EMBO/soda-nel huggingface dataset:
Neutrophils discriminate live from dead bacteria by integrating signaling via formylated peptide receptors and Toll-like receptors (2022)
Figure 4

Fig. 4. Fpr1 or Fpr2 are required for in vivo neutrophil recruitment, Cxcl2 production and antibacterial defenses during GBS infection.

(A-D) Kinetics of neutrophil influx (A), bacterial burden (B) and Cxcl1/2 production (C and D) in peritoneal lavage fluid samples after i.p. challenge with live GBS (2x107 CFU).

(E), Kaplan-Meier analysis of survival rates in WT, Fpr1 and Fpr2 KO mice after i.p. challenge with 2x105 bacteria. Data are the cumulative results from two experiments, each involving 8 animals per group. **P < 0.01, ****P < 0.0001 vs WT mice, as determined by Kaplan-Meier analysis.

(F-G) Neutrophil influx (F) and bacterial burden (G) in peritoneal lavage fluid samples collected from Fpr2-/- mice at 3 h after i.p. challenge with live GBS (2x107 CFU). At 1h post-challenge mice were inoculated i.p. with 1 μg of recombinant Cxcl1 or Cxcl2 or with vehicle.

Data information: In (A-D), means + SDs of three duplicate determinations, each conducted in a different animal. In (F and G), individual data points and means. *P < 0.05, **P < 0.01, ***P < 0.001 versus WT mice (A, C, and D) or versus vehicle treated mice (F and G), determined by unpaired t test (A, C, D and F) or Mann-Whitney U test (B and G).
Neutrophils discriminate live from dead bacteria by integrating signaling via formylated peptide receptors and Toll-like receptors (2022)
Figure 5

Fig. 5. Bacterial signal peptides activate neutrophils and synergize with TLRs in Cxcl2 induction.

(A), Neutrophils from WT or Fpr-deficient mice were allowed to migrate towards GBS signal peptide sequences (fPep1-15, 1µM), positive control peptides (f-MIFL or WKYMVM, 1µM) or vehicle (0.2% DMSO).

(B), Analysis of intracellular calcium flux in neutrophils stimulated with signal peptides as monitored by flow cytometry.

(C and D), Cxcl2 release in neutrophil cultures stimulated with combinations of HK-bacteria, GBS fMet peptides or TLR agonists. Neutrophils were exposed to a 50 µM concentration of Fpr agonists (fMIFL, WKYMVM or fPep8 and fPep10) for 1 h before the addition of the indicated TLR agonists, which included HK-GBS (10 μg/ml), LPS (1 µg/ml) and RNA (1 µg /ml). In some cases, neutrophils were exposed to graded doses (10, 20 and 50 µM) of the indicated Fpr agonists alone.

Data information: Data are expressed as means + SD from three independent experiments, each conducted in duplicate. **P < 0.01, ***P < 0.001, as determined by unpaired t-test.
Neutrophils discriminate live from dead bacteria by integrating signaling via formylated peptide receptors and Toll-like receptors (2022)
Figure 6

Fig. 6. ROS production requires the synergistic activation of Fprs and TLRs.

(A), Effect of actinonin on Cxcl2 production by neutrophils. Neutrophils were stimulated with live GBS (MOI 5) in the presence or absence of the formyl peptidase inhibitor actinonin (32µg/ml).

(B and C), ROS production in neutrophils from WT, MyD88 or Fpr1/2-deficient mice stimulated with different doses of live bacteria.

(D), Effect of treatment with ROS scavengers/inhibitors on Cxcl2 production. Neutrophils were treated with the singlet oxygen scavenger MnTBAP (100 μM), the hydrogen peroxide scavenger DMTU (10 mM), the NADPH oxidase inhibitor DPI (20 μM) or the ASK1 inhibitor GS-444217 (1 μM) for 1 h before the addition of live GBS (MOI of 5).

Data information: Data are means + SD from three independent experiments conducted in duplicate. *P < 0.05, **P < 0.01, ***P < 0.001, as determined by unpaired t-test.
Neutrophils discriminate live from dead bacteria by integrating signaling via formylated peptide receptors and Toll-like receptors (2022)
Figure 8

Fig. 8. The Tak1 and p38 kinases and the AP-1 and NF-κB transcription factors are involved in Cxcl2 responses to live GBS.

(A), Time course of p38-MAPK activation in lysates of neutrophils stimulated with live GBS, as detected by phospho-specific antibodies using Western Blot analysis. Data are from one representative experiment of three showing similar results.

(B), Time course of AP-1 DNA-binding activity in neutrophils stimulated with live GBS (MOI 5). Data are means + SD from three independent experiments conducted in duplicate.

(C), Effect of p38MAPK inhibition on the activation of AP-1 family members in neutrophils stimulated with live GBS. Neutrophils were pretreated with SB202190 (5μM), stimulated for 90min with live GBS (MOI 5), lysed and assayed for AP-1 activation. Data are means + SD from three independent experiments conducted in duplicate. *P < 0.05, ***P < 0.001, as determined by unpaired t-test.

(D), Effect of the absence of Fpr1, Fpr2 or the TLR adaptor MyD88 on the activation of AP-1 family members in neutrophils. Neutrophils from WT, Fpr- or MyD88-deficient mice were stimulated for 90 min with live GBS (MOI 5), lysed and assayed for AP-1 activation. Data are means + SD from three independent experiments conducted in duplicate. *P < 0.05, ***P < 0.001 vs WT mice, as determined by unpaired t-test.

(E), Effect of TAK1 inhibition on p38 MAPK activation in neutrophils stimulated with live GBS (MOI 5).

(F), Effect of the absence of Fpr1, Fpr2 or the TLR adaptor MyD88 on p38MAPK activation in neutrophils stimulated with live GBS (MOI 5). Shown is a blot representative of three separate experiments.
Esrrb extinction triggers dismantling of naïve pluripotency and marks commitment to differentiation (2018)
Figure 1

Figure 1. Nanog acts upstream of Esrrb in ESCs.

A, B: Immunofluorescent detection of Nanog and Esrrb protein in wild-type E14Tg2a ESCs cultured for three days in GMEMβ/FCS/LIF. (A) Widefield images of a representative colony. (B) Quantification of the mean Nanog and Esrrb fluorescence levels measured in nuclei identified by automatic segmentation of single optical plane images obtained by confocal microscopy. Values are expressed in Arbitrary Units (AU). Oct4 negative cells, also identified by immunostaining, were excluded from the analysis. The red line represents the moving average of the data distribution. Representative of three independent experiments each including at least 1000 nuclei.

C: Colonies of TNG E-tdT ESCs showing GFP expression from Nanog and Esrrb-tdTomato fusion protein expression from Esrrb after three days in GMEMβ/FCS/LIF.

D: Flow cytometric analysis of Esrrb and Nanog fluorescent reporter expression in SSEA1+ TNG E-tdT ESCs cultured for three days in GMEMβ/FCS/LIF with or without puromycin.

E: Immunofluorescent detection and quantification (as in panel B) of total Nanog and Esrrb protein in E14Tg2a ESCs overexpressing Nanog (left) or Esrrb (right) cultured for three days in GMEMβ/FCS/LIF. Parental E14Tg2as are shown as a reference. The red lines indicate the negative thresholds defined by staining ESCs differentiatied for 3 days in the absence of LIF and presence of retinoic acid.

F: Flow cytometric analysis of Esrrb-GFP and Nanog-mCherry fusion protein expression in SSEA1+ NER ESCs cultured for three days in GMEMβ/FCS/LIF.

G: Histograms showing expression levels of Esrrb-GFP and Nanog-mCherry from the respective targeted endogenous alleles in SSEA1+ NER ESCs overexpressing Nanog (left) or Esrrb (right) cultured for three days in GMEMβ/FCS/LIF. Parental NER ESCs are shown as a reference, and wild-type E14Tg2a set the negative thresholds.

H: Comparative flow cytometric analysis of Esrrb-2a-tdTomato and Nanog:GFP expression in undifferentiated SSEA-1+ TNG E-2a-tdT or Nanog-null ES∆N E-2a-tdT ESCs cultured for three days in GMEMβ/FCS/LIF. Data are shown as dot plots (top) and histogram (bottom).

Data information: For a schematic representation of the reporter allele configuration characteristic of each cell line please refer to Fig EV1.
Esrrb extinction triggers dismantling of naïve pluripotency and marks commitment to differentiation (2018)
Figure 2

Figure 2. Decreased Esrrb expression marks ESC commitment

A: SSEA-1+ TNG E-tdT ESCs were cultured for three days in GMEMβ/FCS/LIF and sorted into GFP+/tdThigh (Nanoghigh/Esrrbhigh) or GFP-/tdTlow fractions (Nanoglow/Esrrblow). The purity of the sorted populations (>99.5%) is shown (day 0). After replating in GMEMβ/FCS/LIF, SSEA-1+ cells were analysed daily for Esrrb-tdTomato and Nanog:GFP expression.

B,C: Immunofluorescent detection of Nanog and Esrrb protein in wild-type E14Tg2a ESCs cultured in N2B27/LIF/2i and released in GMEMβ/FCS/LIF for 1, 2 or 3 days. Quantification of the mean Nanog and Esrrb fluorescence levels measured in nuclei identified by automatic segmentation of single optical plane images obtained by confocal microscopy. Oct4 negative cells, also identified by immunostaining, were excluded from the analysis. (B) The red vertical and horizontal lines indicate the negative thresholds defined by staining ESCs differentiatied for 3 days in the absence of LIF and presence of retinoic acid. The red trendline represents the moving average of the data distribution at day 3. Representative of two independent experiments each including at least 1000 nuclei. (C) Histogram plots of the data presented in B.

D: Sorted SSEA-1+ and Esrrb-GFPhigh/Nanog-mCherryhigh NER ESCs were replated in GMEMβ/FCS/LIF and SSEA-1+ cells were analysed daily for Esrrb-GFP and Nanog-mCherry expression. Wild-type E14Tg2a set the negative thresholds.

E: SSEA-1+ TNG E-tdT ESCs from the sorted populations used above (panel A, day 0) were replated at clonal density in GMEMβ/LIF/FCS or N2B27/LIF/2i and stained for alkaline phosphatase activity after 6 days. Error bars: standard deviation of the number of colonies observed in two independent experiments.

Data information: For a schematic representation of the reporter allele configuration characteristic of each cell line please refer to Fig EV1.
Esrrb extinction triggers dismantling of naïve pluripotency and marks commitment to differentiation (2018)
Figure 3

Figure 3. Naïve pluripotency gene expression is lost upon Esrrb downregulation

A: Flow cytometric analysis of GFP expression in SSEA-1+ E-GFPd1 ESCs cultured in N2B27/LIF/2i or GMEMβ/LIF/FCS compared to wild-type E14Tg2a ESCs.

B: SSEA-1+ E-GFPd1 ESCs sorted into EsrrbHi, EsrrbMed or EsrrbNeg subpopulations. Purity of the sorted populations, assessed by flow cytometry, is indicated above the sorting gates.

C: Quantitative Esrrb mRNA expression analysis of populations sorted in (B). Error bars: standard deviation of gene expression values measured in four independent experiments.

D: Populations sorted in (B) were replated at clonal density in GMEMβ/LIF/FCS (top) or N2B27/LIF/2i (bottom) and scored for alkaline phosphatase after 6d. Error bars: standard deviation of the number of colonies observed in two independent experiments.

E: Quantitative mRNA expression analysis of populations sorted in (B). Error bars: standard deviation of gene expression values measured in four independent experiments.

F: Microarray analysis of populations sorted in (B) represented as a heatmap ordered by expression change between EsrrbHi and EsrrbNeg. Expression levels were measured from three independent experiments and all genes expressed in at least one sample are shown (n = 14,553). The central line plot summarises the average fold change per cell population compared to EsrrbHi cells (log2 scale) with the colour coding indicated below the heat map. Previously identified Nanog and Esrrb (Festuccia et al., 2012) targets are indicated on the right.

G: Pearson correlation coefficients of the gene expression profiles of EsrrbH (H), EsrrbM (M), Esrrb- (Neg) E-GFPd1 ESCs and embryo derived (late bud stage) TNG E-2a-tdT EpiSCs (EpiSC) compared to those of naïve ESC and EpiLC from (Buecker et al., 2014).

H: Pearson correlation coefficients in gene expression obtained comparing EsrrbHi (H), EsrrbMed (M), EsrrbNeg (Neg) E-GFPd1 ESCs and embryo derived (late bud stage) TNG E-2a-tdT EpiSCs (EpiSC) to epiblast/ectoderm cells from embryos dissected at successive developmental stages (CAV, cavity; PS, prestreak; ES, early-streak; MS, midstreak; LMS, late midstreak; LS, late streak; OB/EB, no bud/early bud; LB, late bud). This analysis was restricted to the top 500 differentially expressed gene identified at successive developmental stages sorting by Hotelling T² score, as described in (Kojima et al., 2014).

Data information: For a schematic representation of the reporter allele configuration characteristic of each cell line please refer to Fig EV1.
Esrrb extinction triggers dismantling of naïve pluripotency and marks commitment to differentiation (2018)
Figure 4

Figure 4. Different kinetics of loss of TF binding and accumulation of CpG methylation at key regulatory elements of the pluripotency network.

A: Percentage of methylated CpG dinucleotides profiled across the Esrrb and Nanog enhancer and the Nanog promoter in sorted SSEA-1+ / EsrrbHi, EsrrbMed or EsrrbNeg E-GFPd1 ESCs and TNG E-2a-tdT EpiSC. CpG methylation was assessed by measuring protection from digestion of the HpaII, AciI, Hin6I or TaqI restriction sites (indicated by vertical lines in the gene structure maps derived from the mouse reference genome (mm9) and expressed relative to the TSS in ESCs (Chambers, 2004, Festuccia et al., 2012). Values represent total CpG methylation levels (5mC + 5hmC). Error bars: standard deviation of the measures in four independent experiments.

B: Methylated CpG dinucleotides across the Esrrb enhancer were assessed as in (A). SSEA-1+ / EsrrbHi E-GFPd1 ESCs were sorted and placed back in culture overnight before resorting and methylation analysis relative to Esrrb- E-GFPd1 ESCs. Error bars: standard deviation of the measures in three independent experiments.

C: The sorted E-GFPd1 ESCs populations used in (A) were assessed by immunoblot for Esrrb and Nanog (left) or Oct4 (right) by fluorescence-based detection (Licor).

D: Quantitative ChIP-PCR analysis of OCT4, NANOG and ESRRB binding and histone modifications at the Esrrb and Nanog enhancers and the Nanog promoter in the sorted populations used in (A). Error bars: standard deviation of the measures in two (NANOG, ESRRB, H3K27ac) or three (OCT4, H3K4me3, H3K4me1) independent ChIP experiments, each performed on pooled chromatin from at least three independently sorted samples. The diagrams at the bottom show the approximate position of the regions analysed for transcription factor binding or histone modifications (in color or black), along with the relative control genomic locations (grey).
Esrrb extinction triggers dismantling of naïve pluripotency and marks commitment to differentiation (2018)
Figure 5

Figure 5. Global profiles of OCT4 and NANOG chromatin binding during Esrrb downregulation

A-D: (A,C) Venn diagram showing overlap of (A) OCT4- or (C) NANOG-bound regions (See main text for peak calling strategy) in sorted EsrrbHi (EH) and EsrrbNeg (EN) E-GFPd1 ESCs. (B, D) Average binding profile of (B) OCT4 or (D) NANOG in binding peaks where binding in EsrrbNeg ESCs was kept, lost or gained relative to EsrrbHi E-GFPd1 ESCs. Read counts per base pair were normalised by library size and by subtracting the background (IgG) signal. EsrrbHi (EH), EsrrbMed (EM) and EsrrbNeg (EN)

E: Venn diagram showing the overlap of NANOG- and OCT4- bound regions in sorted EsrrbHi and EsrrbNeg E-GFPd1 ESCs.

F: Pie chart showing the number of genomic regions co-bound by NANOG and OCT4 in EsrrbHi E-GFPd1 ESCs that lose binding of NANOG and OCT4 (Class I) or that lose binding of NANOG but retain OCT4 (Class II) during transition to EsrrbNeg. A few regions keep NANOG binding but lose OCT4 (n = 16) or keep binding of both (n = 60).

G: Boxplots showing the fold change in expression levels of genes proximal to Class I elements, Class II elements or both Class I and Class II elements in EsrrbHi (EH), EsrrbMed (EM) or EsrrbNeg (EN) E-GFPd1 ESCs and EpiSC (Epi). Boxes span the inter-quartile range (IQR) from the first to the third quartile. The line indicates the median. Whiskers extend up to 1.5 IQR, and outliers are plotted.

H, I: Binding profiles of OCT4 and NANOG in the proximity of two genes representative of (H) Class I (Tet2) or (I) Class II (Sall1) in EsrrbHi, EsrrbMed or EsrrbNeg E-GFPd1 ESCs. The relative occupancies of NANOG and OCT4 at Tet2 or Sall1 (positions highlighted by hatched boxes) alongside expression levels of Tet2 and Sall1 mRNAs in EsrrbHi (EH), EsrrbMed (EM) or EsrrbNeg (EN) GFPd1 ESCs and EpiSC (Epi) is shown at the bottom. Oct4 binding data from EpiSC is from (Buecker et al., 2014).
Esrrb extinction triggers dismantling of naïve pluripotency and marks commitment to differentiation (2018)
Figure 6

Figure 6. Differential TF binding and activity of Class I and Class II elements in ESC and EpiLC.

A: Average binding profile of ESRRB, KLF4, NANOG, OCT4 and SOX2 to Class I (green), Class II (orange) or all Nanog and Oct4 bound (grey dashed line) elements in ESCs. Data from (Chen et al., 2008). Read counts per base pair were normalised by library size and by subtracting the background (IgG) signal.

B: Average FAIRE profile or ChIP-Seq profiles for H3K27ac, OCT4, Ep300 or OTX2 in ESCs or EpiLC for Class I and Class II elements. Data from (Buecker et al., 2014).

C: Results of a differential motif analysis (Thomas-Chollier et al., 2011) in Class I and Class II peaks.
Functional and physical interaction between Bcl‐XL and a BH3‐like domain in Beclin‐1 (2007)
Figure 1

Physical interaction between Bcl‐XL/Bcl‐2 and the BH3‐like domain of Beclin‐1. (A) Selection of Beclin‐1 fragments that interact with Bcl‐XL/Bcl‐2 in the yeast‐two‐hybrid system. Black lines indicate the fragments of Beclin‐1 (grey) that interact with Bcl‐XL (upper part of the graph) or with Bcl‐2 (lower part). ▪, CEMC7 library; •, human thymocyte library; ▴, placenta library and ★, human brain library. The minimal interacting domain required for interaction with Bcl‐XL/Bcl‐2 is situated between aa 112 and 159. (B) Alignment of the BH3‐like domain of Beclin‐1 with established BH3 domains. L116A Beclin‐1 and F123A Beclin‐1 describe two Beclin‐1 mutant peptides. (C) Fluorescence polarization assays demonstrating that a peptide containing the BH3‐like domain of Beclin‐1 interacts with Bcl‐XL. Recombinant Bcl‐XL ΔTM was incubated with carboxyfluorescein‐labeled Bak BH3 peptide, in the absence or presence of wild‐type (WT) Beclin‐1 BH3 or either of the two mutant peptides described in B. (D) Affinity determination of the interaction between recombinant Bcl‐XL ΔTM and fluorescent Beclin‐1 BH3, by fluorescence polarization. A comparison between WT Bcl‐XL(squares) and G138A Bcl‐XL (triangles) is shown (means±s.d., n=3 separate experiments). (E) Co‐immunoprecipitation of Bcl‐XL and WT or mutant Beclin‐1. HeLa cells were transfected with the indicated constructs, followed by immunoprecipitation of Flag‐tagged Bcl‐XL and immunochemical detection of Beclin‐1. (F) Co‐immunoprecipitation of Beclin‐1 with‐WT and mutant (G138A) Bcl‐XL. Results are representative of at least three experiments yielding similar results.
Functional and physical interaction between Bcl‐XL and a BH3‐like domain in Beclin‐1 (2007)
Figure 2

Inhibition of the interaction between Beclin‐1 and anti‐apoptotic Bcl‐2 proteins by ABT737. (A) Competition between Beclin‐1 BH3 and ABT737 for Bcl‐XL binding. A fluorescent 25‐mer peptide containing the BH3‐like domain of Beclin‐1 (Figure 1D) was docked to recombinant Bcl‐XL ΔTM protein in the absence or presence of ABT737. The IC50 of ABT737, as measured in the presence of 15 nM of peptide and 100 nM of Bcl‐XL ΔTM, was 1.7 μM. (B, C) Abolition of the interaction between Bcl‐XL and Beclin‐1 by ABT737 in intact cells. Co‐immunoprecipitation assays were performed on HeLa cells transfected 48 h earlier with the indicated constructs as in Figure 1E. Sixteen hours before the immunoprecipitation, cells were exposed to ABT737. Similar results were obtained for co‐transfected Bcl‐XL and Beclin‐1 (B) and for endogenous Beclin‐1 interacting with Flag‐tagged Bcl‐XL (C). (D, E) Differential effect of ABT737 on the interaction between Bcl‐2 (D) or Mcl‐1 (E) and Beclin‐1. This experiment was designed as (B). All experiments have been performed at least three times, with similar results.
Functional and physical interaction between Bcl‐XL and a BH3‐like domain in Beclin‐1 (2007)
Figure 3

Beclin‐1‐dependent autophagy stimulated by ABT737. (A, B) Detection of autophagic vacuoles by LC3‐GFP and their modulation by ABT737 and by Beclin‐1‐specific siRNAs. HeLa cells were transfected with control or Beclin‐1‐specific siRNAs, 24 h later re‐transfected with LC3‐GFP, cultured in complete medium (CM) for 24 h, and finally kept 12 h either in CM or in nutrient‐free (NF) conditions, in the presence or absence of 1 μM ABT737. Representative microphotographs of cells cultured in NF medium are shown in (A) and the percentage (means±s.d., n=3 separate experiments) of LC3‐GFP‐transfected cells bearing LC3‐GFP aggregates in the cytoplasm (LC3‐GFPvac) are quantified in (B). The insert in (B) demonstrates the efficiency of the Beclin‐1‐specific siRNAs, as quantified by immunoblot. (C, D) Detection of cytoplasmic vacuoles using chloromethylfluorescein diacetate (CMFDA). Cells were transfected with control or Beclin‐1‐specific siRNAs, cultured for 48 h in CM, washed, cultured in CM (D) or NF (C, D) for 12 h, stained with CMFDA, and either photographed (C) or subjected to the quantification of the cells that bear at least one discernible cytoplasmic vacuole (arrow head) (means±s.d., n=3 separate experiments). (E) Ultrastructure of autophagic vacuoles induced by ABT737. Transmission electron microphotographs are shown. (F) Detection of dead and dying cells in the cultures. Cells treated as in (A) were stained with the ΔΨm‐sensitive dye DiOC6(3) and the vital dye propidium iodide (PI). The black portions of the columns refer to the DiOC6(3)low PI+ population (dead) and the remaining part of the column corresponds to the DiOC6(3)low PI− (dying) population. Results are means±s.d. of three independent experiments.
Functional and physical interaction between Bcl‐XL and a BH3‐like domain in Beclin‐1 (2007)
Figure 4

Quantification of autophagic vacuoles induced by ABT737. HeLa cells transfected with the indicated siRNAs (specific for Emerin or for Beclin‐1 at 0 h) were re‐transfected with LC3‐GFP finally cultured in nutrient‐free (NF) conditions (60–72 h), in the presence or absence 1 μM ABT737 and then subjected to electron microscopic detection of immature (AV1) or mature (AV2) autophagic vacuoles. Representative pictures are shown in (A). The number of AV1 and AV2 was determined for a minimum of 50 cells (means±s.e.m.) (B).
Functional and physical interaction between Bcl‐XL and a BH3‐like domain in Beclin‐1 (2007)
Figure 5

Impact of the BH3‐like domain on Beclin‐1‐induced autophagy. (A–D) Modulation of Beclin‐1‐induced autophagy by BH3 mutants and ABT737. Cells were transfected with GFP‐LC3 together with an empty control vector or Beclin‐1 (WT, L116A, F123A) and cultured for 48 h, followed by overnight culture in CM (B) or NF (A, B) in the absence or presence of 1 μM ABT737. Representative microphotographs are shown in (A) and the LC3‐GFP‐positive vacuoles per cell are quantified in (B) (means±s.d.; n=3 separate experiments). Alternatively, cells were not transfected and stained with CMFDA to detect vacuoles, as shown in (C) and (D), yielding similar results as for the LC3‐GFP method. (E) BH3‐dependent modulation of Beclin‐1‐induced autophagy by anti‐apoptotic Bcl‐2 proteins. Cells were transfected simultaneously with Beclin‐1 (or control vector) and equivalent amounts of plasmids coding for WT Bcl‐XL, Bcl‐XL G138A, Bcl‐2 or Mcl‐1 (or control vector), as well as GFP‐LC3. The culture in CM or NF in the absence or presence of ABT737 was performed during the last 12 h of the 60 h‐long experiment. The percentage of cells exhibiting the accumulation of LC3‐GFP in vacuoles (LC3‐GFPvac) is quantified as means±s.d. (n=3 separate experiments).