EMBO/soda-nel huggingface dataset:
ALK ligand ALKAL2 potentiates MYCN-driven neuroblastoma in the absence of ALK mutation (2020)
Figure 2

Figure 2 - ALKAL stimulation of downstream ALK signaling in NB cells at the posttranscriptional level.

A. Differential protein expression and phosphorylation was determined in NB1 cells in response to ALKAL2 stimulation. See Table S2 for detailed results.

B. Volcano plots showing differential protein expression 1hr and 24hr after ALKAL2 +/- lorlatinib stimulation as indicated. Dashed lines indicate differential expression thresholds. Differentially expressed proteins indicated in blue. Most pronounced responding proteins indicated in black and labelled.

C, D. Immunoblot analysis of VGF protein in NB cells. (C) NB1 cells after 24h stimulation with ALKAL2 in the presence or absence of lorlatinib. (D) CLB-BAR and CLB-GE cells after 24h inhibition with lorlatinib.

E. Volcano plots showing differential phosphorylation. Labelling colors as in (B).

F. GSEA network graph with nodes representing the enriched reactome pathways (at 25% FDR). Node sizes correlate to the normalized enrichment scores, node colors indicate P values (as in color legend) and edge widths correspond to the number of overlapping genes between the connected nodes.

G. Graphical representation of FOXO3 dynamics, indicating S253 phosphorylation and total FOXO3 protein levels in response to ALKAL2 stimulation, in the presence or absence of lorlatinib.

H-I. Immunoblot validation of FOXO3a and STAT3 in response to ALKAL2 stimulation in the presence or absence of lorlatinib as indicated. The slower migrating FOXO3a band in SDS-PAGE in (H) likely reflects FOXO3a phosphorylation that is not seen in the presence of lorlatinib. Proteomic analysis was performed using 3 biological repeats. Immunoblots are representative of at least 3 independent experiments.
ALK ligand ALKAL2 potentiates MYCN-driven neuroblastoma in the absence of ALK mutation (2020)
Figure 3

Figure 3 - Alk-F1178S collaborates with Th-MYCN to drive NB in mouse models.

A. Hematoxylin and eosine staining of longitudinal sections of P9 pups at the central part of the left celiac ganglion.

B. Quantification of the area of celiac ganglia cross-section. Largest sections from the central part of left celiac ganglions of different individuals were chosen for the analysis. **p<0.01, one-way ANOVA followed by Tukey multiple comparison test. Data shown represents mean ± SD.

C. Hyperplasia quantification in central sections of P9 left celiac ganglions shown as a percent of hyperplastic regions areas per ganglion cross-section. **p<0.01, ****p<0.0001, one-way ANOVA followed by Tukey multiple comparison test. Data shown represents mean ± SD.

D. Kaplan-Meier survival curve of mice resulting from intercrosses of Th-MYCN hemizygotes and Alk-F1178S heterozygote mice (p<0.0001; log-rank test). Wild type littermates were excluded.

E. Gross appearance, hematoxylin and eosin as well as Ki67 stained sections of representative Th-MYCN and Alk-F1178S;Th-MYCN tumours. Scale bars indicate 250μm.

F. Ki67 immunohistochemically staining of P9 celiac ganglia in mice of the indicated genotype (quantified in G).

G. Quantification of hyperplastic areas (shown as percent of hyperplastic regions per ganglion cross-section) and Ki67 expression (shown as positive for Ki67 staining areas per total area of the section through the ganglion central part at P9). (*p<0.05, ***p<0.001, ****p<0.0001, one-way ANOVA followed by Tukey multiple comparison test. Data shown represents mean ± SD).
ALK ligand ALKAL2 potentiates MYCN-driven neuroblastoma in the absence of ALK mutation (2020)
Figure 4

Figure 4 - ALKAL2 collaborates with MYCN to drive NB in mouse models.

A. The oncogenic activity of MYCN is potentiated by overexpression of ALKAL2. Kaplan-Meier survival curves for Rosa26_Alkal2;Th-MYCN, Alk-F1178S;Th-MYCN and Th-MYCN mice. Also shown are Rosa26_Alkal2N, Alk-F1178S and control (Ctrl) mice. Comparison of survival of Th-MYCN alone and Rosa26_Alkal2;Th-MYCN curves showed a significant difference (p=0.003; log-rank test).

B-E. Tumours harvested from Rosa26_Alkal2;Th-MYCN, Alk-F1178S;Th-MYCN and Th-MYCN mice express NB markers. Tumours from all three genotypes were large, in most cases filling the abdominal cavity (B). Dissection post-mortem revealed that the majority of Rosa26_Alkal2;Th-MYCN (18/20) and Alk-F1178S;Th-MYCN (7/10) tumours did not involve the adrenal glands (C). Histological examination of Rosa26_Alkal2;Th-MYCN, Alk-F1178S;Th-MYCN and Th-MYCN tumours revealed positive staining for NCAM1, synaptophysin (SYP) and Chromogranin A (CGA). Scale bars indicate 100μm. (D), that was confirmed for NCAM1 and SYP along with MYCN, ALK and ALKAL2 by immunoblotting (E). Immunoblots are representative of 3 independent technical analyses.

F. Accumulated Kaplan-Meier survival curves are shown for all monitored Rosa26_Alkal2;Th-MYCN, Alk-F1178S;Th-MYCN and Th-MYCN mice over time, estimating tumour penetrance (p<0.001; log-rank test).
ALK ligand ALKAL2 potentiates MYCN-driven neuroblastoma in the absence of ALK mutation (2020)
Figure 5

Figure 5 - ALKAL2-driven tumours share a transcriptional signature with ALK-F1178S-driven NB.

RNA-Seq based differential gene expression analysis of tumours arising in Rosa26_Alkal2;Th-MYCN (Alkal2) [n=6], Alk-F1178S;Th-MYCN (AlkF1174S) [n=6] and Th-MYCN (MYCN) mice [n=4]. See Table S4 for detailed results.

A. Read coverage of the codon-optimised Alkal2 transgene, confirming Alkal2 expression in Alkal2 tumours.

B. Principal component (PC) analysis of the expression signature of human neuroblastoma (NB) and 5 other human cancers (BRCA: breast adenocarcinoma; COAD: colon adenocarcinoma; LUAD: lung adenocarcinoma; KIRC: kidney renal clear cell carcinoma; GBM: glioblastoma multiforme) with mice tumour samples mapped independently using PC coordinates. MYCN amplified NB samples are indicated by circles and non-amplified samples are indicated by squares.

C-D. Volcano plot showing differential expression (DE) between Alkal2 and MYCN tumours. Differentially expressed genes are shown in blue (DE in Alkal2 tumours only) or black (DE in both Alkal2 and AlkF1174S tumours, as shown in (D)). Top ranked genes labelled. Dashed lines represent DE cut-offs.

E. DE heatmap based on unsupervised hierarchical clustering of 400 DE genes (rows) and 16 samples (columns, as indicated on top). Sample color legend as in (A). Color key shown on top left.

F. Boxplot showing Vgf expression in the 3 tumour types as indicated. Box plots indicate median values and lower/upper quartiles with whiskers extending to 1.5 times the interquartile range. P values calculated using Wald test as reported by DESeq2.

G. Histological examination of Th-MYCN, Rosa26_Alkal2;Th-MYCN and Alk-F1178S;Th-MYCN tumours revealing positive staining for VGF. Scale bars indicate 100μm.

H. Kaplan-Meier relapse free (RF) and event free (EF) survival probability curves from two different NB cohorts, the Versteeg 88 cohort (upper panel) and the Kocak 649 cohort (lower panel), as derived from the R2 platform. Patients with higher VGF expression are highlighted in blue, whereas patients with lower expression are highlighted in red. The log-rank test P values are indicated.
ALK ligand ALKAL2 potentiates MYCN-driven neuroblastoma in the absence of ALK mutation (2020)
Figure 6

Figure 6 - Cell lines derived from ALKAL2-driven NB respond to ALK TKI treatment.

Murine NB cell lines were generated from tumours arising in Rosa26_Alkal2;Th-MYCN (#3540) and Alk-F1178S;Th-MYCN (#3456) mice.

A. Alkal2 expression in cells derived from Rosa26_Alkal2;Th-MYCN (#3540) and Alk-F1178S;Th-MYCN (#3456) NB. Immunoblotting analysis for ALKAL2 and tubulin in the indicated mouse NB cell lines. Whole cell lysates (30μg) were analysed in each lane.

B. The effect of increasing concentrations of brigatintib on cell confluence was analyzed by IncuCyte Live Cell Analysis of both Rosa26_Alkal2;Th-MYCN (#3540) and Alk-F1178S;Th-MYCN (#3456) cell lines. Data is presented as mean ± SEM from three independent experiments. *p<0.05, **p<0.005; two-tailed paired student t-test.

C-D. Brigatinib suppressed tumour spheroid formation and spheroid viability. Cells (#3456 or #3540) were treated with brigatinib (0, 150nM) for 4d in ultra-low attachment plates. The spheroid number was analyzed by IncuCyte® Live Cell Analysis Systems. Data are presented as means ± SEM from three independent experiments. *p<0.05, two-tailed unpaired student t-test. Scale bar (C) is 200μm.

E. Tumour spheroids formed from either Rosa26_Alkal2;Th-MYCN (#3540) or Alk-F1178S;Th-MYCN (#3456) were formed at indicated cell number in ultra-low attachment plates for 3d and followed by brigatinib (0, 150nM) for 10d. Inhibitor was re-fed every other day. Cell viability was determined by CellTiter-Glo 3D cell viability kit and data is presented as mean ± SEM from five independent experiments. *p<0.05, ****p<0.0001, two-tailed unpaired student t-test.

F. Cell viability. Mouse tumour derived cell lines #3540 (Rosa26_Alkal2;Th-MYCN) and #3456 (Alk-F1178S;Th-MYCN) were treated with brigatinib (125nM and 500nM) and viability was evaluated by using a resazurin-based assay. Data is presented as mean ± SEM from three independent experiments. *p<0.05, **p<0.005; two-tailed paired student t-test.

G. Brigatinib treatment (0 or 150 nM) for 6h resulted in inhibition of ALK phosphorylation, and of activation of downstream signaling (ERK1/2), as well as MYCN expression. Cell lysates (Rosa26_Alkal2;Th-MYCN (#3540) and Alk-F1178S;Th-MYCN (#3456)) were immunoblotted with the indicated antibodies.
Structure and functional mapping of the KRAB-KAP1 repressor complex (2022)
Figure 4

Figure 4. Effects of point mutations in the KRAB binding site and KAP1 binding and silencing. See also Fig EV3.

(A) Surface plasmon resonance (SPR) KAP1-KRAB binding assay. MBP-KRAB was immobilized on the chip. WT or R311E KAP1 RBCC were flowed over the chip. Data points are shown in dark red or blue; fits are shown as light red or blue lines. See Fig EV3 for binding kinetics constants.

(B) Position of the mutations in the KRAB-KAP1 interface.

(C) LINE-1 reporter repression with single point mutants and a previously described KRAB-binding deficient KAP1 variant containing four mutations in the CC domain (CCmut; V293S/K296A/M297A/L300S).

(D) SVA reporter repression with the same set of mutants as in (B). Data were normalized to KAP1 KO cells transfected with an empty vector (EV).

Data information: In (C) and (D), data are presented as fold-repression of reporter luciferase luminescence. Error bars represent standard error of the mean between measurements (n = 3). Data are representative of at least three independent (biological replicate) experiments. Lower panels: Western blots of cell lysates from KAP1 KO HEK293T cells transfected with each of the variants or empty vector. Uncropped blots available in Source Data.
Exploiting dominant-negative toxins to combat Staphylococcus aureus pathogenesis (2016)
Figure 1

Figure 1. Deletions of the glycine-rich motifs of LukE and LukD inactivate the toxin and make the mutant LukED dominant-negative.

A. Viability of primary human PMNs following treatment with purified recombinant bi-component leukocidins using the viability dye CellTiter. Bars indicate Mean ± SEM, with n = 5 donors.

B. Half maximal effective concentrations (EC50) of native and heterologous toxin pairings on primary human PMNs using the viability dye CellTiter. E = LukE, D = LukD, A = HlgA, B = HlgB, C = HlgC, S = LukS-PV, F = LukF-PV. Bars indicate Mean ± SEM, with n = 6 donors.

C. Leukocidin octamer highlighting stem domain motifs deleted from LukE and LukD (LukEmut1, LukEmut2, and LukDmut), as shown from the side view (left), and top view (right) of the pore. The monomeric structures of LukE (3ROH) and LukD (4Q7G) were obtained from the Protein Data Bank, and superimposed and colored on the structure of the HlgA-HlgB octameric pore (3BO7) using PyMOL. The location of where the deleted residues would be in the pore structure is shown from a side view on the left, and the location within an oligomeric pre-pore structure are shown from a top view on the right.

D. Viability of primary human PMNs following incubation with WT and mutant toxin pairings using the viability dye CellTiter. Insert: 2 μg per lane of purified recombinant toxins visualized by Coomassie Blue staining on a 12% SDS-PAGE gel. E = LukE, D = LukD, Emut1 = LukEmut1, Dmut = LukDmut. Bars indicate Mean ± SEM, with n = 4 donors.

E. Viability of primary human PMNs following simultaneous incubation with a lethal dose of WT LukED (78.12 nM per subunit) and a dose response of WT or dominant-negative (DN) toxin subunits using the viability dye CellTiter. E = LukE, D = LukD, Emut1 = LukEmut1, Emut2 = LukEmut2, Emut1Dmut = LukEmut1LukDmut, Emut2Dmut = LukEmut2LukDmut . Bars indicate Mean ± SEM, with n = 6 donors.
Exploiting dominant-negative toxins to combat Staphylococcus aureus pathogenesis (2016)
Figure 2

Figure 2. The glycine-rich motifs within the stem domain are required for the cytotoxic activity of other leukocidins.

A-B. Amino acid sequences of toxin S-subunits depicting (A) Mut1 (∆112-117) and (B) Mut2 (∆130-134) domains, based on ClustalW alignment.

C. Viability of primary human PMNs following incubation with mutant toxin pairings using the viability dye CellTiter. Bars indicate Mean ± SEM, with n = 3 donors.

D-F. Viability of primary human PMNs following incubation with WT or mutant toxin pairings. Insert: 2 μg per lane of purified recombinant toxins visualized by Coomassie blue staining using a 12% SDS-PAGE gel. Amut1 = HlgAmut1, Bmut = HlgBmut, Cmut1 = HlgCmut1, Smut1 = LukS-PVmut1, Fmut = LukF-PVmut. Bars indicate Mean ± SEM, with n = 4 donors (D), n = 4 donors (E), and n = 5 donors (F).
Exploiting dominant-negative toxins to combat Staphylococcus aureus pathogenesis (2016)
Figure 3

Figure 3. Dominant-negative toxins exhibit cross-neutralizing effects over other WT leukocidins.

A-D. Viability of primary human PMNs following incubation with a LD90 of LukED (78.12 nM), HlgAB (78.12 nM), HlgCB (39.1 nM), or LukSF-PV (19.53 nM) and the indicated dominant-negative toxins using the viability dye CellTiter. Open white symbols indicate no treatment. Filled black symbols indicate treatment with dominant-negative toxins. Emut1Dmut = LukEmut1LukDmut, Amut1Bmut = HlgAmut1HlgBmut, Cmut1Bmut = HlgCmut1HlgBmut, Smut1Fmut = LukS-PVmut1LukF-PVmut. Bars indicate Mean ± SEM, with n = 5 donors.

E. Viability of primary human PMNs following incubation with filtered culture supernatants collected after 8 hours from CA-MRSA USA300 strain LAC using the viability dye CellTiter. Bars indicate Mean ± SEM, with n = 6 donors.

F. Viability of primary human PMNs following incubation with 1.25% filtered culture supernatants and a dose response of the indicated dominant-negative (DN) toxins using the viability dye CellTiter. Emut1Dmut = LukEmut1LukDmut, Amut1Bmut = HlgAmut1HlgBmut, Cmut1Bmut = HlgCmut1HlgBmut, Smut1Fmut = LukS-PVmut1LukF-PVmut. Bars indicate Mean ± SEM, with n = 3 donors.

G. Viability of primary human PMNs during a 3-hour ex vivo infection with the S. aureus Newman toxinless strain overproducing the indicated toxins using the CytoTox-One Homogenous Membrane Integrity Assay to measure lactate dehydrogenase (LDH) release. Bars indicate Mean ± SEM, with n = 3 donors.

H. Viability of primary human PMNs during a 4-hr ex vivo infection with a MOI of 50 of S. aureus Newman toxinless strain overproducing the indicated toxins in the presence of a dose response of the DN toxins using the CytoTox-One Homogenous Membrane Integrity Assay to measure lactate dehydrogenase (LDH) release. Emut1Dmut = LukEmut1LukDmut, Amut1Bmut = HlgAmut1HlgBmut, Cmut1Bmut = HlgCmut1HlgBmut, Smut1Fmut = LukS-PVmut1LukF-PVmut. Bars indicate Mean ± SEM, with n = 3 donors.
Exploiting dominant-negative toxins to combat Staphylococcus aureus pathogenesis (2016)
Figure 4

Figure 4. Deletions of the glycine-rich motifs within the stem domain LukAB protect against S. aureus infection.

A. LukAB octamers (4TW1) highlighting the location of where the deleted residues would be in the pore structure (left- LukABmut1, right- LukABmut2), as shown from the side view of the pore. The octameric LukAB structure was obtained from the Protein Data Bank and the motifs labeled using PyMOL.

B. Exoproteome analysis of LukAB mutants produced by S. aureus strain Newman in a toxinless background. Samples were electrophoresed on a 12% SDS-PAGE gel and visualized by staining with Coomassie blue. A representative of two experiments is shown.

C. Exoproteome and immunoblot analyses of wildtype S. aureus strain Newman transformed with empty plasmid (p-) or with plasmids containing the mutated lukAB loci. A representative of two independent experiments is shown.

D. Viability of primary human PMNs during a 2-hour ex vivo infection with the indicated S. aureus strains using the CytoTox-One Homogenous Membrane Integrity Assay to measure lactate dehydrogenase (LDH) release. Bars indicate Mean ± SEM, with n = 3. Statistical significance is indicated as follows: MOI of 100: **p = 0.0026 for p- vs LukABmut1 and ***p = 0.0003 for p- vs LukABmut2; MOI of 50: ****p <0.0001 for p- vs LukABmut1 and ****p <0.0001 for p- vs LukABmut2; MOI of 25: ***p = 0.0007 for p- vs LukABmut1 and ****p < 0.0001 for p- vs LukABmut2 using two-way ANOVA with Tukey’s post hoc test correction for multiple comparisons.
Exploiting dominant-negative toxins to combat Staphylococcus aureus pathogenesis (2016)
Figure 5

Figure 5. Dominant-negative toxins act by forming defective pores on the target host cell plasma membrane.

A. Schematic depicting competition assay using GFP-LukE and unlabeled WT or mutant toxins.

B. Competition assay using GFP-LukE (312.5 nM) and a dose response of unlabeled WT or dominant-negative (DN) toxin subunits evaluated by flow cytometry on the surface of primary human PMNs. E = LukE, Emut1 = LukEmut1, A = HlgA, Amut1 = HlgAmut1, S = LukS-PV, C = HlgC. Data is presented as total fluorescence emitted from PMNs. A representative of three independent experiments each done with hPMNs isolated from different donors is shown.

C. Schematic depicting recruitment assay using GFP-LukD and unlabeled WT or dominant-negative toxins.

D. Recruitment assay using GFP-LukD (156.3 nM) and a dose response of unlabeled dominant-negative (DN) toxin subunits evaluated by flow cytometry on the surface of primary human PMNs. Emut1 = LukEmut1, Amut1 = HlgAmut1. Data is presented as total fluorescence emitted from GFP-positive PMNs. A representative of three independent experiments each done with hPMNs isolated from different donors is shown.

E. Immunoblot using anti-His antisera to detect dimer and oligomer formation by purified polyhistidine (6xHis)-tagged WT or dominant-negative LukED toxins in the absence or presence of 2-methyl-2,4-pentanediol (MPD, 40% v/v). Emut1 = LukEmut1, Dmut = LukDmut. A representative of two independent experiments is shown.
Exploiting dominant-negative toxins to combat Staphylococcus aureus pathogenesis (2016)
Figure 6

Figure 6. Dominant-negative toxins are active in vivo.

A. Survival of Swiss-Webster mice following intravenous administration of purified recombinant toxins: LukED (10 μg per subunit), HlgAB (5 μg per subunit), HlgCB (35 μg per subunit) and LukSF-PV/PVL (35 μg per subunit). Buffer: n = 2 mice; LukED: n = 3 mice; HlgAB: n = 3 mice; HlgCB: n = 2 mice; PVL: n = 3. A representative of two independent experiments is shown.

B. Survival of Swiss-Webster mice following prophylactic intravenous administration of dominant-negative mutants, followed by intravenous challenge with a lethal dose of WT LukED. Emut1Dmut = LukEmut1LukDmut, Amut1Bmut = HlgAmut1HlgBmut, Cmut1Bmut = HlgCmut1HlgBmut, Smut1Fmut = LukS-PVmut1LukF-PVmut. n = 6 mice per treatment. ***p = 0.0005 for Buffer vs Emut1Dmut, Buffer vs Amut1Bmut, and Buffer vs Cmut1Bmut. p = 0.1050 (not significant, ns) for Buffer vs Smut1Fmut. Statistics performed using Log-rank (Mantel-Cox) test. A representative of two independent experiments is shown.

C. Survival of Swiss-Webster mice following prophylactic intravenous administration of dominant-negative mutants, followed by intravenous challenge with a lethal dose of WT HlgAB. Emut1Dmut = LukEmut1LukDmut, Amut1Bmut = HlgAmut1HlgBmut, Cmut1Bmut = HlgCmut1HlgBmut, Smut1Fmut = LukS-PVmut1LukF-PVmut. n = 6 mice per treatment. ***p = 0.0004 for Buffer vs Emut1Dmut and Buffer vs Amut1Bmut. **p = 0.0015 for Buffer vs Cmut1Bmut. p = 0.7955 (not significant, ns) for Buffer vs Smut1Fmut. Statistics performed using Log-rank (Mantel-Cox) test. A representative of two independent experiments is shown.

D. Bacterial burden 96 hours post infection in the liver of mice infected systemically with ~1.5 x 107 CFU of S. aureus strain Newman and intravenously treated with buffer or LukEmut1LukDmut (Emut1Dmut, 10 μg per subunit) at t = 0, 24, and 48 h.p.i. Bars indicate Mean ± SEM, with n = 20 mice (Buffer) and n = 19 mice (Emut1Dmut2) combined from two independent experiments. *p = 0.0303 by unpaired, parametric, two-tailed Student’s t-test.
Src-dependent impairment of autophagy by oxidative stress in a mouse model of Duchenne muscular dystrophy (2014)
f1

(a) Nox2-specific ROS production was assessed using the Nox2 redox biosensor p47-roGFP redox biosensor Cat: catalase, PEG-Cat: pegylated catalase. (b) Measurement of intracellular glutathione redox potential with Grx1-roGFP2. (c) Analysis of Rac1 and (d) Src. (e) Immunoblot of precipitated p47phox probed with an anti-phosphoserine or anti-p47phox antibody. (f) Nox2-specific intracellular ROS production was measured using p47-roGFP redox biosensor. (g) Extracellular ROS production was assessed using Amplex-red dye. (h) Plasma membrane calcium influx was measured by analysing the Fura-2 fluorescence quench rate upon addition of extracellular Mn2+. (i) Intracellular RNS generation was measured using DAF-FM. Bars represent average±s.e.m. from n=15 individual fibres for each condition in (a,b,f,g,i,j). Markers of autophagy were analysed in isolated fibres (incubated with or without PP2) from FDBs. (k) Autophagosome formation was analysed using fluorescence microscopy (scale bar=100 μm) and illustrated LC3 localization and autophagosome formation. (l) Confocal microscopy detected p62-LC3 localization in single fibres from FDBs (scale bar=140 and 50 μm for white box areas). All immunoblots were performed with isolated proteins from FDBs and probed with antibodies as indicated. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was detected as a loading control. Representative images are shown. Bars represent average ±s.e.m. from n=3 independent biological experiments. Statistical differences between groups were determined using analysis of variance (ANOVA) with Tukey’s post hoc test. *P0.05 and **P0.01.
Src-dependent impairment of autophagy by oxidative stress in a mouse model of Duchenne muscular dystrophy (2014)
f2

(a) FDB lysates were analysed using immunoblotting with antibodies as indicated. (b) Confocal microscopy shows the effect of Nox2 inhibition on p62-LC3 localization in single FDB fibres. Representative images of independent biological experiments (n=3) are shown (scale bar=100 and 35 μm for white box areas). (c) Autophagic flux was analysed by immunoblotting with anti-LC3B and anti-p62 antibodies. (d) Effect of gp91 ds and PP2 on LC3-LAMP1 colocalization was analysed using confocal microscopy. Representative images from independent biological experiments (n=3) are shown (scale bar=140 and 40 μm for red box areas). (e) Immunoblot analysis of LAMP1, (f) PARP-1 and caspase3 activity. (g) Immunoblot of Caspase3 activity. All immunoblots were performed with isolated proteins from FDBs and probed with antibodies as indicated. GAPDH was detected as a loading control. Representative images are shown. Bars represent average ±s.e.m. from n=3 independent biological experiments. Statistical differences between groups were determined using ANOVA with Tukey’s post hoc test. *P0.05 and **P0.01.