EMBO/soda-nel huggingface dataset:
TFAM loss induces nuclear actin assembly upon mDia2 malonylation to promote liver cancer metastasis (2022)
Figure 7

Figure 7. Downregulated TFAM induced malonylation of mDia2 to promote its nuclear translocation.

(A) Schemes of glucose metabolism alternation detected by targeted metabolomics. The fold change of metabolites in mitochondria and cytoplasm between TFAM knockdown and WT SNU-368 cells were shown (n =6 per group). Increased and decreased metabolites are indicated by red and green circles, respectively.

(B) The fold change of intracellular acetyl-CoA, malonyl-CoA and lactate in SNU-368 cells with TFAM knockdown was evaluated by ELISA or enzymatic reaction (n = 3 independent experiments).

(C) Extracellular acidification rate (ECAR) was measured in TFAM knockdown and WT SNU-368 cells with serial addition of glucose, oligomycin and 2-DG (n = 3 independent experiments). The panel shows the calculated glycolytic parameters, including glycolysis and maximal glycolysis. Glycolysis was calculated as maximum rate measurement before Oligomycin injection minus last rate measurement before glucose injection. While glycolytic capacity was calculated as maximum rate measurement after Oligomycin injection minus last rate measurement before glucose injection.

(D) Western blot analysis of anti-mDia2 IPs and whole-cell lysates (WCL) derived from HCC cells and mouse tumor tissues (n = 3 independent experiments). NT, non-tumor liver tissues; MT, metastatic tumor tissues; NMT, non-metastatic tumor tissues.

(E) The level of malonylated mDia2. Equal amount of immunoprecipitated mDia2 either from TFAM knockdown or control cells was loaded in Western blot assay.

(F) Western blot analysis for the distribution of mDia2 in SNU-368 cells incubated with malonyl-CoA (5 μM) or ND-630 (10 μM) (n = 3 independent experiments).

(G) Bioluminescent images for lung metastasis in mice inoculated with HCC cells. n =10. Ten days after orthotopic implantation of SNU-368 shTFAM cells, ND-630 (3 mg/kg) was intravenously injected in mice every 2 days until euthanasia was performed.

(H) Representative H&E images of lung metastasis in mice used in (G). Scale bars, 100μm.

(I) Number of metastatic nodules per lung in mice used in (G).

(J) Kaplan-Meier survival curves for mice inoculated with orthotopic HCC cells as indicated. n=10 per group.

(K and L) Representative merged images of mDia2-GFP (Green) and DAPI staining (blue) (K) and Phalloidin (Red) and DAPI staining (blue) (L) in HCC cells transfected with different mutant mDia2-GFP constructs as indicated (n = 3 independent experiments). Scale bars, 20μm. K12E and K27E are malonylation-mimetic mutants at lysine residues 12 and 27. K12Q and K27Q are acetylation-mimetic mutants at lysine residues 12 and 27.

(M) Statistical analysis of transwell migration and invasion assays for HCC cells with treatment as indicated (n = 3 independent experiments).

Data information: Graphs show mean ± s.e.m., two-tailed unpaired t-test for (B, C, I). One-way ANOVA for (M). *P < 0.05; **P < 0.01.
TFAM loss induces nuclear actin assembly upon mDia2 malonylation to promote liver cancer metastasis (2022)
Figure 8

Figure 8. Down-regulation of TFAM and nuclear F-actin were associated with poor prognosis in HCC patient.

(A) IHC scores of TFAM in 429 paired tissues from HCC patients. The relative expression ratio of tumor to non-tumor was log2-transformed. T, tumor tissues; NT, non-tumor liver tissues.

(B) mRNA level of TFAM in 50 HCC patients' paired tissues from TCGA database. The relative expression ratio of tumor to non-tumor was log2-transformed.

(C) Representative images of IHC staining of TFAM (top panel) and scores (bottom panel) in HCC tumor tissues. One-way ANOVA. *P < 0.05; **P < 0.01. MT, metastatic tumor tissues; NMT, non-metastatic tumor tissues. Scale bars, 25μm. Boxes: first quartile to third quartile; Whiskers: minimum value to maximum value; Central band: median. (D and E) Kaplan-Meier curves for overall survival and the recurrence of HCC patients stratified by TFAM expression.

(F and G) Kaplan-Meier curves for overall survival and the recurrence of HCC patients stratified by nuclear F-actin positive rates.

(H) Representative images of IHC staining of TFAM, FN1 and ITGB3 from two HCC patients. Scale bars, 25μm.

(I) Spearman correlation analysis between the levels of TFAM and FN1, IL-6, TGFB1 or ITGB3.
Disulfiram bolsters T-cell anti-tumor immunity through direct activation of LCK-mediated TCR signaling (2022)
Figure 1

Figure 1. DSF activates TCR signaling in CD8+ T cells.

A FACS screening assay in WT mouse splenic cells.

B FACS analysis of p-S6 in WT mouse splenic CD8+ T cells stimulated for 8 min with α-mouse CD3ε(145-2C11) after pretreatment with DSF (5 µM) for 2 h (n = 3).

C Representative FACS of p-S6 in WT mouse splenic CD8+ T cells stimulated for 8 min with α-mouse CD3ε(145-2C11) after pretreatment with the indicated concentrations of DSF for 2 h (n = 2).

D Western blot analysis of the phosphorylation level of S6 and p70S6K in WT naïve CD8+ T cells stimulated by α-mouse CD3ε(145-2C11) (1 µgml-1) in the presence of α-mouse CD28 (1 µgml-1) for the indicated times after pretreatment with DSF (5 µM) for 2 h. The immunoblot shows normalized expression of p-S6(S235/236) to S6, p-p70S6K to p70S6K, p-Zap70 to Zap70, as well as that of p-LCK(Y394) and P-Lck(Y505) to Lck.

E FACS analysis of p-CD3ζ(PY142) in mouse naïve CD8+ T cells treated with DSF or stimulated with α-mouse CD3ε(145-2C11) in the presence of α-mouse CD28 (1 µgml-1) for 2 h (n = 3).

F Western blot analysis of the indicated proteins in Jurkat T cells treated with the indicated concentrations of DSF for 2 h. The immunoblot analysis shows normalized expression of p-CD3ζ to CD3ζ as well as that of p-Zap70(Tyr319)/Syk(Tyr352), p-LCK(Y394), and p-LAT(Tyr191) to β-actin.

G, H Luciferin assay of NFAT activity (G) and Nfatc1 mRNA expression (H) in J76-NFATRE-luc and TCR052 cells stimulated with α-human CD3 (OKT3) (1 µgml-1) in the presence of α-human CD28 (1 µgml-1) for 4 h or treated with DSF (5 µM) for 2 h. The levels of NFAT activity were normalized with TCR052 cells treated with DMSO (n = 3).

Data information: In (B-H), data are representative of 3 independent experiments, mean ± SD. **P<0.01; ***P<0.001; ****P<0.0001; ns, not significant [unpaired t tests for measurements between the two groups in (B), (C), and (E), Image J analysis in (D) and (F), or two-way analysis of variance (ANOVA) in (G, H)].
Disulfiram bolsters T-cell anti-tumor immunity through direct activation of LCK-mediated TCR signaling (2022)
Figure 2

Figure 2. DSF directly binds to and activates LCK.

A, B MST analysis determined the Kd of DSF towards His-LCK(WT) (86.812 nm) (A) or His-LCK(C20/23S) (22.797 µM) (B) labeled with RED-tris-NTA 2nd Generation dye. Concentration is reported in nanomolar.

C, D MS/MS spectra of the Cys20/23-containing human LCK peptide WMENIDVCENCHYPIVPLDGK (aa 13-33; 2532.87 Da) (LC retention time was 49.16 min; a triplet-charged precursor ion m/z 844.7163 (mass, 2531.1284 Da; ΔM of −0.5 ppm; 10logP = 200) was observed) (C), or of the corresponding LCK peptide after LCK incubation with DSF, which was modified on Cys23 (red) by the diethyldithiocarbamate moiety of DSF (an increase of 147.0176 Da; LC retention time was 56.31 min; a triplet charged precursor ion m/z 893.7295 (mass, 2678.1460 Da; ΔM of 7.7 ppm; −10logP = 116.99) was observed (D).

E Recombinant human LCK proteins left untreated (Ctrl) or incubated with ATP and increasing concentrations of DSF for 15 min. Enzyme-linked immunosorbent assay (ELISA) of LCK kinase activity was analyzed by using α-p-LCK (Y394) antibody. The results show the average values of the optical density at 450 nm (OD450) from five replicates (n = 5).

F Confocal analysis of p-Lck(Y394) (red) in naïve CD8+ T cells treated with DSF (5 µM) or stimulated with α-mouse CD3 and α-mouse CD28 for 2 h (left). The total fluorescence intensity (IntDen) was quantified (right; n= 20 cells). Scale bars, 3 µm.

G Western blot analysis of the active LCK enrichment [assessed by p-Lck (Y394)] in Triton X-100-insoluble fractions after DSF (5 µM) treatment in naïve CD8+ T cells.

H IVP assay of His-tagged CD3εCD peptides was phosphorylated by LCK in the presence of DSF. CD3ε phosphorylation was analyzed by immunoblotting using α-pTyr(PY100).

I Western blot analysis of p-CD3ζ, LCK and p-Zap70 in LCK-deficient cells were infected with the lentivirus collected from human HEK293T cells transfected with control plasmid (Control) (1 µg), LCK(WT) (1 µg), or LCK(C20/23S) (1 µg) with psPAX2 (1 µg) and pMD2.G (1 µg) for 48 h followed by treatment with DSF (5 µM) for 2 h stimulated with α-human CD3 and α-human CD28 for 5 min.

Data information: In (A-I), data are representative of 2 (C-E, H) or 3 (A, B, F, G and I) independent experiments, mean ± SD. **P<0. 01; ***P<0.001; ns, not significant [one-way ANOVA in (E) and (F)].
Disulfiram bolsters T-cell anti-tumor immunity through direct activation of LCK-mediated TCR signaling (2022)
Figure 3

Figure 3. DSF directly activates T cell effector function.

A Schematic depicting treatment of naïve primary OT-I T cells for assessment of T cell activation.

B Functional enrichment analysis of Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways upregulated by DSF compared with DMSO (n = 3).

C Scatter plot comparing global gene-expression profiles of DMSO and DSF (n = 3).

D A heatmap of upregulated genes associated with TCR and T cell activation-related genes in DSF relative to that in DMSO. One column indicates one sample in each condition (n = 3).

E ELISA analysis of the production of IL-2 in primary OT-I T cell supernatants treated with DMSO, DSF (5 µM) or OVA peptide (5 µgml-1) for 4 h (n = 3).

F CFSE dilution of proliferation of primary OT-I T cells treated with DMSO or DSF (5 µM) for 2 h (n = 3).

G FACS analysis of IFNγ and TNFα production in primary OT-I T cells primed by N4 (SIINFEKL, 0.01 μgml-1) and A4 (SAINFEKL, 0.01 μgml-1) for 2 h followed by treatment with DSF (5 µM) for 2 h (n = 3).

H, I Extracellular acidification rate (ECAR) (H) and oxygen consumption rate (OCR) (I) in primary OT-I T cells (n = 3).

Data information: In (B-I), data are representative of 3 independent experiments, mean ± SD. *P<0.05; **P<0.01; ***P<0.001; ****P<0.0001. ns, not significant [unpaired t tests for the measurements between the two groups in (E), (F) and (H), two-way ANOVA in (G) and (I)].
Disulfiram bolsters T-cell anti-tumor immunity through direct activation of LCK-mediated TCR signaling (2022)
Figure 4

Figure 4. DSF enhances the anti-tumor immunity of CD8+ T cells.

A, C Tumor growth [(A), (Vehicle, n = 5; DSF, n = 4, one tumor had disappeared)] and survival curves [(C), (n = 10 mice per group)] in DSF (suspended in 0.5% NaCMC)- and Vehicle (equal volume of 0.5% NaCMC)-treated mice after B16F10 melanoma inoculation.

B Images and tumor weight of subcutaneous mouse B16F10 tumors extracted from mice at day 21 (Vehicle, n = 5; DSF, n = 4, one tumor had disappeared).

D - F Phenotypic (D, F) and immunofluorescence (E) analysis of tumor-infiltrating T cells at day 21 after melanoma inoculation (Vehicle, n = 5; DSF, n = 4, one tumor had disappeared). Scale bars, 20 µm. Statistics data are from 40× field of view.

G Phenotypic analysis of IFNγ and TNFα production in T cells isolated from draining lymph nodes at day 19 after melanoma inoculation (n = 3).

H, I Tumor growth [(H), (n = 5 mice per group)] and survival curves [(I), (n = 5 mice per group)] in Vehicle- and DSF-treated mice injected s.c. with B16F10-melanoma cells followed by intraperitoneal (i.p.) injection with PD-1 antibody on days 0 and 4 and then every third day (n = 5).

Data information: In (A-I), data are representative of 3 independent experiments, mean ± SD. *P<0.05; **P<0.01; ***P<0.001; ****P<0.0001. ns, not significant [multiple t tests between the two groups in (B), (D) to (G), or two-way ANOVA in (A) and (H), log-rank (Mantel-Cox) test in (C) and (I)].
Disulfiram bolsters T-cell anti-tumor immunity through direct activation of LCK-mediated TCR signaling (2022)
Figure 5

Figure 5. DSF bolsters anti-tumor immunity via activating CD8+ T cells.

A Tumor growth in Vehicle- and DSF-treated mice injected s.c. with B16F10 melanoma cells followed by intraperitoneal (i.p.) injection with CD8(2A3) antibody on days −1, 0, and 2 and then every second day (n = 5).

B FACS analysis of cytotoxicity of B16F10-OVA cells after coculture with DMSO- or DSF-treated primary OT-I T cells (n = 3).

C, D Tumor growth [(C), (n = 5 mice per group)] and survival curves [(D), (n = 10 mice per group)] of B16F10-OVA tumor-bearing mice after adoptive transfer of Vehicle- or DSF-treated primary OT-I T cells.

Data information: In (A-D), data are representative of 3 independent experiments, mean ± SD. *P<0.05; **P<0.01; ****P<0.0001. ns, not significant [two-way ANOVA in (A) to (C), log-rank (Mantel-Cox) test in (D), mean ± SD].

Supplementary Figure legends
Autophagosome-Lysosome Fusion in Neurons Requires INPP5E, A Protein Associated with Joubert Syndrome (2016)
Figure 1

Figure 1. INPP5E, an essential factor for autophagy.

A Schematic diagram of human INPP5E. PRD, proline-rich domain; IPPc, inositol polyphosphate phosphatase domain.

B Levels of INPP5E protein 72 h after transfection of N1E-115 cells with control (siControl) or INPP5E-specific siRNAs (siINPP5E #1 or #2), as determined by immunoblot.

C N1E-115 cells treated with siControl or siINPP5Es were cultured for 2 h in growth medium with or without 125 nM bafilomycin A1 (Baf.A1), and then analyzed by immunoblot using anti-p62, anti-LC3, and anti-GAPDH antibodies.

D Quantitation of protein signal intensities from immunoblots in C showing LC3-II (left) or p62 (right) levels, following normalization to the control protein GAPDH. Because Baf.A1 inhibits autophagosome–lysosome fusion and degradation within lysosomes via inhibition of vacuolar-type H+-ATPase, it is possible to measure autophagic flux, i.e., the degradation of autophagic substrates such as p62 and LC3-II, by calculating the difference of the signal intensities of these proteins in the presence and absence of Baf.A1. Results represent the mean ± s.d. of three independent experiments. **, P < 0.01.

E N1E-115 cells treated with siControl or siINPP5Es were cultured in growth medium. Cells were fixed and stained with anti-LC3 antibodies and analyzed by immunofluorescence microscopy. Bar, 10 µm.

F Quantitation of the number of LC3 puncta per cell, as described in E (mean ± s.d.; n > 100 cells from three independent experiments). **, P < 0.01.

G Electron micrographs in N1E-115 cells treated with siControl or siINPP5Es were cultured in growth medium. Arrowheads indicate autophagosomes. Bar, 1 µm.
Autophagosome-Lysosome Fusion in Neurons Requires INPP5E, A Protein Associated with Joubert Syndrome (2016)
Figure 2

Figure 2. INPP5E functions in autophagosome–lysosome fusion.

A N1E-115 cells treated with siControl, siINPP5E, or siAtg2a/2b were cultured in growth medium. The post-nuclear fraction (PNS) was separated into LSP, HSP, and HSS fractions, and then analyzed by immunoblots using anti-p62, anti-LC3, and GAPDH antibodies. The subfractions were treated with proteinase K (Pro. K) with or without Triton X-100. Quantitation of protein signal intensities from immunoblots showing LC3-II (left) or p62 (right) levels, following normalization to the control protein GAPDH. **, P < 0.01; *, P < 0.05.

B NIE-115 cells stably expressing LAMP1-mCherry treated with siControl or siINPP5Es were cultured for 2 h in growth medium containing 200 nM Torin1 with protease inhibitors (10 µg/ml Pepstatin A and 10 µg/ml E-64-d). Because the treatment of protease inhibitors inhibits lysosomal degradation, LC3 puncta persist even if autophagosomes fuse with lysosomes. Cells were fixed and stained with anti-LC3 antibodies, and then analyzed by immunofluorescence microscopy. Insets show the boxed areas at high magnification. Bar, 10 µm.

C Percentages of colocalization of LC3 dots with LAMP1 dots (mean ± s.d.; n > 20 cells from three independent experiments). **, P < 0.01.
Autophagosome-Lysosome Fusion in Neurons Requires INPP5E, A Protein Associated with Joubert Syndrome (2016)
Figure 3

Figure 3. Phosphatase activity and lysosomal localization of INPP5E are needed for autophagy.

A Schematic diagram of human INPP5E and mutants used in this study. D477N, phosphatase-inactive mutant.

B N1E-115 cells stably expressing mSt-INPP5E (WT, D477N, ∆CAAX, or 295-644) treated with siControl or siINPP5E were cultured in growth medium with or without 125 nM Baf.A1 for 2 h, and then analyzed by immunoblot using anti-LC3 and anti-GAPDH antibodies. The mSt-INPP5Es used in this study were resistant to mouse siINPP5E because target sequence of the siRNA is different from the human correspondent sequence.

C Quantitation of protein signal intensities from immunoblots in B, showing differences in LC3-II levels in the presence and absence of Baf.A1 following normalization to the control protein GAPDH. Results represent means ± s.d. of three independent experiments. **, P < 0.01; *, P < 0.05.

D NIE-115 cells stably expressing mSt-INPP5E (WT, D477N, or ∆CAAX) were cultured in growth medium. Cells were fixed and stained with anti-LAMP1 antibodies, and then analyzed by immunofluorescence microscopy. Insets show the boxed areas at high magnification. The number showing to the right side indicates the colocalization rate of LAMP1 with mSt-INPP5E. Bar, 10 µm.
Autophagosome-Lysosome Fusion in Neurons Requires INPP5E, A Protein Associated with Joubert Syndrome (2016)
Figure 4

Figure 4. INPP5E knockdown influences neither lysosomal integrity nor fusion between endosomes and lysosomes.

A N1E-115 cells treated with siControl or siINPP5Es were cultured in growth medium with 20 µg/ml DQ-BSA for 12 h and chased for 2 h. Cells were fixed and stained with anti-LAMP1 antibodies and analyzed by immunofluorescence microscopy. Insets show the boxed areas at high magnification. Bar, 10 µm.

B Quantitation of signal intensities in A showing colocalization of DQ-BSA with LAMP1 (means ± s.d.; n > 50 cells from three independent experiments). Baf.A1 was used as a control for inhibition of delivery and degradation of DQ-BSA via the endocytic pathway. **, P < 0.01; n.s., non significant.

C N1E-115 cells treated with siControl, siINPP5Es, or siCHMP5 were stimulated with 100 ng/ml EGF for the indicated time periods, and then analyzed by immunoblot using anti-EGFR and anti-α-tubulin antibodies. Levels of CHMP5 mRNA 72 h after transfection of N1E-115 cells with siControl or siCHMP5, as analyzed by RT-PCR.

D Quantitation of EGFR degradation ratio in C. Results represent means ± s.d. of three independent experiments. **, P < 0.01.
Autophagosome-Lysosome Fusion in Neurons Requires INPP5E, A Protein Associated with Joubert Syndrome (2016)
Figure 5

Figure 5. PI(3,5)P2 is localized to lysosomes and catalyzed by INPP5E.

A NIE-115 cells stably expressing mSt-2xML1N or -2xPLCδ1 PH were cultured in growth medium with or without 200 nM Torin1. Cells were fixed and stained with anti-LAMP1 or anti-LC3 antibodies, and then analyzed by immunofluorescence microscopy. Insets show the boxed areas at high magnification. Bar, 10 µm.

B NIE-115 cells stably expressing mSt-2xML1N treated with siControl or siINPP5Es were cultured in growth medium. Cells were fixed and stained with anti-LAMP1 antibodies, and then analyzed by immunofluorescence microscopy. Insets show the boxed areas at high magnification. Bar, 10 µm.

C Quantitation of signal intensities in B showing mSt-2xML1N colocalizing with LAMP1 (means ± s.d.; n > 100 cells from three independent experiments). **, P < 0.01; *, P < 0.05.

D N1E-115 cells stably expressing GFP-2xML1N treated with siControl or siINPP5E were transiently transfected with mSt-INPP5E (WT, D477N). Quantitation of signal intensities showing GFP-2xML1N colocalizing with LAMP1 (means ± s.d.; n > 20 cells from three independent experiments). **, P < 0.01; *, P < 0.05.

E NIE-115 cells stably expressing mCherry-2xFYVE treated with siControl or siINPP5Es were cultured in growth medium. Cells were fixed and stained with anti-LAMP1 antibodies, and then analyzed by immunofluorescence microscopy. Insets show the boxed areas at high magnification. Bar, 10 µm.

F Quantitation of signal intensities in E showing mCherry-2xFYVE colocalizing with LAMP1 (means ± s.d.; n > 100 cells from three independent experiments). **, P < 0.01.
Autophagosome-Lysosome Fusion in Neurons Requires INPP5E, A Protein Associated with Joubert Syndrome (2016)
Figure 6

Figure 6. INPP5E enhances actin polymerization on lysosomes through cortactin activation.

A N1E-115 cells treated with siControl or siINPP5Es were cultured in growth medium. Cells were fixed and stained with anti-LAMP1 antibodies and Phalloidin, and then analyzed by immunofluorescence microscopy. Insets show the boxed areas at high magnification. Bar, 10 µm. Quantitation of signal intensities showing Phalloidin colocalizing with LAMP1 (means ± s.d.; n > 40 cells from three independent experiments). **, P < 0.01.

B N1E-115 cells treated with siControl or siINPP5Es were cultured in growth medium. Cells were fixed and stained with anti-phospho-Y421 cortactin and anti-LAMP1 antibodies, and then analyzed by immunofluorescence microscopy. Insets show the boxed areas at high magnification. Bar, 10 µm. Quantitation of signal intensities showing cortactin (pY421) colocalizing with LAMP1 (means ± s.d.; n > 40 cells from three independent experiments). **, P < 0.01.

C N1E-115 cells were treated with 0.5 µM Latrunculin A (Lat A) in growth medium for 6 h. Cells were fixed and stained with anti-LC3 antibodies, and then analyzed by immunofluorescence microscopy. Bar, 10 µm. Quantitation of the number of LC3 puncta per cell. (mean ± s.d.; n > 100 cells from three independent experiments). **, P < 0.01.

D NIE-115 cells stably expressing LAMP1-mCherry were treated with DMSO or 0.5 µM Lat A in growth medium for 4 h, subsequently cultured with 200 nM Torin1 and protease inhibitors (10 µg/ml Pepstatin A and 10 µg/ml E-64-d) for 2 h (Lat A treatment for total 6h). Cells were fixed and stained with anti-LC3 antibodies, and then analyzed by immunofluorescence microscopy. Insets show the boxed areas at high magnification. Bar, 10 µm. Percentages of colocalization of LC3 dots with LAMP1 dots (mean ± s.d.; n > 20 cells from three independent experiments). **, P < 0.01.
Autophagosome-Lysosome Fusion in Neurons Requires INPP5E, A Protein Associated with Joubert Syndrome (2016)
Figure 7

Figure 7. Joubert syndrome–associated INPP5E mutations compromise autophagy. A Joubert syndrome–associated INPP5E mutations (Bielas et al, 2009) used in this study.

B N1E-115 cells stably expressing the indicated mutants treated with siControl or siINPP5E were cultured in growth medium with or without 125 nM Baf.A1 for 2 h, and analyzed by immunoblot using anti-LC3 and anti-GAPDH antibodies. The mutants were resistant to mouse siINPP5E because target sequence of the siRNA is different from the human correspondent sequence.

C Quantitation of protein signal intensities from immunoblots in B showing differences in LC3-II levels in the presence and absence of Baf.A1 following normalization to the control protein GAPDH. Results represent means ± s.d. of three independent experiments. **, P < 0.01; *, P < 0.05.