EMBO/soda-nel huggingface dataset:
High grade mesenchymal pancreatic ductal adenocarcinoma drives stromal deactivation through CSF-1 (2020)
Figure 3

Fig 3: Mesenchymal-like PDAC cells deactivate PSCs.

(A) Gene expression level of stromal activation markers COL1A1, ACTA2, SPARC, FN1 and FAP in PS-1 cells which were exposed for 72 hrs to CM from control (CM of PS-1 cells ), epithelial (blue) or mesenchymal (red) PDAC cell lines using qPCR. Values were normalized to control and n=6 per group, 3 biological replicates for each cell line.

(B) Heatmap representation of COL1A1 and ACTA2 relative gene expression levels measured with qPCR in PS-1 cells after treatment indicated in panel A using a large cohort of PDAC cell lines and primary cell cultures 67 and 53m. Values were Log2 transformed and normalized to Suit-2 expression.

(C) Brightfield images of PS-1 cells exposed to 10 days of treatment with 1 and 10 µM all-trans retinoic acid (ATRA). Scale bar represents 100 µm.

(D) Gene expression levels of COL1A1 and ACTA2 in PS-1 cells after treatment indicated in panel C using qPCR, values were normalized to control. n=3 technical replicates per group.

(E) Brightfield images of primary human CAFs exposed to 72 hrs of treatment with CM from control (CM of primary CAFs) or mesenchymal PDAC cell lines. Scale bar represents 100 µm.

(F) Relative gene expression levels of COL1A1 and ACTA2 in primary CAFs after treatment indicated in panel E using qPCR, values were normalized to control. n=3 technical replicates per group.

Data information: (A-F), data are represented as mean±SD, Student's t-test.
High grade mesenchymal pancreatic ductal adenocarcinoma drives stromal deactivation through CSF-1 (2020)
Figure 4

Fig 4: PSC deactivation is mediated by colony stimulating factor 1.

(A) Brightfield images of PS-1 cells after 72 hrs of treatment with unfiltered, <100 kDa or <30 kDa filtered CM of indicated cell lines. Scale bar represents 100 µm.

(B) Gene expression levels of COL1A1 and ACTA2 in PS-1 cells after treatment indicated in panel A using qPCR. Data was normalized against control CM. Statistical significance was tested against unfiltered CM samples. Graph shows n=3 technical replicates of an example experiment that was performed three times.

(C) Graphical representation of 64 cyto- and chemokines present in CM of epithelial and mesenchymal PDAC cell lines using forward-phase protein array. Significantly differential expression of proteins (triangles) and genes (green triangles) are identified between epithelial and mesenchymal cell lines. Dotted line indicates P=0.05.

(D) Brightfield images of PS-1 cells after 72 hrs of treatment with CM of control (CM of PS-1s) or epithelial PDAC cells supplemented with recombinant human CSF-1. Scale bar represents 100 µm.

(E) Gene expression levels of COL1A1 in PS-1 cells after treatment indicated in panel D using qPCR. n=3 biological replicates per group.

(F) PS-1 cells were starved and stimulated with 500 ng/ml rh-CSF-1 at indicated time points. Activation of pathways involved in CSF1R signaling was analyzed using western blot. Samples were derived from the same experiment and blots were processed in parallel.

Data information: (B-E), data are represented as mean±SD, Student's t-test.
High grade mesenchymal pancreatic ductal adenocarcinoma drives stromal deactivation through CSF-1 (2020)
Figure 5

Fig 5: Targeting CSF-1 in mesenchymal-like PDAC cells abolishes PSC deactivation.

(A) Brightfield images of PS-1 cells after 72 hrs of treatment with CM of control (CM of PS-1s) or mesenchymal PDAC cell lines supplemented with anti-CSF-1. Scale bar represents 100 µm.

(B) Gene expression levels of COL1A1 in PS-1 cells after treatment indicated in panel A using qPCR. Graph shows n=3 technical replicates of an example experiment that was performed three times.

(C) Brightfield images of PS-1 cells after 72 hrs of treatment with PANC-1 CM of pLKOctrl or shCSF1 (#560-562). Scale bar represents 100 µm.

(D) Correlation graph of CSF1 expression in PANC-1 pLKOctrl or shCSF1 (#560-562) and how these various CM affect COL1A1 expression in PS-1 cells using qPCR, values were normalized for RPLPO expression. n=3 biological replicates for all individual experiments, error bars indicated SD. R2 and P-value were analyzed with linear regression.

(E) Brightfield images of PS-1 cells after 72 hrs of treatment with CM of Capan-2 cells with control vector or Capan-2 cells overexpressing CSF1. Scale bar represents 100 µm.

(F) Gene expression level of CSF1 in Capan-2 cells after transduction with control vector or overexpressing CSF1. n=3 technical replicates per group.

(G) Gene expression levels of COL1A1 in PS-1 cells after treatment indicated in panel E using qPCR. Graph shows n=3 technical replicates of an example experiment that was performed three times.

Data information: (B, F-G), data are represented as mean±SD, Student's t-test.
High grade mesenchymal pancreatic ductal adenocarcinoma drives stromal deactivation through CSF-1 (2020)
Figure 6

Fig 6: High-grade CSF-1-positive PDAC features deactivated stroma.

(A) IHC staining of CSF-1 in PDAC tissue following surgical resection (same cohort as in Fig 1A). A representative image of CSF-1 expression in grade 1 and grade 3 PDAC is depicted. Scale bar represents 100 µm.

(B) For each tumor of panel A, CSF-1 expression in ductal adenocarcinoma cells was quantified. Student's t-test. Shown is mean±SD. n=15 primary PDAC samples.

(C) A validation cohort of PDAC patients (n=21) that underwent direct resection was stained for CSF-1 (scale bar represents 100 µm), PSR (scale bar represents 500 µm) and α-SMA (scale bar represents 200 µm). OK8 is low in CSF-1 expression and high in PSR and α-SMA expression, while the opposite accounts for OK7.

(D) Correlation of % of PSR in the tumor and CSF-1 expressing tumor cells, as indicated in panel C. R2 and P-value were analyzed with linear regression. n=21 primary PDAC samples

(E) Correlation of % of α-SMA in the tumor and CSF-1 expressing tumor cells, as indicated in panel C, R2 and P-value were analyzed with linear regression. n=21 primary PDAC samples

EXPANDED VIEW FIGURE AND TABLE LEGENDS
Characterization of circulating breast cancer cells with tumorigenic and metastatic capacity (2020)
Figure 1

Figure 1. CTC cell line establishment from peripheral blood of an mBCa patient

A Scheme of the breast cancer patient's clinical status and therapies. Course of disease progression (blue) and treatment scheme (green) of the patient giving rise to CTC-ITB-01 are indicated. Timeline of progression and treatment indicated in years and months (mo). Drugs were administered at standard dosage according to indicated pattern. The red star represents the time point of blood sample collection. More detailed information is available the in Appendix Supplementary Materials and Methods.

B Representative pictures of different CTCs from the initial CellSearch® analysis of the metastatic breast cancer patient who gave rise to the breast CTC line. The detected tumor cells display clear keratin and DAPI staining, CD45-negativity as well as lack of, or very weak (4, 8), ERBB2 expression. Cells of small (about 5 µm in diameter, 1, 2) and large size (larger than 10µm in diameter, 2,3) were detected. While some CTCs displayed dot-like perinuclear keratin signals (1, 2), the majority showed diffuse keratin staining. Additionally, CTC clusters of 4-8 cells were present (5, 6). Some CTCs showed multiple/lobed nuclei (4, 7, 8).

C Bright field images of CTC-ITB-01 cells growing adherently.

D Bright field images of CTC-ITB-01 cells growing non-adherently (relation between adherent and non-adherent cells: 80/20 %).

Data information: White scale bars represent 10µm. Black scale bars represent 40 µm.
Characterization of circulating breast cancer cells with tumorigenic and metastatic capacity (2020)
Figure 4

Figure 4. CTC-ITB-01 phenotype in culture:

A Western blot analysis of selected protein markers, including ERα, EGFR, ERBB2, EpCAM, K18, K19, K8, E-cadherin, N-cadherin, vimentin, CD44, CD24, SNAIL, SLUG, TWIST and α-tubulin (as a loading control) (n=3 replicates). CTC-ITB-01 was compared to more mesenchymal ER- Hs578t and epithelial ER+ MCF-7 breast cancer cell lines.

B ICC staining of CTC-ITB-01 for pan-keratin (orange), ER (green), and DAPI (blue). The scale bar corresponds to 20 µm. Two representative panels are shown for CTC-ITB-01 (1) and (2). MCF-7 cells are depicted as reference cell line.

C PAM50 classifier results showing probabilities of pooled CTC-ITB-01 cells matching specific molecular breast cancer subtypes. Starting from lowest probability, CTC-ITB-01 was classified as 1.02% (s=2.5%) normal-like, 3.11% (s=6.6%) Basal-like, 13.11% (s=9.3%) ERBB2-positive, 16.77% (s=23.7%) luminal A, and 65.22% (s=16.6%) luminal B breast cancer subtype. Data was generated from n=3 replicates.

D PAM50 classifier results showing probabilities of the non-adherent and adherent CTC-ITB-01 fractions matching molecular subtypes. Selected bars are not visible due to extremely low probability (close to zero). Both fractions show greatest alignment with a luminal B subtype. Starting from lowest probability, the adherent fraction of CTC-ITB-01 was classified as 0% (±0%) normal-like, 6.22% (±9.0%) Basal-like, 17.85% (±8.6%) ERBB2-positive, 0% (s=0%) luminal A, and 75.92% (s=0.7%) luminal B breast cancer subtype. The suspension cell fraction of CTC-ITB-01 was classified as 2.04% (s=3.5%) normal-like, 0% (s=0%) Basal-like, 17.32% (s=9.7%) ERBB2-positive, 33.5% (s=23.8%) luminal A, and 68.67% (s=18.6%) luminal B breast cancer subtype, respectively. Data was generated from n=3 replicates.

(E) ALDH activity measurement on viable cells via flow cytometry using the ALDEFLUOR™ assay. CTC-ITB-01 is compared to A549 as recommended control cell line. An internal (DEAB) control is also depicted. 77.92% of the gated CTC-ITB-01 population are ALDH+.
Characterization of circulating breast cancer cells with tumorigenic and metastatic capacity (2020)
Figure 5

Figure 5. Epithelial adhesion and EMT analysis of CTC-ITB-01

A ICC staining of CTC-ITB-01 for ß-catenin (green), E-cadherin (orange), and DAPI (blue). The scale bar corresponds to 20 µm. Two representative panels are shown for CTC-ITB-01.

B ICC staining of CTC-ITB-01 for α-catenin (green), visualization of actin filaments by phalloidin (orange), and nuclei by DAPI (blue). The scale bar corresponds to 20 µm. Two representative panels are shown for CTC-ITB-01.

C Generic EMT score of the adherent and non-adherently growing CTC-ITB-01 fractions in relation to tumor samples from different cancer entities. Scaling ranges from 1 (completely epithelial) to -1 (entirely mesenchymal). The adherent fraction is shown with a black dot, the non-adherent fraction with a black triangle, and the mean score per cancer entity by a black line.

D Generic EMT score of the adherent and non-adherently growing CTC-ITB-01 fractions in relation to established cancer cell lines. Scaling ranges from 1 (completely epithelial) to -1 (entirely mesenchymal). The adherent fraction is shown with a black dot, the non-adherent fraction with a black triangle, and the mean score per cancer entity by a black line.
Characterization of circulating breast cancer cells with tumorigenic and metastatic capacity (2020)
Figure 6

Figure 6 - Xenograft of CTC-ITB-01 in immunodeficient mice confirms tumorigenic and metastatic potential:

A In vivo bioluminescence imaging of CTC-ITB-01 in four NSG mice. Blue indicates low radiance, red indicates high radiance.

B Logarithmic plotting of in vivo radiance increase over time. Curves represent means ± SEM of measurements performed on multiple glands (n=8).

C H&E staining of xenografted mouse mammary glands show formation of primary tumors.

D Stereoscope image of a mammary gland of immunodeficient NSG mouse. Fluorescently tagged CTC-ITB-01 cells in green.

E IHC staining for ER, confirms retained in vivo ER-positivity of formed tumors. Representative images of ER staining in higher (left panel) and lower (right panel) magnification of 2 different areas of a xenografted mammary gland.

F Representative ex vivo luminescence images of indicated organs from engrafted mice. Organs include brain (1), lung (2), liver (3) and bones (4).

G Scatter blot showing ex vivo radiance intensity of metastatic organs in three different mice (M1, M2, M3), indicating highest intensity in liver and bone. Data represent means ± SD of measurements performed on metastatic organs derived from three different mice.
DNA hypomethylation leads to cGAS-induced autoinflammation in the epidermis (2021)
Figure 1

Figure 1: Epidermal DNA hypomethylation results in a severe skin phenotype.

(a) Immunofluorescence labeling of wildtype mouse dorsal skin samples (Dnmt1f/f, skin from P3) shows expression of DNMT1 throughout epidermal layers whereas keratinocyte-specific knockout (Dnmt1Δ/Δep) results in the almost absence of a specific staining in the epidermis and in hair follicles. Dashed lines indicate the dermal-epidermal border and dotted lines indicate hair follicles.

(b-c) Deletion of DNMT1 was also confirmed in isolated epidermis by analysis of RNA expression ((b), data are mean ± SEM, two-tailed t-test, **** p≤0.001, n=3-4 mice) and Western blotting (c).

(d) Knockout of Dnmt1 results in lower levels of DNA methylation in the epidermis and in hair follicles as determined by anti 5-mC labeling of P3 skin. Dashed lines indicate the dermal-epidermal border and dotted lines indicate hair follicles.

(e) HPLC analysis of cytosine methylation in the epidermis of Dnmt1Δ/Δep mice compared to control littermates at different time points after birth. Values are percentages of methylated CpGs compared to total number of CpGs in the genome. P3: n=2, P7: n=2, P8/9: n=3. Data are shown as mean ± SEM.

(f-g) Images of control (Dnmt1f/f, Dnmt1f/+) and Dnmt1Δ/Δep littermates at P3, P5, P7 and P9. An enlargement of the skin surface is shown on the right (g).

(h) Hematoxylin and Eosin (H&E) staining, immunolabeling for KERATIN 10 (KRT10), LORICRIN (LOR) and KERATIN 6 (KRT6) expression of dorsal skin sections (P5) of control and Dnmt1Δ/Δep mice.

Data information: Scale bars, 50 µm.
DNA hypomethylation leads to cGAS-induced autoinflammation in the epidermis (2021)
Figure 2

Figure 2: Epidermal DNA hypomethylation results in induced immune response.

(a) Representative immunostaining for CD45 of dorsal skin sections from control and Dnmt1Δ/Δep mice at P7. Dashed lines indicate the dermal-epidermal border.

(b) Flow cytometric analysis of CD45 positive cells isolated from the epidermis of control and Dnmt1Δ/Δep mice of different postnatal age. Cells are shown as percent of viable cells. Data are mean ± SD, two-tailed t-test, ** p≤0.01, *** p≤0.001, n≥4 mice.

(c) Flow cytometric analysis of the respective immune cell populations from the epidermis of control and Dnmt1Δ/Δep mice of different postnatal age. Cells are shown as percent of viable cells. Data are mean ± SEM, two-tailed t-test. ns, not significant, * p≤0.05, ** p≤0.01, **** p≤0.0001, n=3 (P3), 4 (P5) and 4 (P6 + P7).

(d) Relative mRNA expression levels of immune related genes in control and Dnmt1Δ/Δep mice at P3 (n=3), P5 (n=5) and P7 (n≥3). Data are mean ± SEM. P values are calculated using one-way ANOVA with post hoc Tukey multiple comparison test, * p≤0.05, ** p≤0.01, *** p≤0.001 , **** p≤0.0001.

(e) Gene ontology analysis of significantly up- and down-regulated genes (P-value<0.05) obtained from epidermal keratinocytes of control and Dnmt1Δ/Δep mice (n=3 mice). Keratinocytes were isolated from 3 day old mice and cultured for 72 h.

(f) Relative mRNA expression of representative immune related genes found to be deregulated by RNA-sequencing in keratinocytes cultured for 72 h and isolated from 3 day old control and Dnmt1Δ/Δep mice (n=3 per group). Data are mean ± SEM, two-tailed t-test, ** p≤0.01 *** p≤0.001.

Data information: Scale bars, 50 µm.
DNA hypomethylation leads to cGAS-induced autoinflammation in the epidermis (2021)
Figure 3

Figure 3: Epidermal deletion of DNMT1 results in de-repression of transposable elements and activation of immune genes with minor impact of the MAVS pathway.

(a) Relative mRNA expression levels of epidermal Intracisternal A-type particle (IAP) during different postnatal time points (P0 n≥3, P3 n=3, P5 n=5, P7 n≥3 mice), which were compared using one way-ANOVA with post hoc Tukey multiple comparison test. Data are mean ± SEM. ns not significant, * p≤0.05, ** p≤0.01.

(b) IAP protein was determined from epidermal isolates of two control and two Dnmt1Δ/Δep mice. A bracket indicates the size of group-specific antigen (gag) proteins.

(c) Relative expression of immune related genes of the epidermis from P7 of control (n≥7), Dnmt1Δ/Δep, (n≥8), MAVS knockout (Mavs-/-, n=7) and Dnmt1Δ/Δep Mavs-/- (n=6) mice were compared using one way-ANOVA with post hoc Holm-Sidak multiple comparison test. Data are mean ± SEM. ns not significant, * p≤0.05, ** p≤0.01, *** p≤0.001, **** p≤0.0001.
DNA hypomethylation leads to cGAS-induced autoinflammation in the epidermis (2021)
Figure 4

Figure 4: DNA hypomethylation results in release of cytoplasmic DNA and cGAS activation.

(a) Paraffin skin sections (P5) of control and Dnmt1Δ/Δep mice were stained for DNA using PicoGreen. Enlarged sections are indicated on the right where arrows point to DNA-positive nuclear protrusions. Dashed lines indicate the dermal-epidermal border.

(b) Labeling of dorsal skin sections of control and Dnmt1Δ/Δep mice for cGAS and PicoGreen. Enlarged sections are indicated on the right. Dashed lines indicate the dermal-epidermal border.

(c-f) Immortalized human keratinocytes (NHEK SV-Tert3-5) were treated with 5-aza-2′-deoxycytidine (DAC) or vehicle (PBS). (c, d) PicoGreen staining of PBS- and DAC-treated keratinocytes for 48h detected micronuclei (arrow), which were analyzed in a blinded manner, and compared using two-tailed t-test. Data are mean ± SEM, * p≤0.05 (n=3 biological replicates) (d). (e) Co-labeling of DNA by PicoGreen and by cGAS in PBS and DAC treated keratinocytes. Within enlarged sections on the right arrows point at DNA/cGAS double-positive blebs and micronuclei. (f) Relative expression of CCL5 and IFT2 in keratinocytes treated with PBS or DAC for 45 h or 72 h using one way-ANOVA with post hoc Dunnett multiple comparison test. Data are mean ± SEM. ns not significant, * p≤0.05, ** p≤0.01, *** p≤0.001 (n=4 biological replicates).

Data information: Scale bars, 20 µm (a, d), 50 µm (b, e).
DNA hypomethylation leads to cGAS-induced autoinflammation in the epidermis (2021)
Figure 5

Figure 5: Additional deletion of cGAS attenuates the autoinflammatory Dnmt1Δ/Δep skin phenotype.

(a) Representative images of P7 H&E labeled dorsal skin sections of control (Dnmt1f/f), Dnmt1Δ/Δep, and Dnmt1Δ/Δep Cgas-/- mice.

(b) The histopathology of at least 9 animals per genotype was assessed in a blinded manner and an overall score was determined from individual parameters (see Suppl. Fig. 7 and Suppl. Tab. S3). Comparison was performed using one way-ANOVA with post hoc Holm-Sidak multiple comparison test. Data are mean ± SEM. **** p≤0.0001.

(c) Representative images of CD45 immunofluorescence labeling of P7 dorsal skin sections from Dnmt1f/f, Dnmt1Δ/Δep, and Dnmt1Δ/Δep Cgas-/- mice. Dashed lines indicate the dermal-epidermal border.

(d) Quantification of CD45-positve cells. Tissue sections approximately 1 cm in length were scanned and the area of CD45 positive cells per area of nuclei was quantified using ImageJ. Sections obtained from five mice per genotype were analyzed and calculated values were compared using one way-ANOVA with post hoc Holm-Sidak multiple comparison test. Data are mean ± SEM. ** p≤0.01, *** p≤0.001.

(e) Epidermis obtained from P7 control, Dnmt1Δ/Δep, Cgas-/- and Dnmt1Δ/Δep Cgas-/- mice was analyzed for relative mRNA expression levels of immune related genes using one way-ANOVA with post hoc Holm-Sidak multiple comparison test. Data are mean ± SEM. ns not significant, * p≤0.05, **** p≤0.0001. At least 7 mice per genotype were used.

Data information: Scale bars, 50 µm (a), 100 µm (c).
Small molecules enhance autophagy and reduce toxicity in Huntington's disease models (2007)
figf1

(a) Chemical structures of SMERs 10, 18 and 28. (b) A53T α-synuclein (α-syn) expression was induced in stable PC12 cells for 48 h, then switched off for 24 h. Cells were treated with DMSO (control), or with 0.94 μM, 4.7 μM or 47 μM of SMER10; 0.86 μM, 4.3 μM or 43 μM of SMER18; or 0.9 μM, 4.7 μM or 47 μM of SMER28 added in the switch-off period. A53T α-syn levels were analyzed by immunoblotting with antibody against HA (left) and densitometry analysis relative to actin (right). Error bars denote s.e.m. P = 0.0917, P = 0.009, P = 0.0001 (for increasing concentrations of SMER10); P = 0.0068, P = 0.0023, P = 0.0002 (for increasing concentrations of SMER18); P = 0.0016, P 0.0001, P 0.0001 (for increasing concentrations of SMER28). (c) COS-7 cells transfected with EGFP-HDQ74 construct were treated with DMSO (control), 0.2 μM rapamycin (Rap), 47 μM SMER10, 43 μM SMER18 or 47 μM SMER28 for 48 h, and we assessed the proportions of apoptotic or aggregate-containing transfected cells. Error bars: 95% confidence interval. P 0.0001 (Rap and SMER28), P = 0.013 (SMER10), P = 0.019 (SMER18) (aggregation); P 0.0001 (Rap, SMER18 and SMER28), P = 0.004 (SMER10) (cell death). (d) Wild-type (Atg5+/+) and knockout (Atg5−/−) Atg5 MEFs were transfected with EGFP-HDQ74 and treated with DMSO (control), 47 μM SMER10, 43 μM SMER18 or 47 μM SMER28 for 24 h, and we assessed the percentages of aggregate-containing transfected cells. The control (DMSO-treated) values were fixed at 1 for both cell lines. Error bars: 95% confidence interval. P = 0.039 (SMER10), P 0.0001 (SMER18 and SMER28) (in Atg5+/+ cells); P = 0.092 (SMER10), P = 0.271 (SMER18), P = 0.358 (SMER28) (in Atg5−/− cells). Note that EGFP-HDQ74 aggregation was higher in Atg5−/− than in Atg5+/+ cells (Supplementary Fig. 3a). (e) The percentages of EGFP-positive COS-7 cells with EGFP-HDQ74 aggregates as in c, treated with DMSO (control), 10 μM lactacystin (proteasome inhibitor) or both 10 μM lactacystin along with either 47 μM SMER10, 43 μM SMER18 or 47 μM SMER28 for 48 h, expressed as odds ratios. Error bars: 95% confidence interval. P 0.0001 (control versus Lact), P = 0.014 (SMER10 versus Lact), P 0.0001 (SMER18 versus Lact), P = 0.001 (SMER28 versus Lact). ***P 0.001; **P 0.01; *P 0.05; NS, nonsignificant.
Small molecules enhance autophagy and reduce toxicity in Huntington's disease models (2007)
figf2

(a) COS-7 cells transfected with EGFP-LC3 construct for 4 h were treated with DMSO (control), 0.2 μM rapamycin (positive control), 47 μM SMER10, 43 μM SMER18 or 47 μM SMER28 for 16 h, and analyzed by fluorescence microscopy. The effects of treatment on the percentage of EGFP-positive cells with >5 EGFP-LC3 vesicles are shown. Error bars denote s.e.m. P 0.0001 (all SMERs). (b) HeLa cells stably expressing EGFP-LC3 were treated with DMSO (control), 47 μM SMER10, 43 μM SMER18 or 47 μM SMER28 for 24 h. Confocal sections show cells containing EGFP-positive autophagic vesicles. Nuclei are stained with DAPI. Bar, 20 μM. (c) HeLa cells stably expressing EGFP-LC3 were treated for 4 h with DMSO (control) or 200 nM bafilomycin A1 (Baf), or with 200 nM bafilomycin A1 and 47 μM SMER10, 43 μM SMER18 or 47 μM SMER28. Cells were left untreated or pretreated with SMERs for 24 h before adding bafilomycin A1. Levels of EGFP-LC3-II were determined by immunoblotting with antibody against EGFP (above) and densitometry analysis relative to actin (below). Error bars denote s.e.m. P = 0.0259 (baf), P 0.0001 (SMER10), P = 0.0003 (SMER18 and SMER28) versus control; P = 0.0025 (SMER10), P = 0.0218 (SMER18), P = 0.0195 (SMER28) versus bafilomycin A1. ***P 0.001; **P 0.01; *P 0.05.