EMBO/soda-nel huggingface dataset:
Linc-MYH configures INO80 to regulate muscle stem cell numbers and skeletal muscle hypertrophy. (2020)
Figure 3

Figure 3: linc-Myh restricts proliferation of MuSCs in an INO80-dependent manner.

A-D RNA in situ hybridization proximity ligation assay (rISH-PLA) detects the close proximity of a specific RNA with proteins in situ modified from Roussis et al.. (B-C) rISH-PLA confirms the proximity of linc-MYH to endogenous INO80-V5 in the nucleus of proliferating WT MuSCs (B). The specific signal is lost in linc-MYH deficient MuSCs (C). (n = 3 WT/3 KO biological replicates; Mann-Whitney test, *p = 0.05). Data are mean ± S.E.M.

E-I Representative images of TA muscle sections of 10 weeks old mice stained for PAX7 (green) and EdU incorporation (red). MuSCs without (green arrows) and with EdU-labeling (red arrows) are indicated. (E, F) Deletion of linc-MYH results in increased numbers of muscle stem cells and pronounced increase of the ratio of EdU-positive MuSCs. (G-I) The increase in both the number of MuSCs and EdU positive MuSCs in linc-MYH mutant muscle is abolished by constitutive (G: Pax7-Crepos / INO80-/-, Ino80 KO; H: linc-MYH-/- / Pax7-Crepos / INO80-/-, dKO) and induced (I: linc-MYH-/- / Pax7-CreERT2pos / INO80-/-, dKO CreERT) deletion of INO80 in Pax7 expressing cells.

J-L Statistical evaluation of the number of muscle stem cells observed in TA muscles (J). Statistical evaluation of the ratio of EdU-incorporating MuSCs relative to all Pax7-positive MuSCs (K). Statistical evaluation of the number of myonuclei in myofibers of TA muscles from different genotypes (L); (n = 5 WT/5 linc-MYH KO/4 Ino80 KO/5 dKO/3 CreERT dKO animals in J/K, n = 6 WT/6 linc-MYH KO/5 Ino80 KO/6 dKO/3 CreERT dKO animals in L **p < 0.01, ****p <0.0001, > 31 MSCs /animal in K; ANOVA test with multiple comparisons against WT. Sidak-Holm correction was used. Data are mean ± S.E.M.).
Linc-MYH configures INO80 to regulate muscle stem cell numbers and skeletal muscle hypertrophy. (2020)
Figure 4

Figure 4: Deletion of INO80 prevents linc-MYH dependent changes in MuSC proliferation and gene expression

A-D Isolated linc-MYH KO MuSCs reach confluence much faster than WT MuSCs indicating higher proliferation rates. Deletion of INO80 eliminates the difference between Pax7-Crepos / INO80-/- (Ino80 KO) and linc-MYH-/- / linc-MYH-/-/ Pax7-Crepos / INO80-/- (dKO). Images are representative images for cultures after 13 days in culture.

E Differences in proliferation rates are abolished by additional deletion of INO80 in MuSCs. Cell confluence was monitored by a live cell imaging analysis system for 13 days (n = 7 WT / 6 linc-MYH KO / 3 Ino80 KO / 3 dKO independent preparations of MuSCs).

F Statistical analysis of cell confluence after 13 days of muscle stem cell proliferation (n = 7 WT / 6 linc-MYH KO / 3 Ino80 KO / 3 dKO independent preparations of MuSCs, Student's t test, two-tailed; *p < 0.05, ns = not significant). Data are mean ± S.E.M.

G-I Gene set enrichment analysis (GSEA, broadinstitute.org) of microarray data from proliferating MuSCs. Comparison of linc-MYH-/- vs. WT proliferating MuSC transcriptomes shows enrichment of genes related to cell proliferation (G), enrichment of genes containing cis regulatory elements for YY1 (H) and p53 (I). Enrichments are highly significant using the most conservative familywise-error rate method (FWER). Enrichments were lost in linc-MYH-/- MuSCs after additional deletion of INO80 (dKOs) compared to Pax7-Crepos / INO80-/- MuSCs.

J-L Heat maps of the 10 most upregulated genes of the respective gene sets depicted in G-I in linc-MYH-/- vs. WT MuSCs and in linc-MYH-/-/ Pax7-Crepos / INO80-/- dKO vs. Pax7-Crepos / INO80-/- MuSCs. Expression of up-regulated genes in linc-MYH-/- was normalized after additional deletion of INO80 (dKOs), indicating that effects of linc-MYH completely depend on INO80 (WT n = 4, linc-MYH-/- n = 4, Pax7-Crepos / INO80-/- n = 4, Pax7-Cre dKO n = 3 animals used for independent isolations of MuSCs).
Linc-MYH configures INO80 to regulate muscle stem cell numbers and skeletal muscle hypertrophy. (2020)
Figure 5

Figure 5: linc-MYH does not regulate the function of INO80 during replication stress

A-D γ-H2AX staining of proliferating MuSCs from WT (A), linc-MYH deficient (B), Pax7-Crepos / INO80 KO (C), and linc-MYH/INO80 dKO (D) mice. Deletion of INO80 results in increased γ-H2AX signals while deletion of linc-MYH has no effects (asterisk: low γ-H2AX signals, arrow: γ-H2AX-foci detected in nuclei, arrowhead: pan-nuclear γ-H2AX signal.

E-G Statistical evaluation of the presence of γ-H2AX signals in MuSCs of different genotypes (n = 3/3/3/3 animals used for independent isolations of MuSCs, two technical replicates for each animal and at least 164 cells were counted, Mann-Whitney test, **p<0.01. Data are mean ± s.e.m.).

H-I Gene set enrichment analysis of microarray data obtained for proliferating MuSCs shows enrichment of genes related to activity of ATR in response to replication stress recovery (H) and enrichment of genes involved in chromosome maintenance (I) in WT compared to Pax7-Crepos INO80-/- MuSCs. Enrichment of these or similar gene sets was not observed when comparing WT to linc-MYH-/- MuSCs.

J Model of the role of INO80 and linc-MYH in the recognition of γ-H2AX and DNA repair. The DNA repair function of the INO80 complex does not dependent on its interaction with linc-MYH.
Linc-MYH configures INO80 to regulate muscle stem cell numbers and skeletal muscle hypertrophy. (2020)
Figure 6

Figure 6: linc-MYH modulates INO80 complex composition by reducing incorporation of YY1 and WDR5

A, B Co-Immunoprecipitation of INO80 complex subunits from TA muscles of WT and linc-MYH KO mice followed by Western Blot analysis. (B) Statistical analysis of Co-IP/Western Blot data. The amount of RUVBL1 and RUVBL2 in the INO80 complex is not changed after deletion of linc-MYH, while the amounts of YY1, WDR5 and TFPT pulled down by INO80 increase (n = 3 WT / 3 linc-MYH KO animals; one-way ANOVA, multiple comparisons by Fishers LSD test, *p<0.05). Data are mean ± S.E.M.

C-K Proximity ligation assays between INO80 and YY1 (C, D), INO80 and WDR5 (F, G), and INO80 and RuvBl2 (I, J) in proliferating MuSCs. Red signals indicate close proximity of INO80-V5 and the respective interacting protein. Nuclei were stained using DAPI (blue). Statistical evaluation of proximity ligation assays indicating increased interactions of INO80-V5 and YY1 (E, n = 3/3 animals, 3 independent wells per animal, two-tailed students t-test, *p<0.05) and of INO80-V5 to WDR5 (H, n = 2 (WT)/2 (KO) animals, 3 independent wells per animal, two-tailed students t-test, **p<0.01). No increase in proximity was detected between Ino80-V5 and Ruvbl2 (K, n = 2 (WT)/2 (KO) animals, 3 independent wells per animal, two-tailed students t-test, ns: not significant). Specificity of all analyzed signals was analyzed using single primary or single secondary antibodies in PLA assays. No signals were detected for these controls. Data are mean ± S.E.M.

Table 1 Identification of linc-MYH-dependent INO80 interaction partners

Proteins interacting with INO80 in WT and linc-MYH deficient TA muscle were identified by co-immunoprecipitation in combination with label-free quantitative mass-spectrometry. Three independent Co-IP experiments were performed using muscle tissues from WT (n = 3) and linc-MYH KO (n = 3) animals. A coefficient for relative abundance of individual proteins in WT / linc-MYH KO Co-IP samples was calculated and results were filtered for known INO80 interacting proteins based on the NCBI record of the human INO80 interactome (https://www.ncbi.nlm.nih.gov/gene/?term=INO80). The calculations revealed increased abundance of TFPT, WDR5 and YY1 and reduced abundance of INO80c in INO80 Co-IP experiments. Other core-components of the INO80 complex such as Ruvbl1/2, ACTR5, ACTR8 and NFRKB were unaltered.

Expanded View Figure legends
Intraductal xenografts show lobular carcinoma cells rely on their own extracellular matrix and LOXL1 (2021)
Figure 2

Figure 2. Histopathology of MM134 and SUM44 intraductal xenografts.

A, B Representative micrographs of H&E stained histological sections of xenografted mammary gland from 3 female mice 1 month after injection of MM134 (A) or SUM44 cells (B). Scale bars, 100 μm.

C Representative micrographs of H&E stained histological sections of MM134 xenografts from 3 different female mice 5 months after intraductal injection. Arrows point to indian files. Scale bars, 100 μm.

D Representative micrographs of H&E stained histological sections of SUM44 cell xenografts from 3 different female mice 5 months after intraductal injection. Arrows point to indian files. Scale bar, 50 μm.

E, F Representative micrographs of IHC for Ki67, ER, and PR on histological sections of MM134 (E) and SUM44 (F) xenografts 5 months after intraductal injection counter stained with hematoxylin. Scale bars, 200 μm. Bar plots represent means ± SEM of measurements and indicate the percentage of positive cells for >1000 cells counted on 3 glands.

G Representative micrographs of PR-IHC on glands from 5-month-old MM134 xenografts from hosts treated for 2 months with vehicle (left) or E2 (right) pellets. Scale bars, 200 μm. Right, bar plot showing percentage of PR+ cells in MM134 and SUM44 xenografts with n>1000 cells per condition on glands from 3 different mice. Data represent mean ± SEM unpaired, Student's t-test, two-tailed; *** p < 0.001, **** p < 0.0001.

H Fluorescence stereomicrograph of a mammary gland of a NSG:EGFP+ mouse 2 months after injection with MM134-RFP/luc2 cells. Scale bar, 1 mm.

I, J Micrographs of IF for GFP and E-cadherin (I) and GFP and SMA (J) counterstained with DAPI of MM134 glands 1 month after intraductal injection of NSG:EGFP+ mice, n=4. Scale bars, 50 μm.

K Scheme showing the timeline of lobular carcinogenesis in the intraductal model.
Intraductal xenografts show lobular carcinoma cells rely on their own extracellular matrix and LOXL1 (2021)
Figure 4

Figure 4. ECM-related transcriptome in patient samples.

A Heatmap of unsupervised hierarchical clustering performed on ER+ BC TCGA based on expression of most matrisome collagens and proteoglycans. Tumor identities ILC (blue) and non-ILC (red) are shown.

B Density plot showing the estimated score distributions of ILC and non-ILC tumors based on the expression of matrisome ECM-related genes. The vertical lines identify the medians. Statistical test performed by unpaired Student's t test, two-tailed.

C Heatmap of normalized expression levels of LOX family members and ELN in invasive BCs, in ILC (blue) and non-ILC (red) tumors sorted based on CDH1 expression levels. Right, boxplots showing normalized transcript expression levels. The line in the middle of the box is the median. The edges of the box reflect the 25th and 75th percentiles, vertical lines outside the box end at minimum and maximum values. Outliers are depicted as grey dots. Statistical analysis was by ANOVA analysis of the linear model with tumor subtypes and tumor purity as covariates. Adjusted p-values computed by post-hoc tests. Sample size for NSTs n=490 and ILCs n=127. **** p < 0.0001.

D Representative RNAscope image of sections from patient ILCs (n=5) stained with anti-LOXL1 probes (red) and counterstained with DAPI (blue). Scale bars; 50 μm.

E Representative immunofluorescence image of primary ILC (n=3) stained with anti-LOXL1 (green) and anti-ER (red) antibodies counterstained with DAPI (blue). Scale bars; 50 μm and right inset 30 μm.
Intraductal xenografts show lobular carcinoma cells rely on their own extracellular matrix and LOXL1 (2021)
Figure 6

Figure 6. LOXL1 knockdown and its effects on ILC progression.

A Bar plot showing LOXL1 transcript levels as measured by semi qRT-PCR normalized to 36B4 of scramble and shLOXL1 expressing ILCs cells in vitro. Data represent mean ± SEM (n=3); Student's unpaired t-test, two-tailed.

B Scheme describing the experimental approach used to generate shLOXL1 expressing and control ILC xenograft models. ILC:RFP-luc2 cells were transduced with GFP+ sh-scramble or GFP+ shLOXL1 expressing lentiviruses, selected by FACS and injected into the into contralateral 3rd thoracic and the 4th inguinal gland of NSG females. Xenografted mice were monitored for tumor growth by bioluminescence imaging.

C, D Radiance-based tumor growth curves (mean ± SE) for the scramble and shLOXL1 MM134 (C) and SUM44 (D) xenografts. For statistical analysis, mixed-effects with spline regression models were used on log10(radiance) curves (Fig S5A). Likelihood-ratio tests from the ANOVA function in R were used for model comparison, i.e. to check whether the treatment/group covariate is significant. **p<0.01.

E Representative fluorescent stereo-micrographs of glands injected with MM134:RFP-luc2 cells (left, top and bottom) infected with either GFP:sh-scramble (top) or GFP+ shLOXL1 (bottom), n=5 and 3. Scale bar, 1000 μm.

F Barplot showing the ratio of GFP/RFP signal from fluorescent stereo-micrographs of glands engrafted with MM134 cells, n=8. Statistical analysis by paired Student t-test, two-tailed. *p < 0.05.

G Representative fluorescensce micrographs of GFP:sh-scramble (n=8) and GFP+ shLOXL1 (n=10) MM134 xenografted glands. ILC cells expressing viral transcripts are detected based on GFP expression (green); DAPI nuclear stain (blue). Asterics point to intraductal growth while arrow points to invasive tumor cells. Scale bars, 50 μm.

H Representative fluorescent stereo-micrographs of glands engrafted with SUM44:RFPluc2 cells infected with either GFP:sh-scramble (top) or GFP+ shLOXL1, n=6. Scale bar, 1000 μm.

I Barplot (mean ± SEM) showing quantification of the ratio GFP/RFP from fluorescent stereo-micrographs of SUM44. Statistical analysis by unpaired Student's t-test, two-tailed, n=7 sh-scramble and n=6 for sh-LOXL1, ****p < 0.0001.

J, K Representative photomicrographs of picrosirius red stained histological sections for sh-scramble or sh-LOXL1 MM134 (J) and SUM44 (K) xenografts. Scale bars, 100 μm.
Proteostasis is essential during cochlear development for neuron survival and hair cell polarity (2019)
Figure 1

Figure 1: Elp3 expression in the inner ear is essential for hearing.

(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.

(B) Elp3 specific immunostaining confirms the presence of the protein in HCs and SGNs at birth. Scale bar=10µm.

(C-D) 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. (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).

Data information: #IHC; *OHC1; **OHC2; ***OHC3, OC: Organ of Corti.
Proteostasis is essential during cochlear development for neuron survival and hair cell polarity (2019)
Figure 2

Figure 2: Elp3cKO SGNs are lost before birth as a result of early apoptosis.

(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).
Proteostasis is essential during cochlear development for neuron survival and hair cell polarity (2019)
Figure 3

Figure 3: Aggresome-like structures are formed in SGNs and HCs from Elp3cKO animals.

(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.
Proteostasis is essential during cochlear development for neuron survival and hair cell polarity (2019)
Figure 5

Figure 5: Intrinsic polarity is dependent upon Elongator's function on tRNAs and is impaired upon proteasome inhibition.

(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.

(H-I) 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. (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.
Proteostasis is essential during cochlear development for neuron survival and hair cell polarity (2019)
Figure 6

Figure 6: The presence of aggresome-like structures affects microtubular transport speed in cultured HEK293T cells and cochlear HCs.

(A-B) LGN 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). (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.

(D-G) Microtubular transport speed is reduced by the presence of aggresome-like structures in HEK293T cells and cultured HCs, and is improved upon 4-PBA treatment. The mean velocity of lysosomes was measured on time-lapse recordings of HEK293T cells (D, E) and HCs, at the level of their apical surface (F, G), labelled with LysoTracker®. (D): DMSO/MG-132/MG-132+4PBA n=555/337/453, pooled from 3 experiments, ***p<0.001, **p<0.01. (E): siCtl/+4PBA n=622/677; siElp3/+4PBA n=674/577; siAlkbh8/+4PBA n=460/484, pooled from 4 experiments, ***p<0.001. (F): DMSO/MG-132/MG-132+4PBA n=210/225/266, pooled from 3 experiments, ***p<0.001. (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.