).

F Quantification as in (D) of cells treated as in (E). ** = p < 0.01 *** = p < 0.001 (Student’s unpaired t-test). ns, not significant. Scale bars, 10 µm.
VCP/p97 cooperates with YOD1, UBXD1 and PLAA to drive clearance of ruptured lysosomes by autophagy (2016)
Figure 6

Figure 6. YOD1 functions as a p97-activated ubiquitin sensor through its ubiquitin-binding site S2

A YOD1 can link p97 to ubiquitin. Ubiquitin-GST (Ub-GST) or GST alone was incubated with the indicated purified proteins and isolated. Co-isolated proteins (pulldown) were detected in SDS-gels. Note binding of p97 and YOD1 to Ub-GST only in combination.

B The S2 site of YOD1 is critical for ubiquitin binding. Experiments as in (A) with indicated combinations of p97 and YOD1-wt or the ubiquitin-binding mutant, MutS2.

C Reconstitution of a complex of ELDR components on ubiquitin. Experiments as in (A) with combinations of p97-wt or p97-EQ, YOD1, PLAA and UBXD1 as indicated. Note that PLAA associates with p97 and YOD1 on ubiquitin only in the case of p97-EQ.

D Translocation of YOD1 to lysosomes depends on ubiquitin binding by the S2 site. LLOMe-treated cells expressing GFP-YOD1-CS or the double mutant GFP-YOD1-CS-MutS2 were chased for 2 h and stained for K48 chains and LAMP1. Ratio of GFP signal on K48 vesicles to GFP signal in the whole cell was determined.

E The S2 site is essential for K48-conjugate removal. GFP-YOD1-wt or GFP-YOD1-MutS2 overexpressing cells treated as in (D) were stained for K48 chains. Quantification of the number of K48-positive vesicles per cell. Data represent mean ± SD of three independent experiments. ** = p < 0.01 (Student’s unpaired t-test). Scale bars, 10 µm.
VCP/p97 cooperates with YOD1, UBXD1 and PLAA to drive clearance of ruptured lysosomes by autophagy (2016)
Figure 7

Figure 7. ELDR components remove K48-conjugates from damaged lysosomes to promote autophagosome formation

A Largely mutually exclusive localization of LC3 and K48 chains. GFP-LC3 expressing HeLa cells were fixed 2 h after LLOMe washout and stained for endogenous LAMP1 and K48 or K63 chains. Correlation coefficients were calculated for colocalization of LC3 with K63 or K48. The whiskers represent 10th and 90th percentile. Points below and above the whiskers are drawn as individual dots. The single line indicates the median. *** = p < 0.001 (Mann-Whitney-U test).

B Lipofectamine-coated latex beads damage lysosomes and induce their K48-ubiquitination. Cells expressing mCherry-Gal3 were incubated with Lipofectamine-coated latex beads for 3 h and stained with K48 chain-specific antibodies.

C YOD1 association and dissociation precedes LC3 recruitment. Live cell video microscopy stills of cells co-expressing mCherry-YOD1-wt and GFP-LC3 after treatment with Lipofectamine-coated latex beads for 30 min at 2 min image intervals.

D Stabilization of K48-conjugates on lysosomes impairs recruitment of LC3. Cells expressing GFP-YOD1-CS were treated as in (A) and stained for LAMP1 and p62 or LC3. Percentage of GFP-YOD1 vesicles positive for p62 or LC3 was determined (mean ± SD).

E Depletion of ELDR components impairs LC3 recruitment for autophagosome formation. Cells were transfected with control (Ctrl), p97 or YOD1 siRNA oligos, treated with LLOMe, fixed after 12 h chase and stained for endogenous LC3 and K48 chains. Quantification of the number of K48-positive vesicles per cell and number of LC3-positive K48 vesicles per cell was determined. Data represent mean ± SD of three independent experiments. Scale bars, 10 µm.
Modulation of intracellular ROS levels by TIGAR controls autophagy. (2009)
Figure 1

TIGAR regulates intracellular ROS levels in response to nutrient starvation or metabolic stress. (A) ROS levels in U2OS cells stably over-expressing Flag-tagged-TIGAR (clones TIGAR#5 and TIGAR#7) or control cells (clones Cont#1 and Cont#3) left untreated, after 6 h of nutrient starvation or 24 h of metabolic stress. ROS levels were measured by flow cytometry after DCF treatment. The results are expressed as the mean DCF fluorescence (and standard deviation), from three independent experiments. (B) Basal, nutrient starvation-induced (5 h) or metabolic stress-induced (18 h) ROS levels in U2OS cells in the presence of either scrambled, TIGAR siRNA1 or TIGAR siRNA2, measured by flow cytometry after DCF treatment. The results are expressed as the mean intensity of cell fluorescence (and standard deviation). * represents significant difference from control conditions (P<0.05).
Modulation of intracellular ROS levels by TIGAR controls autophagy. (2009)
Figure 2

TIGAR expression modulates autophagy in response to nutrient starvation or metabolic stress. (A) (Left panel) Confocal microscopic images of the fluorescence in U2OS cells stably over-expressing Flag-tagged-TIGAR (clone TIGAR#7) or control cells (clone Cont#1) and infected with an adenovirus expressing GFP-LC3 for 16 h. Cells were then left untreated, exposed to nutrient starvation for 6 h or to metabolic stress for 24 h. (Right panel) Quantitation of the percentage of GFP-LC3–positive cells displaying GFP puncta from three independent experiments. The mean values with standard deviation are presented. (B) (Left panel) Confocal microscopic images of the fluorescence in U2OS cells stably expressing GFP-LC3 and transfected with scrambled or TIGAR siRNAs. After 48 h transfection, cells were then left untreated, exposed to nutrient starvation for 5 h or to metabolic stress for 18 h. (Right panel) Quantitation of the percentage of GFP-LC3–positive cells displaying GFP puncta from three independent experiments. The mean values with standard deviation are presented. (C) (Left panel) Western blot showing the expression levels of endogenous LC3-I, LC3-II and TIGAR in U2OS cells transfected with scrambled or TIGAR siRNAs, and 48 h later exposed to nutrient starvation for 0, 2.5 and 6 h. (Middle panel) Western blot showing the expression levels of endogenous LC3-I, LC3-II and TIGAR in U2OS stably over-expressing Flag-tagged-TIGAR (clones TIGAR#5 and TIGAR#7) or control cells (clones Cont#1 and Cont#3) and left untreated. (Right panel) Western blot showing the expression levels of p62, COX-IV and TIGAR in U2OS cells transfected with scrambled or TIGAR siRNAs and left untreated. Actin expression was examined as a loading control. (D) Western blot showing the expression levels of endogenous TIGAR in U2OS cells after exposure to nutrient starvation or metabolic stress for 0, 1, 3, 5 and 8 h; * represents significant difference from control conditions (P<0.05); # represents a lack of significant difference from control conditions (P>0.05).
Modulation of intracellular ROS levels by TIGAR controls autophagy. (2009)
Figure 3

TIGAR expression modulates autophagy independently of p53. (A) Quantitation of the percentage of GFP-LC3–positive cells displaying GFP puncta. U2OS cells stably expressing GFP-LC3 were transfected with scrambled or TIGAR siRNAs, and 48 h after transfection, cells were left untreated (t0) or treated with Bafilomycin A1 (100 nM) for 1 or 2 h (t1 and t2). The percentage of cells with GFP-LC3 puncta was calculated at the indicated time points. Data are shown as the mean and standard deviation from three independent experiments. (B) Quantitation of the percentage of GFP-LC3–positive cells displaying GFP puncta. U2OS cells stably expressing GFP-LC3 were cotransfected with scrambled or TIGAR siRNAs, and scrambled or p53 siRNA. After 48 h transfection, cells were left untreated or exposed for 5 h to nutrient starvation. The percentage of cells with GFP-LC3 puncta was calculated, and data are shown as the mean and standard deviation from three independent experiments. (C) Western blot showing the expression levels of endogenous p53 and TIGAR in U2OS cells cotransfected with scrambled or TIGAR siRNAs, and scrambled or p53 siRNA, and 48 h later exposed to nutrient starvation for 5 h. Actin expression was examined as a loading control. (D) Quantitation of the percentage of GFP-LC3–positive cells displaying GFP puncta. U2OS cells stably expressing GFP-LC3 were cotransfected with scrambled or TIGAR siRNAs, and scrambled or DRAM siRNA1/2. After 48 h transfection, cells were left untreated or exposed for 5 h to nutrient starvation. The percentage of cells with GFP-LC3 puncta was calculated, and data are shown as the mean and standard deviation from three independent experiments; * represents significant difference from starved control conditions (P<0.05); @ represents significant difference from untreated control conditions (P<0.05); # represents a lack of significant difference from control conditions (P>0.05).
Modulation of intracellular ROS levels by TIGAR controls autophagy. (2009)
Figure 4

Modulation of ROS by TIGAR correlates with the modulation of autophagy. (A) U2OS cells were left untreated, exposed to nutrient starvation for 6 h or to metabolic stress for 24 h, with or without treatment with AO1 (NAC (2 mM) and L-ascorbic acid (2 mM)) for 24 h. The percentage of cells with GFP-LC3 puncta was calculated, and data are shown as the mean and standard deviation from three independent experiments. (B) (Left panel) ROS levels in U2OS cells left untreated or treated with 0.5 or 1 mM of H2O2 for 24 h. ROS levels were measured by flow cytometry after DCF treatment. The results are expressed as the mean DCF fluorescence (and standard deviation) from three independent experiments. (Right panel) Quantitation of the percentage of GFP-LC3 puncta positive cells for cells treated as described above. Data are shown as the mean and standard deviation from three independent experiments. (C) Quantitation of the percentage of GFP-LC3 puncta positive cells. U2OS cells stably expressing GFP-LC3 were transfected with vector pCHER1A expressing the mCherry gene as control, or expression plasmids for Flag-tagged-TIGAR or HA-tagged-FBPase-2. After 48 h transfection, cells were left untreated, exposed to nutrient starvation for 6 h or to metabolic stress for 24 h, with or without treatment with AO1 (NAC (2 mM) and L-ascorbic acid (2 mM)) for 24 h. The percentage of cells with GFP-LC3 puncta was calculated, and data are shown as the mean and standard deviation from three independent experiments. (D) Quantitation of the percentage of GFP-LC3 puncta positive cells. Cells were left untreated, exposed to nutrient starvation for 6 h with or without treatment with AO1 (NAC (2 mM) and L-ascorbic acid (2 mM)), AO2 (glutathione ethyl ester (4 mM)) or AO3 (ethyl pyruvate (4 mM)) for 24 h. U2OS cells stably expressing GFP-LC3 were transfected with vector pCHER1A expressing the mCherry gene as control, or expression plasmid for Flag-tagged-TIGAR. After 48 h transfection, cells were treated. The percentage of cells with GFP-LC3 puncta was calculated, and data are shown as the mean and standard deviation from three independent experiments. (E) (Left panel) Quantitation of the percentage of GFP-LC3 puncta positive cells. Cells were left untreated, exposed to nutrient starvation for 5 h or to metabolic stress for 18 h, with or without treatment with AO1 (NAC (2 mM) and L-ascorbic acid (2 mM)) for 24 h. U2OS cells stably over-expressing GFP-LC3 in the presence of either scrambled, TIGAR siRNA1 or TIGAR siRNA2. (Right panel) Basal or nutrient starvation-induced (5 h) ROS levels in U2OS cells in the presence of either scrambled, TIGAR siRNA1 or TIGAR siRNA2 with or without treatment with AO1 (NAC (2 mM) and L-ascorbic acid (2 mM)) for 24 h, measured by flow cytometry after DCF treatment. The results are expressed as the mean intensity of cell fluorescence (and standard deviation). * represents significant difference from control conditions (P<0.05); # represents a lack of significant difference from control conditions (P>0.05).
Modulation of intracellular ROS levels by TIGAR controls autophagy. (2009)
Figure 5

TIGAR modulates ROS levels upstream of the autophagic response. (A) Western blot showing knockdown of ATG5 protein expression by ATG5 siRNA. (B) RT–PCR showing knockdown of ATG10 mRNA expression by ATG10 siRNA. (C) Quantitation of the percentage of GFP-LC3 puncta positive cells. U2OS cells stably over-expressing GFP-LC3 in the presence of either scrambled, TIGAR siRNA1 or TIGAR siRNA2, and either scrambled, ATG5 siRNA or ATG10 siRNA. After 48 h, cells were left untreated or exposed to nutrient starvation for 5 h. (D) ROS levels in U2OS cells in the presence of either scrambled, TIGAR siRNA1 or TIGAR siRNA2, and scrambled, ATG5 siRNA or ATG10 siRNA, measured by flow cytometry after DCF treatment. After 48 h, cells were left untreated or exposed to nutrient starvation for 5 h. The results are expressed as the mean intensity of cell fluorescence (and standard deviation). * represents significant difference from control conditions (P<0.05); # represents a lack of significant difference from control conditions (P>0.05).