EMBO/SourceData huggingface dataset:
Figure 1(A-C) Bone marrow derived mouse macrophages (BMDMs) from wild type C57BL/6 mice were treated with 4µ8c (100 µM) or vehicle (dimethyl sulfoxide; DMSO) for 24 hours prior to sample processing for lipidomics analysis: (A) PI(3,4,5)P3 to PIP2; (B) PI(3,4,5)P3 to PI; (C) PI(3,4,5)P3 to PIP ratio are shown. Data represent mean values ± standard error of mean (SEM) of peak areas normalized to internal standards (n=3 biological replicates).(D) BMDMs were treated with 4µ8c (100 µM) or DMSO for 24 hours and protein lysates were analyzed by western blotting using specific antibodies against, pAKTS473, AKT, p70S6KT389, p70S6K, pS6S235/236, S6, p4E-BP1S65, 4E-BP1 and β-actin. Western blot quantifications are shown next to the figure (n=3 biological replicates; a representative blot is shown).(E) Wild type C57BL/6 mice were injected with tunicamycin (1 mg/kg) and 4µ8c (10 µg/kg) for 8 hours prior to sacrifice and collection of thiogylcolate-elicited peritoneal macrophages. Protein lysates were analyzed by western blotting using specific antibodies against, pIRE1S742, IRE1, pAKTS473, AKT, p70S6KT389, p70S6K, pS6S235/236, S6, p4E-BP1S65, 4E-BP1 and β-actin (n=3 biological replicates; a representative blot is shown). Western blot quantifications are shown next to the figure.(F) IRE1+/+ and IRE1-/- BMDMs were treated with PA (500 µM) for 6 hours and protein lysates were analyzed by western blotting using specific antibodies against pIRE1S742, IRE1, p70S6KT389, p70S6K, pS6S235/236, S6, p4E-BP1S65, 4E-BP1 and β-actin. Western blot quantifications are shown next to the figure (n=3 biological replicates, a representative blot is shown).
Figure 2IRE1-/- and IRE1+/+ BMDMs were treated with PA (500 µM) or vehicle for 6 hours prior to RNA isolation for miRNA analysis using a micro-array platform. (B) RNA lysates from the same experiment were analyzed by qRT-PCR for miR-2137 expression.IRE1-/- and IRE1+/+ BMDMs were treated with PA (500 µM) or vehicle for 6 hours prior to RNA isolation for miRNA analysis using a micro-array platform. (C) Protein lysates were analyzed by western blotting using specific antibodies for pIRE1S742, IRE1 and β-actin (n=3 biological replicates).IRE1-/- and IRE1+/+ BMDMs were treated with PA (500 µM) or vehicle for 6 hours prior to RNA isolation for miRNA analysis using a micro-array platform. (D) RNA lysates were analyzed by qRT-PCR for sXBP1 expression (n=3 biological replicates).IRE1-/- and IRE1+/+ BMDMs were treated with PA (500 µM) or vehicle for 6 hours prior to RNA isolation for miRNA analysis using a micro-array platform. (E-F) RNA lysates from the same experiment were analyzed by qRT-PCR for (E) BLOC1S1 and (F) SCARA3 expression (n=3 biological replicates).
Figure 3(A-B) BMDMs were treated with PA, steric acid (SA), palmitoleic acid (PAO) or oleic acid (OA) (500 µM) for 16 hours. RNA lysates were analyzed by qRT-PCR for the expression of (A) miR-2137 and (B) sXBP1 (n=3 biological replicates).(C, D) (C) C57BL/6 (WT) and (D) Apoe-/- mice were fed with chow or WD for 16 weeks followed by peritoneal macrophage isolation. RNA lysates were analyzed by qRT-PCR for miR-2137 expression (WT; n=4-4 mice per group, Apoe-/-; n=5-5 mice per group).(E-F) Apoe-/- mice were fed with chow or WD for 12 weeks and injected with 4µ8c (10 mg/kg/day) or vehicle (DMSO) in the final 6 weeks of the diet (n=5-7 mice per group): (E) RNA lysates from bone marrow were analyzed by qRT-PCR for miR-2137 expression (n=5-7 mice per group). (F) Total RNA isolated from the aortic root plaques was analyzed by qRT-PCR for miR-2137 expression (n=5-7 mice per group).
Figure 4(A) BMDMs were transfected with scrambled (Scr) or IRE1-specific siRNA (40 nM) and treated with palmitate (PA, 500 µM) or vehicle for 6 and 9 hours (hr). RNA lysates were analyzed by qRT-PCR for miR-2137 expression (n=3 biological replicates).(B) BMDMs were treated with PA (500 µM) and 4µ8c (100 µM) or vehicle for 9 hours. RNA lysates were analyzed by qRT-PCR for miR-2137 expression (n=3 biological replicates). (C) BMDM cells were treated with tunicamycin (Tun; 5 µg/ml) and 4µ8c (100 µM) or vehicle (DMSO) for 9 hours. RNA lysates were analyzed by qRT-PCR for miR-2137 expression (n=3 biological replicates). (D) IRE1-/- MEFs were transfected with vector (control), WT-IRE1 or K907A-IRE1 (RNase dead mutant) plasmids for 24 hours, followed by PA (500 µM) treatment for 9 hours. RNA lysates were analyzed by qRT-PCR for miR-2137 expression (n=3 biological replicates).(E) BMDMs were transfected with scrambled or XBP-1 specific siRNA (70 nM) for 24 hours, followed by treatment with PA (500 µM) for 16 hours. RNA lysates were analyzed by qRT-PCR for miR-2137 expression (n=3 biological replicates).(F) XBP1-/- MEFs were transfected with empty vector or sXBP1 plasmid for 24 hours, followed by PA (500 µM) treatment for 9 hours. RNA lysates were analyzed for miR-2137 expression by qRT-PCR (n=3 biological replicates).(G) IRE1 cleavage assay performed using synthetic pre-miR-2137 (50 nM) as substrate with active, recombinant IRE1 (100 ng) at 37℃ for 2 hours, followed by sample separation in Urea-PAGE and detection with SYBR gold staining. M indicates micro RNA marker.(H-I) BMDMs isolated from WT (C57BL6) and DICER-/- mice were treated with PA (500 µM) for 12 hours. RNA lysates were analyzed by qRT-PCR for (H) miR-2137 and (I) sXBP1 expression (n=3 biological replicates).
Figure 5(B) HEK293 cells were co-transfected with an INPPL1 3` UTR-luciferase plasmid and scrambled miRNA mimic (40 nM), miR-2137 mimic (40 nM) or miR-205 mimic (40 nM) for 24 hours. Protein lysates were analyzed for luciferase activity and normalized to secreted embryonic alkaline phosphatase (SEAP) reporter activity (n=3 biological replicates).(C) BMDMs were transfected with scrambled miRNA mimic (40 nM), miR-2137 mimic (40 nM) and miR-205 mimic (40 nM) for 48 hours and protein lysates were analyzed by western blotting using specific antibodies for INPPL1 and β-actin. Protein quantifications are displayed in graph next to the blots (n=3 biological replicates).(D) BMDMs were transfected with negative control antagomir (200 nM) or miR-2137 antagomir (200 nM) for 24 hours, followed by PA (500 µM) treatment for 9 hours. Protein lysates were analyzed by western blotting using specific antibodies for INPPL1 and β-actin. Protein quantifications shown in the graph next to blots (n=3 biological replicates).
Figure 6(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)BMDMs were transfected with (D) scrambled miRNA mimic (40 nM) or miR-2137 mimic (40 nM) for 48 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).BMDMs were transfected (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).
Figure 1(A) In situ hybridizations for Elp3 transcripts in the developing inner ear. Transcripts are present at E12.5 in a ventro-medial region of the developing cochlear duct (CD) and in the cochleo-vestibular ganglion (CVG). At E16.5, transcripts are present in the spiral ganglion neurons (SGNs) and in the newly formed hair cells (HCs) at the base of the cochlea, where they remain strongly expressed at P1. Scale bar=50µm; inset=25µm. Data information: #IHC; *OHC1; **OHC2; ***OHC3, OC: Organ of Corti.(B) Elp3 specific immunostaining confirms the presence of the protein in HCs and SGNs at birth. Scale bar=10µm. Data information: #IHC; *OHC1; **OHC2; ***OHC3, OC: Organ of Corti.Validation of Elp3 depletion from the cochlea in Elp3cKO animals. (C) At P1, Elp3 mRNA is absent from Elp3cKO. Scale bars=50µm. In situ hybridizations were repeated 4 times.Validation of Elp3 depletion from the cochlea in Elp3cKO animals. (D) Western Blots from P1 WT and Elp3cKO cochlear extracts confirm the loss of Elp3 at the protein level (cochleae from 3 different animals are pooled per sample).(E) Representative ABR recording from P19 WT and Elp3cKO animals reveal an absence of sound-evoked potentials in Elp3cKO animals, indicating that they suffer from severe hearing loss. (F) Elp3cKO animals present increased ABR thresholds (WT/Elp3cKO n=5/3. Unpaired two-tailed t-test, **p=0.0017, t=5.369, DF=6, mean±SD).
Figure 2(A) Toluidine blue stainings of semi-thin sections from P15 WT and Elp3cKO cochleae reveal a drastic reduction in the size of the spiral ganglion due to SGN loss. Scale bar=100µm, OC: Organ of Corti.(B) Specific labellings of the pre-synaptic marker Ctbp2 (in IHCs) and the post-synaptic marker GluR2/3 (in SGN terminals) indicate a decrease in the number of double-labelled dots in P15 Elp3cKO whole-mounted cochlea compared to WT. The remaining synapses still show coupling of the pre- and post-synaptic markers, suggesting they may be functional. Scale bar=20µm. (C) The mean number of Ctbp2-GluR2/3 double-positive dots in IHCs from the basal region of P15 Elp3cKO cochleae is reduced by 65% compared to control (n=6 animals per genotype; Unpaired two-tailed t-test, ***p<0.001, t=13.48, DF=10, mean±SD). (D) NF-1 and Mafb stainings on P0 cochlear sections indicate that SGN loss occurs before birth in Elp3cKO cochleae. Scale bar=100µm.(E) SGN quantifications on cochlear sections from different embryonic stages reveal a progressive loss of SGNs from E14.5 onwards (n=3-6 animals; one-way ANOVA, Tukey's multiple comparisons test; ***p<0.001; *p<0.05; F=20.43; DF=7; mean±SD).(F) Detection of apoptotic SGNs from E13.5 cochlear sections with cleaved caspase-3 and B-tubIII stainings. Scale bar=100µm.(G) The number of apoptotic SGN per section is significantly increased in E13.5 and E14.5 Elp3cKO compared to WT cochleae, suggesting SGNs are properly specified but die during their differentiation process (n=3-4 animals; one-way ANOVA, Tukey's multiple comparisons test; ***p<0.001; **p<0.01; F=23.74; DF=7; mean±SD).
Figure 3(A) Proteostat staining of WT and Elp3cKO E13.5 cochlear sections reveals the presence of numerous aggresome-like structures in Elp3-deficient SGNs. Scale bar =100µm.(B) Transmission electron micrographs from E13.5 Elp3cKO SGNs confirm the presence of apoptotic neurons (marked by a red arrowhead) and a large electron dense juxtanuclear structures typical of aggresome (dashed circle). Scale bars =10µm and 2µm (last panel).(C) Chop and Chac1 transcripts are upregulated in E14 Elp3cKO compared to WT cochleae (n=8/6 for WT/KO; unpaired t-test two-tailed; p=0.0001/0.0015/0.0010; t=9.535/4.090/4.307; DF=12/12/12 for Elp3, Chop and Chac1 respectively; mean±SEM presented) indicating that the pro-apoptotic arm of the unfolded protein response (UPR) is activated in Elp3-deficient SGNs.(D-E) Proteostat staining of whole-mounted P0 organs of Corti reveal the presence of aggregates in HCs upon loss of Elp3. Orthogonal slices (Orthog) reveal aggregated proteins localize at the apical surface of Elp3cKO HCs. Scale bars =5µm. #IHC; *OHC1; **OHC2; ***OHC3.(F) Protein extracts from P1 cochleae show increased levels of polyubiquitinated conjugates upon loss of Elp3, confirming proteostasis disruption.(G) The levels of Chop and Chac1 transcripts are unaffected by Elp3 depletion in P0 organs of Corti (n=3 animals per genotype; unpaired t-test two-tailed; p=0.0003/0.4296/0.056; t=11.62/0.8778/2.662; DF=4/4/4 for Elp3, Chop and Chac1 respectively; mean±SEM presented) suggesting that proteostasis disruption in Elp3cKO HCs is not sufficient to induce UPR-mediated apoptosis.
Figure 5(A-B) Polarized enrichment of LGN at the cortices of dividing HEK293T cells is disrupted upon Elp3 or Alkbh8 knockdown. (A) HEK293T cells transfected with siRNAs for Elp3, Alkbh8 or control and immunolabelled with LGN, acetylated alpha-tubulin and DAPI. Scale bar=5µm. (B) The mean fluorescence intensity profile of LGN across the radii perpendicular to division in HEK293T cells was quantified (siRNA Control/Elp3/Alkbh8: n=27/30/27; pooled from 3 independent experiments; mean±SEM).(C-D) The length of the primary cilium is reduced in non-dividing HEK293T cells upon Elp3 or Albh8 knockdown. (C) Control, Elp3 or Alkbh8 siRNA-treated HEK293T cells were immunolabelled for basal body marker ALMS1, axoneme marker Arl13b and DAPI. Scale bar=5µm. (D) Primary cilium length of non-dividing HEK293T was measured and pooled data were plotted (siRNA Control/Elp3/Alkbh8: n=94/81/90; data collected from 3 independent experiments; One-way ANOVA, Tukey's multiple comparisons test, ***p<0.001, F=21.46, DF=4; minimum, first quartile, median, third quartile and maximum presented).(E-F) Elp3 knockdown increases the amount of polyubiquitinated conjugates in HEK293T cells (n=3; One-way ANOVA, Tukey's multiple comparisons test, **p<0.01, *p<0.05, F=16.51, DF=2; mean±SD).(G) Increased formation of aggresome-like structures in Elp3- or Alkbh8-depleted HEK293T. Aggresomes (red); DAPI (blue). Enlarged images depict examples of non-dividing (left) and dividing (right) cells. Scale bar=10µm.Inducing aggresomes with MG-132 disrupts the polarized enrichment of LGN at the cortices of dividing HEK293T cells. (H) HEK293T cells treated with DMSO vehicle or MG-132 and stained for aggresomes (left panels) or LGN, F-actin and DAPI (green, red and blue, respectively in right panels). Scale bar=10µm.Inducing aggresomes with MG-132 disrupts the polarized enrichment of LGN at the cortices of dividing HEK293T cells. (I) Fluorescence intensity profile of LGN across the radii perpendicular to division in dividing HEK293T cells. Mean fluorescence values per percentage of cell length with SEM are plotted. Fluorescence intensity profiles collected from 3 independent experiments (DMSO/MG-132: n= 24/24).(J) Proteostat staining of MG132-treated whole-mounted P0 organs of Corti reveals the presence of aggresome-like structures in HCs. Orthogonal slices (Orthog) reveal aggregated proteins localize at the apical surface of MG132-treated HCs. Scale bars=5µm. #IHC; *OHC1; **OHC2; ***OHC3(K) Localization of LGN at the lateral apical surface of E16.5 WT HCs is disrupted by MG-132. LGN (white/green) and F-actin (red). Scale bars=5µm; magnified views=2µm.
Figure 6LGN and Gαi3 protein levels are not affected by Elp3 loss. (A) Western blots with total lysates from control and Elp3 siRNA-treated HEK293T cells and corresponding quantifications of normalized LGN, Gαi3 and Elp3 levels (n=6 from 2 independent cultures; Unpaired two-tailed t-test, LGN: p=0.749, t=0.328, DF=10, Gαi3: p=0.275, t=1.155, DF=10, Elp3: ***p=0.0003, t=5.438, DF=10, mean±SEM).LGN and Gαi3 protein levels are not affected by Elp3 loss. (B) Western blots on P0 WT and Elp3cKO cochlear extracts and corresponding quantifications of normalized LGN and Gαi3 levels (n=3, each lysate obtained from cochleae pooled from 2 animals; Unpaired two-tailed t-test, LGN: p=0.586, t=0.591, DF=4, Gαi3: p=0.963, t=0.0491, DF=4, mean±SEM).(C) Mislocalized LGN and Gαi3 are not found in protein aggregates formed upon Elp3 depletion. HEK293T transfected with Elp3 siRNA were co-stained for aggresomes, Dapi and LGN or Gαi3. Scale bar=5µm.Microtubular transport speed is reduced by the presence of aggresome-like structures in HEK293T cells and is improved upon 4-PBA treatment. The mean velocity of lysosomes was measured on time-lapse recordings of HEK293T cells labelled with LysoTracker®. (D): DMSO/MG-132/MG-132+4PBA n=555/337/453, pooled from 3 experiments, ***p<0.001, **p<0.01. All data analysed with Kruskal-Wallis ANOVA, Dunn's multiple comparisons test. Box/whisker plots depict minimum, first quartile, median, third quartile, and maximum.Microtubular transport speed is reduced by the presence of aggresome-like structures in HEK293T cells and is improved upon 4-PBA treatment. The mean velocity of lysosomes was measured on time-lapse recordings of HEK293T cells labelled with LysoTracker®. (E): siCtl/+4PBA n=622/677; siElp3/+4PBA n=674/577; siAlkbh8/+4PBA n=460/484, pooled from 4 experiments, ***p<0.001. All data analysed with Kruskal-Wallis ANOVA, Dunn's multiple comparisons test. Box/whisker plots depict minimum, first quartile, median, third quartile, and maximum.Microtubular transport speed is reduced by the presence of aggresome-like structures in cultured HCs, and is improved upon 4-PBA treatment. The mean velocity of lysosomes was measured on time-lapse recordings of HCs, at the level of their apical surface labelled with LysoTracker®. (F): DMSO/MG-132/MG-132+4PBA n=210/225/266, pooled from 3 experiments, ***p<0.001. All data analysed with Kruskal-Wallis ANOVA, Dunn's multiple comparisons test. Box/whisker plots depict minimum, first quartile, median, third quartile, and maximum.Microtubular transport speed is reduced by the presence of aggresome-like structures in cultured HCs, and is improved upon 4-PBA treatment. The mean velocity of lysosomes was measured on time-lapse recordings of HCs, at the level of their apical surface labelled with LysoTracker®. (G): WT/WT+4PBA/Elp3cKO/Elp3cKO+4PBA n=732/763/730/738, pooled from 4 OCs per condition, ***p<0.001, **p<0.01. All data analysed with Kruskal-Wallis ANOVA, Dunn's multiple comparisons test. Box/whisker plots depict minimum, first quartile, median, third quartile, and maximum.
Figure 7Intrinsic polarity is disrupted upon MG-132 treatment of cultured HCs and restored by the chemical chaperone 4-PBA. (A) LGN and F-actin immunostainings of E16.5+1DIV OCs reveal that MG-132 perturbs LGN distribution at the apical surface of HCs, while co-treatment with 4-PBA ameliorates its enrichment at the lateral edge. Scale bars=5µm; magnified views=2µm. Data information: #IHC; *OHC1; **OHC2; ***OHC3Intrinsic polarity is disrupted upon MG-132 treatment of cultured HCs and restored by the chemical chaperone 4-PBA. (B) Acetylated alpha-tubulin immunostaining reveals that the kinocilium is centrally shifted by MG-132 and partially restored by 4-PBA in E16.5+1DIV HCs (yellow arrowheads = kinocilium base; scale bar=2µm).Intrinsic polarity is disrupted upon MG-132 treatment of cultured HCs and restored by the chemical chaperone 4-PBA. (C) Kinocilium position plotted as a fraction of HC diameter for each HC row (DMSO/MG-132/MG-132+4PBA IHC: n=57/67/53, ***p<0.001, F=34.52, DF=2; OHC1: n=61/66/53, ***p<0.001, F=57.88, DF=2; OHC2: n=55/73/58, ***p<0.001, F=124.7, DF=2; OHC3: n=48/66/43, ***p<0.001, F=67.16, DF=2; pooled from 4 explants per condition; mean±SD; One-way ANOVA with Tukey's multiple comparisons test).In vivo administration of 4-PBA improves LGN localization and kinocilium position in Elp3cKO cochlear HCs. Pregnant mice were treated daily with saline or 4-PBA from E13.5 and the cochleae of WT and Elp3cKO embryos were analysed by immunostainings at E17.5. (D) The arc of lateral LGN enrichment remains in many Elp3cKO HCs after 4PBA administration. Scale bars=5µm; magnified views=2µm. Data information: #IHC; *OHC1; **OHC2; ***OHC3In vivo administration of 4-PBA improves LGN localization and kinocilium position in Elp3cKO cochlear HCs. Pregnant mice were treated daily with saline or 4-PBA from E13.5 and the cochleae of WT and Elp3cKO embryos were analysed by immunostainings at E17.5. (E) Acetylated alpha-tubulin immunostaining showing kinocilium position in WT or Elp3cKO OCs in the presence or absence of 4-PBA (yellow arrowheads = base of kinocilium; scale bar=2µm).In vivo administration of 4-PBA improves LGN localization and kinocilium position in Elp3cKO cochlear HCs. Pregnant mice were treated daily with saline or 4-PBA from E13.5 and the cochleae of WT and Elp3cKO embryos were analysed by immunostainings at E17.5. (F) Kinocilium position plotted as a fraction of HC diameter for each HC row (WT/WT+4PBA/Elp3cKO/Elp3cKO+4PBA IHC: n=35/31/35/36, p=0.6812, F=4.510, DF=3; OHC1: n=36/32/37/40, ***p<0.001, **p<0.01, F=27.60, DF=3; OHC2: n=35/33/38/42, ***p<0.001, F=76.62, DF=3; OHC3: n=36/32/38/39, ***p<0.001, *p<0.05, F=27.86, DF=3; pooled from 3 animals per condition; mean±SD; One-way ANOVA with Tukey's multiple comparisons test).
Figure 1.Non-endosomal ubiquitin-positive structures accumulate in ESCRT depleted cells. HeLa cells were left untransfected (A) or transfected with control (B), Hrs (C), Tsg101 (D), Vps22 (E), or Vps24 (F) siRNA for 5 d and processed for immunofluorescence microscopy. Cells were labeled with antibodies against EEA1 (blue), ubiquitin (mono and poly-Ub, red), and Lamp2 (green). Colocalization between EEA1 and Ub is indicated in purple and between Ub and Lamp2 in yellow. Bar, 10 μm. Single channel images of the insets are shown in Fig. S1 (available at http://www.jcb.org/cgi/content/full/jcb.200702115/DC1).(G) Knock-down efficiencies were analyzed by Western blotting. Equal loading was verified by anti-α-tubulin immunoblotting.
Figure 2.p62- and Alfy-positive structures accumulate in cells depleted of Tsg101 or Vps24. HeLa cells transfected with siRNA against Tsg101 or Vps24 were fixed, permeabilized, and stained with antibodies against ubiquitin (red) and p62 (green) (A) or against ubiquitin (green) and Alfy (red) (B). Colocalization is indicated in yellow. Bar, 10 μm. (C) The number and size (area) of p62-positive particles were quantified using ImageJ software. Approximately 100 cells from three different experiments (n = 334 (control), 309 (Tsg101), and 246 (Vps24)) were used for the quantification and the data is presented as average number per 100 cells. Error bars = SEM.
Figure 3.Autophagic degradation is impeded in Tsg101- and Vps24-depleted cells. HeLa cells stably expressing GFP-LC3 (green) were transfected with control (A), Vps24 (B), or Tsg101 (C) siRNA for 5 d and processed for immunofluorescence analysis. Cells were labeled with antibodies against p62 (red) and analyzed by confocal microscopy. Colocalization is indicated in yellow. Bar, 10 μm.(D) The total level of p62 and GFP-LC3 (normalized to control cells) and their degree of colocalization (% of total) in control and Tsg101- and Vps24-depleted cells were quantified using the Zeiss LSM 510 Meta software. 30 cells from three independent experiments were used for quantification. Error bars = SEM.(E) Western blot showing accumulation of LC3-II and p62 in HeLa cells depleted of Vps24 and Tsg101. p62 also accumulates in the insoluble fraction (bottom picture), indicating a shift to a more aggregated form upon depleted of Vps24 and Tsg101.(F) Western blot showing that LC3-II levels were similar in control and Vps24-depleted cells treated with Bafilomycin A.
Figure 4.Formation of autolysosomes is inhibited in cells depleted of Tsg101 or Vps24. (A) The double-tagged LC3 protein (mCherry-GFP-LC3) will emit yellow (green merged with red) fluorescence in non-acidic structures and appear as red only in the autolysosomes due to quenching of GFP in these acidic structures. (B-F) HeLa cells were transfected with control (B), Tsg101 (C), or Vps24 (D) siRNA for 4 d and then with mCherry-GFP-LC3 for another 24 h before confocal microscopy analysis. Bar, 5 μm(E) The mCherry-LC3 (red) signal was quantified in ∼30 cells with similar expression levels from three independent experiments using the Zeiss LSM 510 Meta software and the data are presented as % of total mCherry-GFP-LC3. Error bar = SEM.(F) Degradation of [14C]-valine- labeled proteins in cells incubated in complete medium (control), serum- and amino acid-deficient medium (starve), or starvation media in the presence of 3-methyl adenine (3-MA) to inhibit autophagic degradation. Average degradation per hour from four experiments done in duplicates ±1 SEM are shown.