EMBO/soda-nel huggingface dataset:
CRIP1 suppresses BBOX1-mediated carnitine metabolism to promote stemness in hepatocellular carcinoma (2022)
Figure 7

Figure 7. Clinical significance of the CRIP1-BBOX1-β-catenin axis in HCC.

(A) IHC analysis of CRIP1, BBOX1, β-catenin, CD133, OCT4, and NANOG using TAM (left). Scale bars: 20 μm. Correlation analysis of CRIP1 expression with BBOX1, β-catenin, CD133, OCT4, and NANOG expression in HCC tissues (right).

(B) In vivo bioluminescence images of in mice with CRIP1-overexpressing Hep3B cells. EX527 (30 mg/kg/day) was intraperitoneally administered daily for 20 days.

(C) The growth curves of tumors in the indicated groups (n = 4).

(D) IHC analysis of CRIP1, BBOX1, β-catenin, CD133, OCT4, and NANOG in tumor xenograft samples. Scale bar: 20 μm.

(E) IHC analyses of Ki-67 in the indicated groups.

(F) Schematic depicting the mechanism by which CRIP1 downregulation promotes cancer cell stem-like properties via STUB1-induced BBOX1 proteasomal degradation, leading to decrease of β-catenin acetylation and nuclear accumulation of β-catenin.

Data information: Data represent the mean ± SD of at least three independent experiments.. **P <0.01 and ***P < 0.001. Chi-square test was performed to determine the correlation (A). Differences were tested using an unpaired 2-tailed Student's t-test (C, E).
Inhibition of autophagy by TAB2 and TAB3 (2011)
Figure 1

Identification of novel Beclin 1 interactors. (A) Beclin 1 (BCN1) interactors identified by yeast two‐hybrid technology. Proteins binding to BCN1 are listed and their interacting domains are indicated by black bars. Numbers refer to amino‐acid positions. (B) Identification of TAB2 and TAB3 fragments interacting with BCN1 in the yeast two‐hybrid system. Blue lines depict the fragments of TAB2 or TAB3 that were found to interact with BCN1 (numbers on the right refer to the amount of yeast clones identified for each fragment). The minimal domain required for the interaction is referred to as Beclin‐binding domain (BBD). (C) Constructs derived from TAB2 and TAB3 used in this study. (D, E) Co‐immunoprecipitation of TAB2 or TAB3 with BCN1. The indicated constructs, namely HA‐tagged and T7‐tagged TAB2, and TAB3 constructs in (D) and (E), respectively, and Flag‐tagged Beclin 1 (Flag–BCN1) were transfected into HeLa cells alone or in combination. Twenty‐four hours later, TAB2 and TAB3 were immunoprecipitated with antibodies specific for HA (D) or T7 (E) and the precipitate was separated by SDS–PAGE and revealed with an antibody specific for Flag. (F) Immunoprecipitation of endogenous BCN1 with endogenous TAB2 or TAB3. HeLa cells were subjected to autophagy induction with starvation conditions, 1 μM rapamycin or 30 μM pifithrin α (PFTα) for the indicated time and then processed for TAB2 or TAB3 immunoprecipitation followed by the immunodetection of BCN1, TAK1, TAB2 and TAB3. Results in (E) and (F) are representative for three independent experiments.
Inhibition of autophagy by TAB2 and TAB3 (2011)
Figure 2

Reduced interaction between Beclin 1, TAB and TAB3 in conditions of autophagy induction. (A, B) Inhibition of autophagy by dominant‐negative (DN) TAK1. HeLa cells were co‐transfected with a GFP–LC3‐encoding construct plus pcDNA3.1 (empty vector), or plasmids for the expression of WT TAK1 (TAK1WT) or the DN TAK1K63W mutant. One day later, cells were either left untreated (control) or driven into autophagy by starvation or by the administration of 1 μM rapamycin or 30 μM pifithrin α (PFTα), followed by immunoblotting for the detection of TAK1 and endogenous LC3 (A) or immunofluorescence microscopy for the quantification of cells with cytosolic GFP–LC3 puncta (GFP–LC3VAC cells) (B) (mean values±s.d., n=3; *P0.01 versus control cells). GAPDH levels were monitored to ensure equal loading. (C, D) Inhibition of autophagy by knockdown of VPS34, Beclin 1 (BCN1) and TAK1. siRNAs that effectively deplete VPS34, BCN1 and TAK1 were co‐transfected with a GFP–LC3‐encoding plasmid in HeLa cells. Autophagy was then induced as in (A) and the frequency of GFP–LC3VAC cells (mean values±s.d., n=3; *P0.01 versus control cells) was determined. (E, F) The same setting shown in (C, D) was performed with U2OS cells and FYVE–RFP (mean values±s.d., n=3; *P0.01, **P0.001 versus control cells).
Inhibition of autophagy by TAB2 and TAB3 (2011)
Figure 3

Induction of autophagosomes by depletion of TAB2 or TAB3. (A, B) Detection of autophagic GFP–LC3+ puncta. HeLa or U2OS cells stably expressing GFP–LC3 were transfected with siRNAs targeting TAK1, TAB1, TAB2 or TAB3 or with a control siRNA (siUNR). One day later, the subcellular localization and abundance of GFP–LC3 or immunostained TAB2 or TAB3 was determined by epifluorescence microscopy. Representative images are shown in (A) (HeLa cells) and quantitative results (mean values±s.d., n=3; *P0.01 versus siUNR‐transfected cells) are depicted in (B) (U2OS cells). (C) Lipidation of LC3 induced by TAB2 or TAB3 knockdown. Representative immunoblots showing the conversion of non‐lipidated LC3 (LC3‐I) to its lipidated variant (LC3‐II) as well as SQSTM1/p62 protein levels are shown. GAPDH levels were monitored to ensure equal loading. (D, E) Quantification of autophagosomes and autophagolysosomes by transmission electron microscopy. Representative images of HeLa cells transfected with siUNR or with TAB2‐ or TAB3‐targeting siRNAs are shown in (D), and quantitative results are depicted in (E) (mean values±s.d., n=3; *P0.01 versus siUNR‐transfected cells). (F) Epistatic analysis of the effects of TAB2 and TAB3 depletion on autophagy. HeLa cells stably expressing GFP–LC3 were transfected with siRNAs specific for TAB2 or TAB3 and/or with cDNAs coding for full‐length HA‐tagged TAB2 (HA–TAB2) or TAB3 (HA–TAB3). Twenty‐four hours later, the frequency of cells exhibiting >5 GFP–LC3+ cytosolic puncta (GFP–LC3VAC cells) was determined. Results are mean values±s.d. (n=3; *P0.01 versus siUNR‐transfected cells). (G) U2OS cells stably expressing FYVE–RFP were transfected with siUNR, or with siRNAs specific for TAB2, TAB3, VPS34, Beclin 1 (BCN1) and TAK1, in the indicated combinations. Forty‐eight hours later, the percentage of cells with RFP–FYVE+ puncta cells was determined. Results are mean values±s.d. (n=3; *P0.01 versus siUNR‐transfected cells).
Inhibition of autophagy by TAB2 and TAB3 (2011)
Figure 4

Mechanisms of autophagy induced by depletion of TAB2 or TAB3. (A–D) Impact of bafilomycin A1 (BafA1) on the induction of GFP–LC3+ puncta by TAB2 and TAB3 depletion. HeLa cells stably expressing GFP–LC3 were transfected with a control siRNA (siUNR) or with siRNAs targeting TAB2 and TAB3 for 24 h. During the last 12 h of this period, BafA1 was optionally added. After fixation and permeabilization, LAMP2 was detected by immunofluorescence. Representative confocal microphotographs for the TAB2 siRNA are shown (A), together with the profiles of colocalization of fluorescent signals (B) along the indicated direction (α–ω). Columns in (C) represent the percentage of colocalization of GFP–LC3 and LAMP2 (mean values±s.d.; *P0.01 versus siUNR‐transfected cells), as quantified in at least 50 cells for each condition. The frequency (mean±s.d.) of cells with >5 GFP–LC3+ cytosolic puncta (GFP–LC3VAC cells) is plotted in (D). (E) Impact of BafA1 on LC3 lipidation. MEFs with the indicated genotypes were cultured in complete medium supplemented with BafA1 for 12 h and the proportion of LC3‐I/LC3‐II was determined by immunoblotting. GAPDH levels were monitored to ensure equal loading. (F, G) Impact of autophagy‐relevant proteins and of the TAK1‐IKK signalling axis on GFP–LC3 aggregation induced by the depletion of TAB2 or TAB3. HeLa cells stably expressing GFP–LC3 were transfected with siUNR or with siRNAs targeting the indicated proteins, alone or in combination, and 48 h later GFP–LC3VAC cells were quantified (mean values±s.d., n=4; *P0.01 versus siUNR‐transfected cells). (H) Inhibition of autophagy by dominant‐negative (DN) TAK1. HeLa cells stably expressing GFP–LC3 were co‐transfected with pcDNA3.1 (empty vector) or with plasmids encoding WT (TAK1WT) or a DN TAK1 variant (TAK1K63W) together with the indicated siRNAs for 24 h, followed by the quantification of GFP–LC3VAC cells (mean values±s.d., n=3, *P0.01 versus siUNR‐, pcDNA3.1‐transfected cells).
Inhibition of autophagy by TAB2 and TAB3 (2011)
Figure 5

Inhibition of autophagy by full‐length TAB2 and TAB3 but induction by their C‐terminal fragments. (A) Effects of full‐length TAB2 and TAB3 or their deletion mutants (as in Figure 1C) on autophagy. HeLa cells stably expressing GFP–LC3 were transfected with pcDNA3.1 (empty vector) or with plasmids encoding the indicated TAB2 and TAB3 variants for 24 h, then driven into autophagy by starvation or by the administration of 1 μM rapamycin or 30 μM pifithrin α (PFTα) for 4 h. Finally, the frequency (mean±s.d., n=3) of cells with >5 GFP–LC3+ cytosolic puncta (GFP–LC3VAC cells) was assessed (*P0.01 versus control cells transfected with the same plasmid; #P0.01 versus pcDNA3.1‐transfected cells treated with the same pro‐autophagic trigger). (B) U2OS cells stably expressing FYVE–RFP were co‐transfected with pcDNA3.1 or with vectors encoding TAB2C or TAB3C together with a control siRNA (siUNR) or with siRNAs specific for VPS34, Beclin 1 (BCN1) or TAK1 for 24 h, followed by the quantification of cells exhibiting FYVE–RFP+ dots (FYVE–RFP+). Results are mean values±s.d. (n=3; *P0.01 versus siUNR‐, pcDNA3.1‐transfected cells). (C) Inhibition of the interaction between endogenous TAB2, TAB3 and BCN1 by C‐terminal fragments of TAB2 and TAB3. Forty‐eight hours after transfection with pcDNA3.1 or plasmids coding for the indicated proteins, cells were lysed, TAB2 or TAB3 was immunoprecipitated and BCN1 was immunodetected. GAPDH levels were monitored to ensure equal loading. This experiment has been done three times, yielding comparable results. (D) Mechanisms of autophagy induction by the Beclin‐binding domain (BBD)‐containing C‐terminal fragments of TAB2 and TAB3. HeLa cells were transfected with pcDNA3.1, TAB2C‐ or TAB3C‐encoding plasmids in combination with the indicated siRNAs for 24 h, followed by the quantification of GFP–LC3VAC cells (mean values±s.d., n=3; *P0.01 versus siUNR‐, pcDNA3.1‐transfected cells).
Inhibition of autophagy by TAB2 and TAB3 (2011)
Figure 6

Induction of autophagy by a Beclin 1 fragment that disrupts the interaction between endogenous Beclin 1, TAB2 and TAB3. (A) Determination of the TAB‐binding domain (TBD) within Beclin 1 (BCN1). Yeast two‐hybrid technology was used to screen for positive (+) or negative interactions (−) between BCN1 fragments and full‐length TAB2. (B) Schematic presentation of the constructs used in subsequent experiments (BBD, Beclin‐binding domain). (C) Confirmation of the TBD by immunoprecipitation. The indicated constructs were transfected into HeLa cells, followed by lysis, immunoprecipitation of HA‐tagged full‐length TAB2 or TAB3 and detection of His‐tagged constructs. (D, E) Inhibition of the interaction between endogenous BCN1, TAB2 and TAB3 by a BCN1 fragment corresponding to the TBD. HeLa cells were transfected with pcDNA3.1 (empty vector) or with plasmids encoding the TBD or a His‐tagged Beclin 1 variant lacking the TBD (His–BCN1ΔTBD), followed by co‐immunoprecipitation of endogenous TAB2 (D) or TAB3 (E) and detection of BCN1. (F) Induction of autophagy by the TBD. HeLa cells stably expressing GFP–LC3 were transfected with pcDNA3.1 or constructs encoding the indicated BCN1 variants. After 24 h, Flag‐tagged proteins were detected by immunoblotting and the frequency of cells with >5 GFP–LC3+ cytosolic puncta (GFP–LC3VAC cells) was assessed (mean values±s.d., n=3; *P0.01, **P0.001 versus pcDNA3.1‐transfected cells). (G, H) Mechanisms of autophagy induction by the TBD. (G) U2OS cells stably expressing RFP–FYVE were transfected pcDNA3.1 or plasmids for the expression of TBD, BCN1 or BCN1ΔTBD in combination with a control siRNA (siUNR) or siRNAs that effectively deplete VPS4, BCN1 and TAK1. Forty‐eight hours later, the percentage of cells exhibiting FYVE–RFP+ dots (FYVE–RFP+) was assessed. (G). Alternatively, HeLa cells stably expressing GFP–LC3 were transfected with pcDNA3.1 or with a TBD‐encoding plasmids plus siUNR or siRNAs specific the indicated proteins. Forty‐eight hours later, the percentage of GFP–LC3VAC cells was determined (H). Results are mean values±s.d. (n=3, *P0.01 versus siUNR‐, pcDNA3.1‐transfected cells). (I) Inhibition of TBD‐induced autophagy by dominant‐negative (DN) TAK1. Cells were co‐transfected pcDNA3.1 or vectors encoding WT TAK1 (TAK1WT) or a DN TAK1 variant (TAK1K63W) alone or together with a TBD‐encoding plasmid for 24 h, followed by the quantification of GFP–LC3VAC cells (mean values±s.d., n=3; *P0.01 versus pcDNA3.1‐transfected cells).
PINK1 deficiency sustains cell proliferation by reprogramming glucose metabolism through HIF1 (2014)
f1

The rate of glucose uptake (a,e), lactate released after 4 h of incubation (b,f), glycolytic flux (c,g) and pentose–phosphate flux (d,h) were assessed in MEF and mouse cortical neurons in primary culture, as indicated, obtained from either WT or Pink1 KO (Pink1−/−) mice. Blood concentrations of glucose (i) and lactate (j), and the rate of 14C-lactate appearance in the blood from acutely injected [U-14C]glucose in the tail vein (k) of WT and Pink1 KO mice were analysed. Data are expressed as mean±s.e.m. *P0.05 (Student’s t-test; n=3–4 independent experiments).
PINK1 deficiency sustains cell proliferation by reprogramming glucose metabolism through HIF1 (2014)
f2

Quantitative real-time PCR (RT–qPCR analysis of the relative mRNA abundances of Glut1, Glut3, Hk2 and Gapdh in WT and Pink1 KO mice MEF (a) or neurons (c). ß-Actin mRNA was used for normalization, and the Pink1 KO data are expressed as the change relative to WT. Western blot analysis of the protein abundances of PINK1, GLUT1, GLUT3 (not detectable in MEF), HK2, GAPDH and HIF1α in WT and Pink1-null mice MEF (b) or neurons (d); ß-Actin was used as loading control. (e) Western blot against PINK1, GLUT1, GLUT3 and HIF1α in WT and Pink1 KO mice skeletal muscle samples; α-Tubulin was used as loading control. Only representative western blots are shown per condition; the replicates and the semi-quantitative estimation of the band intensities are shown in the Supplementary Information. The mean±s.e.m. values of the mRNA data were calculated from the fold change of each ß-Actin-normalized transcript abundance in the Pink1 KO samples versus that in the WT. Thus, in all cases, the WT values were considered=1.00. *P0.05 versus the corresponding WT (Student’s t-test; n=3–4 independent experiments).
PINK1 deficiency sustains cell proliferation by reprogramming glucose metabolism through HIF1 (2014)
f3

An siHif1α was used to evaluate its involvement in the glycolytic phenotype of Pink1 KO cells. (a) RT–qPCR analysis of Hif1α mRNA abundance in MEF transfected with siHif1α, or siControl (100 nM), after 72 h. (b) siHif1α effectively knockdowns expressed Hif1α cDNA WT, but not a Hif1α cDNA silent mutant form refractory to siHif1α (Hif1α cDNA REFR.) in mice MEF. (c) RT–qPCR analysis of the relative mRNA abundances of Glut1, Glut3, Hk2 and Gapdh in WT and Pink1 KO mice MEF transfected with siControl or siHif1α. ß-Actin mRNA was used for normalization, and all data are expressed as the change relative to WT-siControl. (d) Western blot against HIF1α in WT and in Pink1 KO mice MEF transfected with siHif1α or siControl shows the efficacy of the siHif1α at knocking down endogenous HIF1α; ß-actin was used as loading control. As shown, siHif1α partially prevented the increase in GLUT1, HK2 and GAPDH protein abundances. The increased rate of lactate released after 4 h of incubation (e) and glycolytic flux (f) observed in the Pink1 KO MEF was prevented by siHif1α. siHif1α was also effective at preventing the endogenous increased HIF1α protein (g), as well as the increased glycolytic flux (h) observed in Pink1 KO primary neurons. Only representative western blots are shown; the replicates and the semi-quantitative estimation of the band intensities are shown in the Supplementary Information. The mean±s.e.m. values of the mRNA data were calculated from the fold change of each ß-actin-normalized transcript abundance in the Pink1 KO samples versus that in the WT (a) or WT-siControl (c); thus, in all these cases, the WT (a) or WT-siControl (c) values were considered=1.00. Only the statistical analysis results of the siHif1α versus siControl (in KO) values (mean±s.e.m.) are shown in c,e,f,h, since the comparison KO versus WT under control conditions was previously shown in Fig. 2a, Fig. 1b,c,g, respectively. *P0.05 (Student’s t-test; n=3–4 independent experiments).
PINK1 deficiency sustains cell proliferation by reprogramming glucose metabolism through HIF1 (2014)
f4

(a) Mitochondrial respiratory chain complexes activity values normalized to citrate synthase (CS), and (b) mitochondrial inner membrane potential (Δψm) in WT and Pink1 KO mice MEF. (c) Western blot against PDK1 and HIF1α in WT and Pink1 KO MEF transfected with siHif1α (or its siControl); ß-actin was used as loading control. (d) PDH complex activity in WT and Pink1 KO MEF transfected with siHif1α or siControl. Rate of whole-cell H2O2 (e) and mitochondrial O2·− (f) detection in WT and Pink1 KO MEF. (g) Detection of mitochondrial O2·− using the mitochondrial-specific probe MitoSox in MEF incubated in the absence (−) or in the presence (+) of the plasma membrane permeable GSH-EE, or by expressing mitochondrial-tagged forms of glutamate-cysteine ligase, (+mitoGCL; empty vector, pIRES2–EGFP, denoted as −) or catalase (+ mitoCAT; empty vector denoted as −), shows effective mitochondrial ROS removal. (h) Western blot against HIF1α in Pink1 KO MEF incubated with (or without) GSH-EE, or transfected with empty vector, mitoGCL or mitoCAT, showing reduction in HIF1α protein abundance by the treatments that remove mitochondrial ROS; α-TUBULIN was used as loading control. Only representative western blots are shown; the replicates and the semi-quantitative estimation of the band intensities are shown in the Supplementary Information. Data are expressed as mean±s.e.m. *P0.05 (Student’s t-test; n=3–4 independent experiments).
PINK1 deficiency sustains cell proliferation by reprogramming glucose metabolism through HIF1 (2014)
f5

(a) Proportion of BrdU incorporation into DNA and cell cycle phases, and (b) cell growth in WT and Pink1 KO MEF after 72 h in the absence (denoted as -2DG) or in the presence (+2DG) of the glucose-metabolizing inhibitor, 2-deoxyglucose (2DG, 1 mM). (c) Proportions of BrdU incorporation into DNA and cell cycle phases, and (d) cell growth in WT and Pink1 KO MEF 72 h after transfection with siHif1α (+siHif1α) or siControl (denoted as -siHif1α). (e) Ki67, Sox2 and DAPI immunostaining in the subventricular zone of WT and Pink1 KO mouse brain, and the corresponding quantification. *P0.05; #P0.05 versus all other conditions (analysis of variance followed by Bonferroni test; n=3–4 independent experiments). All data are expressed as mean±s.e.m. Scale bar, 100 μm.
PINK1 deficiency sustains cell proliferation by reprogramming glucose metabolism through HIF1 (2014)
f6

(a) Rate of mitochondrial O2·– detection in WT and Pink1 KO mice primary neurons. (b) Proportion of BrdU incorporation into DNA of WT and Pink1 KO neurons. (c) Proportion of apoptosis in WT and Pink1 KO neurons, as assessed by the proportion of annexin V-positive/7-AAD-negative cells, determined by flow cytometry. Data are expressed as mean±s.e.m. *P0.05 (Student’s t-test; n=3–4 independent experiments).
TAK1 activates AMPK‐dependent cytoprotective autophagy in TRAIL‐treated epithelial cells (2009)
Figure 1

TRAIL induces cytoprotective autophagy in breast epithelial cells. (A) MCF10A–eGFP–LC3 cells were left untreated (Control) or treated with 5 μM rapamycin (Rapa), 500 ng/ml TRAIL or 10 ng/ml TNF for 24 h. When indicated, 10 mM 3‐MA or 5 μg/ml TRAIL‐R2 antagonist antibody was added 1 h before the drugs. Representative confocal images (20 μm scale bars) and the percentages of cells with LC3 translocation are shown. (B) MCF10A–eGFP–LC3 cells were transfected with the indicated siRNAs for 48 h and analysed by immunoblotting (left), or stimulated with 500 ng/ml TRAIL for 24 h and analysed for LC3 translocation (right). (C) MCF10A cells left untreated (Control) or treated with 500 ng/ml TRAIL or 5 μM rapamycin (Rapa) for 24 h were analysed for LC3‐I, LC3‐II and tubulin by immunoblotting. Pepstatin A and E64d were added to the cells 4 h before harvesting. Similar results were obtained in two independent experiments. (D) MCF10A cells were treated with 5 μM rapamycin (Rapa) or indicated concentrations of TRAIL for 24 h, and the increase in the degradation of long‐lived proteins as compared with the untreated cultures (Control) was measured. (E) MCF10A–eGFP–LC3 cells transfected with the indicated siRNAs were left untreated 48 h later (Control) or treated with 500 ng/ml TRAIL for 24 h and analysed for the DNA content by flow cytometry. The percentage of apoptotic cells with sub‐G1 DNA content is shown. The values represent mean±s.d. for three (A, D) or four (B, E) independent experiments. *P‐value 0.05, **P‐value 0.01 and ***P‐value 0.001 as compared with control sample (A—left panel and D), with TRAIL‐treated cells without anti‐TRAIL‐R2 (A—right panel) or with TRAIL‐treated cells without siRNA (B, E).
TAK1 activates AMPK‐dependent cytoprotective autophagy in TRAIL‐treated epithelial cells (2009)
Figure 2

TRAIL activates AMPK and AMPK‐dependent cytoprotective autophagy. (A) Protein lysates from MCF10A cells left untreated or treated with 500 ng/ml TRAIL or 5 μM rapamycin (Rapa, positive control) for 24 h were analysed by immunoblotting for phosphorylated p70S6K (P‐p70S6K), total p70S6K and GAPDH (loading control). (B) Immunoblot analysis of the levels of P‐ACC and tubulin (loading control) in lysates from MCF10A cells left untreated or treated with 500 ng/ml TRAIL for 24 h, 1 mM AICAR for 4 h (positive control) or 5 μM rapamycin for 24 h (negative control). (C) Immunoblot analysis of the level of P‐ACC and GAPDH (loading control) in lysates from MCF10A cells treated with 500 ng/ml TRAIL for the indicated times. (D) MCF10A cells were transfected with indicated siRNAs for 48 h and analysed by immunoblotting for the indicated proteins immediately (upper panel) or after additional 24 h (middle panel) or 2 h (lower panel) incubation with (+) or without (−) 500 ng/ml TRAIL. (E) MCF10A–eGFP–LC3 cells transfected with indicated siRNAs for 48 h and incubated for an additional 24 h with or without 500 ng/ml TRAIL were analysed for LC3 translocation (top) and the DNA content (bottom). The values represent mean±s.d. for four independent experiments. *P‐value 0.05 and ***P‐value 0.001 as compared with TRAIL‐treated cells without siRNA. Similar results were obtained in two (A, B and D) or three (C, D) independent experiments.
TAK1 activates AMPK‐dependent cytoprotective autophagy in TRAIL‐treated epithelial cells (2009)
Figure 3

LKB1 and CaMKKβ do not have an important function in TRAIL‐induced AMPK activation and autophagy. (A) MCF10A–eGFP–LC3 cells were transfected with indicated siRNAs for 48 h and analysed for the indicated mRNAs (top) and proteins (bottom by RT–PCR and immunoblotting, respectively. Similar results were obtained in three independent experiments. (B) MCF10A–eGFP–LC3 cells transfected with indicated siRNAs for 48 h, and left untreated or treated with 500 ng/ml TRAIL for 2 h, 10 μM ionomycin for 24 h or starved for amino acids and glucose for 24 h (starvation) were analysed for P‐ACC and tubulin (loading control) expression by immunoblotting. Similar results were obtained in two independent experiments. (C) Cells transfected as in (B) and left untreated or treated with 500 ng/ml TRAIL, 10 μM ionomycin or starved for amino acids and glucose for 24 h were analysed for LC3 translocation. The values represent mean±s.d. of three independent experiments. ***P‐value 0.001 as compared with cells treated in a same way, but transfected without siRNA.