EMBO/soda-nel huggingface dataset:
ATG5 is essential for ATG8-dependent autophagy and mitochondrial homeostasis in Leishmania major. (2012)
Figure 5

<italic>L. major</italic> Δ<italic>atg5</italic> promastigotes have a dysfunctional mitochondrion.

(A) Enlarged (EM) and swollen (WM) mitochondria seen by transmission electron microscopy (TEM) in Δatg5 promastigotes under standard growth conditions. WT is shown at bottom right. Scale bar, 500 nm. (B) Fluorescent intensity from MitoTracker Red (MTR, 0.1 µM) and MitoTracker Green (MTG, 0.2 µM) in 2×106 promastigotes after 30 min incubation at 26°C. Values shown are the means ± SD from three independent experiments. * and **, fluorescence was significantly different between WT and Δatg5 (p<0.05). (C) Types of mitochondrial morphology observed by fluorescence microscopy of Δatg5 promastigotes expressing the mitochondrial marker protein MUP-GFP. Scale bar, 10 µm. (D) Differential staining of promastigotes with both MTR (0.1 µM) and MTG (0.2 µM). Scale bar, 10 µm. (E) Viability, as measured by Alamar Blue reduction, of promastigotes. All data are means ± SD from three independent experiments. *, Alamar blue reduction was significantly different (p<0.05). (F) Spectrometric analyses of the DCF fluorescence intensity resulting from incubating promastigotes at 2×106/ml with H2DCFDA at 0.1 mM for 2 h at 26°C. Values shown are the means ± SD from three independent experiments. *, DCF fluorescence was significantly different (p<0.05).
ATG5 is essential for ATG8-dependent autophagy and mitochondrial homeostasis in Leishmania major. (2012)
Figure 6

Phospholipid accumulation in Δ<italic>atg5</italic> promastigotes.

Negative ion survey scans (650–900 m/z) of WT (A) and Δatg5 (B) promastigotes extracted for lipids and analysed by ES-MS, as described in Materials and Methods. a = (alkylacyl).
ATG5 is essential for ATG8-dependent autophagy and mitochondrial homeostasis in Leishmania major. (2012)
Figure 7

Promastigote differentiation and infectivity.

(A) Proportion of metacyclic promastigotes in stationary phase cultures, assessed using the PNA assay. Values shown are the means ± SD from three independent experiments. *, differed significantly (p<0.05). (B) Western blot analysis of extracts of 107 promastigotes at stationary phase of growth probed with α-HASPB. α-Cysteine Synthase was used as a loading control [56]. (C) Infectivity and survival of promastigotes in peritoneal macrophages in vitro, infected at a ratio 5∶1, with the infection rates being assessed after 1 and 5 days. *, differed significantly (p<0.01). (D) Lesion progression in BALB/c mice inoculated with 5×105 stationary phase promastigotes. Values shown are the means ± SD from 5 mice. *, infection level of Δatg5 differed significantly from WT (p<0.01) and Δatg5::ATG5 (p<0.05). (E) Morphologies of cells isolated from mouse lesions and analysed by SEM. Scale bar: 2 µm.
New insight into the significance of KLF4 PARylation in genome stability, carcinogenesis and therapy (2020)
Figure 1

Figure 1. Establishment of KLF4 loxp/loxp coupled AAV-Cre inducible mouse to determine the impact of KLF4 on governing genome stability and tumorigenesis. (A) Schematic diagram of establishment of KLF4 loxp/loxp coupled AAV7-Cre inducible mouse. (B) Genotyping of KLF4loxp/loxp coupled AAV-Cre inducible mouse. 2x1011 particles of AAV7-Cre-mCherry or AAV7-mCherry were intraperitoneally injected into KLF4 loxp/loxp mice (6~8 weeks). Five weeks later, mice tails were cut and collected for DNA extraction and PCR analysis. PCR results were analyzed by 1% agarose gel. (C-D) Validation of inducible knockout of KLF4 in mouse intestine. Five weeks after injection of AAV7-Cre-mCherry, mouse intestine was removed and followed by the preparation of tissue section. The KLF4 expression in the intestine was then detected by Western blot (C) and immunohistochemistry (D). Scale bars, 100 μm. (E) Kaplan-Meier survival curves of KLF4 loxp/loxp mice with intraperitoneal injection of AAV7-Cre-mCherry or AAV7-mCherry followed by the treatment with 8-Gy (total-body) γ-irradiation 5-weeks after then. AAV7-mCherry, n=10 per group; AAV7-Cre-mCherry, n=12 per group. p = 0.0023, log-rank test. (F) Histological analysis of intestinal epithelium of KLF4 loxp/loxp mice with AAV7-mCherry or AAV7-Cre-mCherry intraperitoneal injection followed by 8-Gy (total-body) γ-irradiation 5-weeks after then. Tissues were collected from the sham mice and mice at different time after exposure to γ-irradiation. Scale bars, 100 μm. (G-H) Immunofluorescent staining of γ-H2AX, 53BP1 and Cleaved Caspase-3 in the intestinal epithelium of KLF4 loxp/loxp mice with injection of AAV7-mCherry or AAV7-Cre-mCherry followed by treatment of γ-irradiation. Tissues were collected from sham mice and mice at different time after exposure to γ-irradiation and then staining with indicated antibodies. (G) Quantification of γ-H2AX, 53BP1 and active Caspase-3 -positive cells based on the Immunofluorescent staining results presented in H. Data are mean ± SEM; n=4 per group; p = 0.0023 (r-H2AX), p = 7.9x10-4 (53BP1) p = 5.5x10-3 (Active Caspase-3). one-way ANOVA was used for the statistical analysis. Scale bars, 60 μm.
New insight into the significance of KLF4 PARylation in genome stability, carcinogenesis and therapy (2020)
Figure 2

Figure 2. Identification of KLF4 PARylation by PARP1 in KLF4-mediated DNA damage response. (A). Purification of KLF4 protein complex in the presence and absence of DNA damage based on TAP-KLF4 stable expression cells (U2OS). Proteins that interacted with KLF4 were purified from U2OS cells expressing FLAG and HA-tagged KLF4 in the absence and presence of 5 Gy radiation at 4h after the treatment. The accumulated bind induced in response to radiation was isolated for mass spectrometry analysis. PARP1 was identified as a binding partner for KLF4. (B). The sequences of mass spectrometry analysis for identification of PARP1 (P09874) to be an interacting partner of KLF4. The identified peptides were labeled in yellow. (C). The purified complex was further confirmed by Western blot detected by FLAG-KLF4, PARP1(lane 1). The interaction between KLF4 and PARP1 was significantly increased in response to γ-radiation detected by pulldown experiment (lane 3 and 4). (D) Validation of interaction between ectopically expressed KLF4 and PARP1. 293T cells were transfected with FLAG-KLF4 and Myc-PARP. Whole cell lysates or IP complex pulled down by anti-FLAG antibody were analyzed by Western blotting. (E) Validation of interaction between endogenous KLF4 and PARP1 in cytosolic lysate and nuclear lysate using immunoprecipitation and Western blotting in MDA-MB-231 cells. The Histone 3 (H3) is the control for nuclear portion, and actin is the control for cytosol portion. (F) Co-immunoprecipitation of PARP1 with endogenous KLF4 is independent on DNA. The DNA binding inhibitor EtBr was added to the MDA-MB-231 cell lyses followed by immunoprecipitation of KLF4 complex. (G-H). Co-localization analysis for endogenous PARP1 and KLF4 (G), or ectopic expressed GFP-PARP1 and DsRed-KLF4 (H) in MDA-MB-231 cells. PARP1 and KLF4 are co-localized in the nucleus, and this co-localization is increased by in response to radiation. Scale bars, 5 μm. (I). Validation of the interaction between endogenous KLF4 and PARP1 by in situ proximity ligation assay (PLA). No positive staining in KLF4/Rabbit IgG antibody PLA assay. Scale bar, 100 µm. The right panel show the blow-up. (J) KLF4 was poly(ADP-ribosyl)ated. KLF4 was immunoprecipitated and detected by anti-PAR and anti-KLF4 antibodies in MDA-MB-231 cells. (K) The PARylation of KLF4 was increased after exposure to DNA damage. KLF4 complex was purified using immunoprecipitation from MDA-MB-231 cells that were treated with 5 µM doxorubicin and were then collected at different times (1h, 2h, 4h and 8h). PARylation was detected by using antibody against PAR. (L) Loss of PARP1 attenuates KLF4 PARylation. PARP1+/+ and PARP1-/- MEFs cells were transfected with FLAG-KLF4 followed by 4h after 5 Gy radiation, and then the PARylation of KLF4 was detected by pulldown. (M) PARP1 inhibitors decrease KLF4 PARylation. U2OS cells were pretreated with 10 µM various PARP1 inhibitor niraparib, Olaparib and rucaparib for 1hr followed by exposure to10 Gy radiation for 4 hours. KLF4 was pulled down and the PARylation was detected.
New insight into the significance of KLF4 PARylation in genome stability, carcinogenesis and therapy (2020)
Figure 3

Figure 3. Abnormal KLF4-PARP1 axis correlates with breast cancer poor prognosis. (A) Expression of PARP1 and KLF4 in mammary gland epithelial cell and various types of breast cancer cell lines. (B). Accumulation of PARP1 and KLF4 protein were detected in human breast cancer tissues with 183 breast invasive ductal carcinoma and 10 pairs of primary cancer and adjacent normal tissue specimens by IHC in comparison with adjacent normal breast tissues. Scale bars, 100 μm. (C & D) Statistic results of PARP1 and KLF4 IHC staining. Normal (n=10), tumor (n=183); p = 4.36x10-7 (KLF4) p = 0.0473 (PARP1). one-way ANOVA was used for the statistical analysis. (E) Statistical analysis of PARP1 staining among normal (n=10), ER/PR positive (ER/PR) (n=68), HER2 positive (n=62), and triple negative breast cancer (TNBC) (n=48). The p values were labeled in figure, one-way ANOVA was used for the statistical analysis. (F) Statistical analysis of KLF4 protein staining among normal (n=10), ER/PR positive (n=68), HER2 positive (n=62) and TNBC (n=47). The p values were labeled in figure, one-way ANOVA was used for the statistical analysis. (G & H) Elevated expression of PARP1 and KLF4 are significantly correlated in 183 breast invasive ductal carcinoma human breast cancer tissue specimens (same batch of staining as (B)). Represents of paired IHC staining of PARP1 and KLF4 (case 1-3) are shown (G). Scale bars, 100 μm. (H) Statistic analysis of IHC staining indicates that PARP1 expression is positively correlated with KLF4 expression in breast cancer. n=183, r=0.434, p=8.68x10-10, Pearson correlation coefficients. (I) Survival analysis of KLF4 protein expression in 117 breast cancer patients. Compared to patients with low KLF4 protein expression, patients with high KLF4 levels had an inferior cumulative survival rate (Kaplan-Meier assay, LogRank P=0.012); Low, staining weak and moderate; High, staining strong. (J) Survival analysis of both KLF4 and PARP1 protein expression in 66 breast cancer patients. Compared to patients with both low KLF4 and PARP1 protein expression levels, patients with both high KLF4 and PARP1 levels had lower cumulative survival rate (Kaplan-Meier assay, LogRank P=0.022).
New insight into the significance of KLF4 PARylation in genome stability, carcinogenesis and therapy (2020)
Figure 4

Figure 4. Identification of molecular motifs on KLF4 and PARP1 that mediate KLF4 PARylation. (A) Schematic diagram of human KLF4 functional domains and strategy for engineering a series of KLF4 deletion mutants. (B) Identification of amino acid stretch 411- 441 on the repression domain of KLF4 as a segment mediating the interaction with PARP1 in 293T cells.. (C-D) Mapping of poly(ADP-ribosyl)ation domain on KLF4. (C) Schematic diagram of human KLF4 functional domains and strategy for engineering a set of KLF4 deletion mutants. (D). Identification of amino acid stretch 411- 441 on the Zinc 2 domain of KLF4 involved in mediating its PARylation modification in 293T cells.. PARylation for Flag tagged KLF4 deletion mutants were analyzed, respectively, by pulldown using PAR antibody followed by Western blotting using antibody against Flag. (E) Dissection of the PARylation region of KLF4 into three zinc finger motif-containing domains, Zinc 1, Zinc 2 and Zinc 3. While no effect was observed on Zinc 1 and Zinc 3 domains, deletion of Zinc 2 domain led to significant attenuation of KLF4 PARylation. (F) Schematic diagram of human PARP1 functional domains and strategy for engineering a series of PARP1 deletion mutants. (G) Identification of amino acid stretch 829-1014 on the C-terminal segment of PARP1 to be involved in mediating its interaction with KLF4 in 293T cells.
New insight into the significance of KLF4 PARylation in genome stability, carcinogenesis and therapy (2020)
Figure 7

Figure 7. Synergism of KLF4 and PARP1 in breast cancer treatment. (A) KLF4+/+, KLF4-/- MEFs, KLF4-/- MEF with wild-type KLF4 or KLF4-Zinc 2-YYR/AAA mutant were treated with 5-10 µM cisplatin for 48 hours followed by measuring the expression of PARP1, p21 and Bax. Loss of PARylation on KLF4 impairs KLF4-mediated inhibition of apoptosis in the presence of genotoxic stress. (B) The apoptotic response in U2OS cells with expression of wild-type and mutant KLF4. U2Os cells with wildtype (pLenti-tet-on-KLF4WT) or mutant (pLenti-tet-on-KLF4YYR/AAA) KLF4 were incubated with 10 ng/ml doxycycline for 24h and then treated with 10uM doxorubicin (Dox) or cisplatin (CDDP) for additional 24h. While expression of wild-type KLF4 decreases Bax expression and inhibits drug-induced PARP1 cleavage, expression of PARylation-deficient KLF4 leads to failure in inhibiting genotoxic-induced Bax expression and PARP1 cleavage. (C) Disruption of KLF4 PARylation leads to failure in inhibiting temperature-induced senescence in BTR model (RAS V12 -induced senescence). (D-F) Disruption of KLF4 PARylation abolishes the KLF4-promoted cellular transformation in MDA-MB-231 (D), MCF10A (E) and U2OS (F) measured by soft agar analysis. n=3, p values were labeled in figure, one-way ANOVA assay. (G-H) KLF4 sensitizes cell to Olaparib. The asterisk in the panels represents the significant difference (p<0.05). (G) The representative of MEFs clonogenic assay. (H) is the summary of G. (I) Disruption of KLF4 PARylation reduces KLF4 effect on Olaparib efficacy. (J-K) While elevated KLF4 inhibits PARP1 inhibitor efficacy in killing MDA-MB-231 cells, disruption of KLF4 PARylation partially rescue the KLF4-mediated resistance to Olaparib. (J) The representative of MEFs clonogenic assay. (K) is the summary of J. (L) Elevated KLF4 expression increases resistance to Olaparib in MDA-MB-468 (BRCA-proficient) cells, while disruption of KLF4 PARylation attenuates KLF4-driven resistance to Olaparib. (M-N) Elevation of mutant KLF4 shows the same effect as WT KLF4 to Olaparib in BRCA1 mutant cell lines MDA-MB-436 (M) and HCC1937 (N) cells. No significant difference between expression of wild-type KLF4 and KLF4 PARylation-deficient mutant was observed in both MDA-MB-436 and HCC1937 cells. (O-P) Modulating KLF4 by knockdown or overexpression affects cellular response of MDA-MB-231 (O) and MCF7 (P) to Olaparib in clonogenic assay. (Q-R) Modulating KLF4 by knockdown or overexpression significantly affects synergism on Olaparib/Doxorubicin or Olaparib/Cisplatin in MDA-MB-231(Q) or MCF7 (R). Data are mean ± SEM; one-way ANOVA was used for the statistical analysis. The asterisk in the panels represents the significant difference (p<0.05). The exact p-Values were supplied in Appendix Table S4.
New insight into the significance of KLF4 PARylation in genome stability, carcinogenesis and therapy (2020)
Figure 8

Figure 8. Suppression of KLF4 sensitizes triple negative breast tumor to Olaparib. (A) Depletion of KLF4 significantly enhances the efficacy of Olaparib in killing TNBC cells (4T1). 4T1 cells with stable expression of shKLF4 were implanted into mammary fat pad of BALB/c Nude mice. Drug treatment was started at 10th day. Placebo or Olaparib at the dose of 100 mg/kg was administrated daily for twelve days. Tumor volumes were measured every other day. Tumor volumes were measured every other day. n=9 per group, p values were labeled in figure, one-way ANOVA assay. (B & C) The image of 4T1 xenograft tumors (B) and tumors were weighed and summarized (C). n=9 per group, p values were labeled in figure, one-way ANOVA assay. (D & E) The heatmap of synergizes score distribution between Olaparib with ABT-263 (D) and dasatinib (E). The synergy score was calculated based on Bliss model according to a model that permits synergistic interaction between Olaparib with ABT-263 and dasatinib. (F-I) The combination effect when combine of Olaparib with ABT-263 and dasatinib in suppression MDA-MB-231 xenograft tumor growth. 8 weeks SCID/Beige female mice were injected 8x106 MDA-MB-231 cells in matrigel (1:1 volume). When tumor reach to 50mm3, 100mg/kg Olaparib (dissolve in 2%DMSO+30%PEG400+saline) was administrated by oral gavage daily, 30mg/kg ABT-263 (dissolve in 5%DMSO) was administrated by intraperitoneal injection every other day, 50mg/kg Olaparib (dissolve in 2%DMSO+30%PEG400+saline) or placebo (2%DMSO+30%PEG400+saline) were administrated by oral gavage every other day. Mice were treated with single or combination drugs for four weeks. Tumor volumes were measured every other day (F). The image of MDA-MB-231 xenograft tumors (G). The tumors were weighed and summarized (H). Placebo (n=7), ABT263 (n=9), Dasatinib (n=8), Olaparib (n=6), Olaparib+ABT263 (n=9), Olaparib+Dasatinib (n=10), p values were labeled in figure, one-way ANOVA assay. (I) Staining of H & E, KLF4, PARP1, p21 and Bax in MDA-MB-231 xenograft tumors. Scale bars, 100 μm. Combination of ABT-263 or dasatinib with Olaparib treatment decrease KLF4 and p21expression, but increase Bax expression in xenograft tumors.
Two Beclin 1-binding proteins, Atg14L and Rubicon, reciprocally regulate autophagy at different stages (2009)
figf1

(a) MCF7 cells stably expressing Myc–TEV–Flag–Beclin 1 (MEF–Beclin 1) and parental controls were lysed and subjected to MEF-tag-based purification. Proteins bound to MEF–Beclin 1 were isolated and detected by SDS–PAGE and silver staining. Seven bands specific for MEF–Beclin 1 are numbered. (b) Six bands were identified by nanoflow LC-MS/MS and MASCOT software. (c) MCF7 cells were lysed and subjected to immunoprecipitation using the indicated antibodies. (d) Differential subcellular fractionation. Post-nuclear supernatant of A549 cell lysates were spun at 20,000g for 10 min and the supernatant was further spun at 100,000g for 60 min. Equivalent quantities of each supernatant and pellets from the same number of cells were subjected to western blotting with the indicated antibodies, including anti-Sec61β (ER), cathepsin D (lysosome), EEA1 (early endosome), transferrin receptor (plasma membrane and recycling endosome) and GAPDH (cytosol). (e) Gel filtration analysis. The supernatant fraction of A549 cell (or Atg14L knockdown) lysates were subjected to a Superose 6 column and each fraction was immunoblotted with the indicated antibody (see Supplementary Information, Fig. S2e). Relative amounts of each fraction determined by densitometry were plotted. Vo, void fraction. The elution pattern of each protein was reproduced in several experiments. Full scans of the gel and blots in a, c and d are available in Supplementary Information, Fig. S6.
Two Beclin 1-binding proteins, Atg14L and Rubicon, reciprocally regulate autophagy at different stages (2009)
figf2

(a) A549 cells were infected with adenovirus harbouring shRNA targeting Atg14L, Rubicon, UVRAG or a control (luciferase). Cell lysates were subjected to immunoblotting with the antibodies indicated. The asterisk in the Rubicon blot represents a non-specific cross-reacting band. (b) Quantification of GFP-positive dots per cell. Images were taken from cells shown in c and the number of GFP-positive dots was determined. At least 140 cells were counted per experiment and the values are mean ± s.d. of three independent experiments. (c) A monoclonal A549 cell line stably expressing GFP–LC3 was infected with adenovirus harbouring shRNA targeting Atg14L, Rubicon or control (luciferase). The cells were cultured under nutrient-rich or starvation conditions. GFP was observed by confocal laser microscopy. Scale bar, 10 μm. The full scan of the blot in a is available in Supplementary Information, Fig. S6.
Two Beclin 1-binding proteins, Atg14L and Rubicon, reciprocally regulate autophagy at different stages (2009)
figf3

(a) Southern blot analysis of genomic DNA isolated from wild-type (WT, mAtg14L+/+) , mAtg14L+/− and knockout (KO, mAtg14L−/−) ES cells after digestion with PstI. Details of the probe are shown in Supplementary Information, Fig. S3. (b) Analysis of Atg14L protein in knockout cells. Lysates from WT, mAtg14L−/− ES cells and mAtg5−/− mouse ES cells were subjected to western blotting with the antibodies indicated. (c) The effect of Atg14L knockout on p62 turnover. WT, mAtg14L−/−, mAtg14L−/− + Atg14L, and mAtg5−/− ES cells were cultured in nutrient-rich (N) or starvation medium (S) for 3 h. Cell lysates were subjected to western blotting with the antibodies indicated. (d) Degradation of long-lived proteins is reduced in Atg14L knockout cells. WT, mAtg14L−/−, and mAtg5−/− ES cells were cultured in N or S medium or S medium with wortmannin (W, 100 nM) for 3 h and degradation of long-lived proteins was measured. The data are the mean ± s.d. of three independent experiments. (e) The effect of Atg14L knockout on LC3 turnover. WT, mAtg14L−/− and mAtg5−/− ES cells were cultured in N or S medium for 3 h in the presence or absence of E64d (50 μg ml−1) and pepstatin A (50 μg ml−1). Cell lysates were subjected to western blotting with the antibodies indicated. Full scans of the blots in b, c and e are available in Supplementary Information, Fig. S6.
Two Beclin 1-binding proteins, Atg14L and Rubicon, reciprocally regulate autophagy at different stages (2009)
figf4

(a) The effect of Atg14L knockout (mAtg14−/−) on Atg16L dot formation. Wild-type (WT) and mAtg14−/− ES cells were cultured in nutrient-rich or starvation medium for 3 h. Cells were fixed and subjected to immunofluorescence with an anti-Atg16L antibody. Images of Atg16L staining (grey-scale) and Atg16L merged with Hoechst staining (colour) are shown. (b) The effect of Atg14L knockout on LC3 dot formation. WT and mAtg14L−/− ES cells were cultured in nutrient-rich or starvation medium for 3 h. Cells were fixed and subjected to immunofluorescence with anti-LC3 antibody. Images of LC3 staining (grey-scale) and LC3 merged with Hoechst staining (colour) are shown. Scale bars, 20 μm. (c) Quantification of the number of Atg16L dots in a. The data are the mean ± s.d. of three independent experiments. (d) Quantification of the number of LC3 dots in b. The data are the mean ± s.d. of three independent experiments. For each of the experiments in c and d at least 13 cells were counted.
Two Beclin 1-binding proteins, Atg14L and Rubicon, reciprocally regulate autophagy at different stages (2009)
figf5

(a) Immunofluorescence using an anti-Atg16L antibody. A549 cells were transfected with adenovirus expressing GFP–Atg14L and cultured in nutrient-rich or starvation medium for 4 h. Cells were fixed and stained with an anti-Atg16L antibody. (b) Immunofluorescence with an anti-LC3 antibody. A549 cells were transfected with adenovirus expressing GFP–Atg14L and cultured in starvation medium for 4 h. Cells were fixed and stained with an anti-LC3 antibody. Scale bar, 10 μm. (c) Colocalization efficiency in a and b. The percentage of colocalization between Atg14L and Atg16L or LC3-positive dot is shown. For example, (Atg14L×Atg16L)/Atg16L means the ratio of colocalization of Atg14L and Atg16L puncta to all of the Atg16L-positive puncta. For colocalization between Atg14L and Atg16L the data are mean ± s.d. of 25 cells. For colocalization between Atg14L and LC3 the data are mean ± s.d. of 20 cells. d) Immunofluorescence with an anti-calnexin (ER) antibody. A549 cells transfected with adenovirus-based GFP–Atg14L. After 48 h, the cells were fixed and immunostained with calnexin (ER).Scale bar, 10 μm. (e) Immuno-EM analysis of GFP–Atg14L. A549 cells transfected with adenovirus-vector based GFP or GFP–Atg14L were subjected to immuno-EM analysis using an anti-GFP antibody. Scale bar, 500 nm. The arrows indicate the ER membrane and the arrowheads indicate the autophagosome membrane.