Monoclonal antibodies against Flag (cat. no. ab205606) and DNAJA1 (cat. no. ab192904), were purchased from Abcam. AMPKα2 (cat. no. A7339), BAX (cat. no. A12009), Bcl-2 (cat. no. A0208) and α-tubulin (cat. no. AC012) antibodies were purchased from ABclonal Biotech Co., Ltd. The streptavidin-peroxidase antibody (cat. no. SA00001-0) was obtained from Proteintech Group, Inc. CCCP (cat. no. C6700) was purchased from Beijing Solarbio Science & Technology Co., Ltd. PVDF membranes were obtained from MilliporeSigma. Dynabeads^™ MyOne^™ streptavidin C1 (cat. no. 65001), biotin (cat. no. B20656) and liposomal transfection reagent (Lipofectamine^® 2000; cat. no. 11668500) were obtained from Thermo Fisher Scientific, Inc. RIPA buffer (cat. no. G2002-100ML), DAPI (cat. no. G1012-100ML) and anti-fluorescence quenching sealing agent (cat. no. G1401-25ML) were purchased from Wuhan Servicebio Technology Co., Ltd. The anti-DYKDDDDK (Flag) affinity gel (cat. no. 20585ES03), lentivirus concentration solution (cat. no. 41101ES50) and protein silver staining kit (cat. no. 36244ES25) were purchased from Shanghai Yeasen Biotechnology Co., Ltd. A DNA purification kit (cat. no. DP204) and a high purity plasmid extraction kit (cat. no. DP104) were obtained from Tiangen Biotech Co., Ltd. Lastly, inorganic salts were purchased from Sinopharm Chemical Reagent Co., Ltd.

293T (human embryonic kidney epithelial) and U251 (malignant astrocyte) cell lines were obtained from American Type Culture Collection. U251 and 293T cells were cultured in DMEM (Gibco; Thermo Fisher Scientific, Inc.) containing 10% FBS (Gibco; Thermo Fisher Scientific, Inc.) and 0.5% penicillin-streptomycin. All cells were cultured in a CO₂ incubator at 37˚C with 5% CO₂.

TurboID cDNA was synthesized and integrated into a plenti-cytomegalovirus (CMV)-EGFP (Beijing Tsingke Biotech Co., Ltd.) plasmid replacing the EGFP fragment (plenti-CMV-TurboID). This was conducted by Beijing Tsingke Biotech Co., Ltd. The AMPK gene was amplified using human cDNA as a template and the following primers: AMPK forward, 5'-atagaagacaccgactctagaATGGCTGAGAAGCAGAAGCAC-3'; AMPK reverse, 5'-catacgcgtgatatccccgggACGGGCTAAAGTAGTAATCAG-3' (the lowercase letters indicate restriction enzyme sites and protective bases, and the uppercase letters indicate the gene-specific sequences that anneal to the template). The thermocycling conditions were as follows: Initial denaturation at 94˚C for 5 min; followed by 30 cycles of denaturation at 94˚C for 30 sec, annealing at 56˚C for 30 sec and extension at 72˚C for 2 min; with a final extension at 72˚C for 8 min. The plasmid plenti-CMV-TurboID was double digested using XbaI and SmaI and the AMPK fragment was purified using a DNA purification kit and combined with the linearized plenti-CMV-TurboID plasmid. Positive clones were screened following transformation into E. coli TOP10 cells and selection on ampicillin (100 µg/ml)-containing LB agar plates. Plasmids were extracted using a high purity plasmid extraction kit, and then the extracted plasmids were processed through enzymatic digestion and sent to Beijing Qingke Biotechnology Co., Ltd. for sequencing of the insert. The selected plasmid was named AMPK-TurboID and was tagged with Flag.

Firstly, 293T cells were cultured in a 10-cm dish until their cell density reached 80-90% for transfection. At the beginning of transfection, the plasmids were diluted with serum-free high-glucose medium. A total of two 1.5 ml Eppendorf (EP) tubes were prepared, one EP tube with 15 µg of psPAX2 (packaging plasmid), 6 µg of pMD2.G (envelope plasmid) and 10 µg of AMPK-TurboID (transfer plasmid) and the other EP tube with 15 µg of psPAX2, 6 µg of pMD2.G and 10 µg of plenti-CMV-EGFP (transfer plasmid). Subsequently, an appropriate amount of liposomal transfection reagent (~60 µl per 10-cm dish) was diluted with serum-free medium and incubated at room temperature for 5 min. Next, the diluted DNA plasmid and the diluted liposomal nucleic acid transfection reagent were gently mixed and incubated at room temperature for 30 min. Subsequently, the incubated DNA liposome complexes were evenly dropped into 293T cell culture dishes that had been replaced with pre-warmed complete high-glucose DMEM (containing 10% FBS) without antibiotics DMEM and incubated overnight at 37˚C in a cell culture incubator with 5% CO₂. The next day, the medium was replaced with fresh medium and incubation continued for 48-72 h when the cell supernatant was collected. The collected medium supernatant was filtered by aspiration with a 5-ml syringe onto a 0.45-mm membrane filter, after which the filtrate was mixed with lentivirus concentration reagent in a 4:1 ratio and incubated overnight at 4˚C. After incubation, the supernatant was removed by centrifugation (4˚C; 6,580 x g; 40-60 min) and fresh medium was added to the sediment and dispensed into a number of 1.5 ml EP tubes.

U251 cells were grown in 10-cm cell culture dishes and when cell confluence reached 80-90%, an appropriate number of cells (2x10⁵) were seeded in 24-well cell culture plates. When the cell density reached 70-80%, lentivirus concentrate carrying either the AMPK-TurboID plasmid or the plenti-CMV-EGFP plasmid was dropwise added to different wells of the plate, and the cells were infected (multiplicity of infection of 10) for 6-8 h at 37˚C and replaced with fresh medium. The fluorescence intensity of plenti-CMV-EGFP plasmid-infected cell culture plates was visualized using an inverted fluorescence microscope. after 48 h. Successful infection was demonstrated when stronger fluorescence appeared. Resistance screening was performed with medium containing puromycin (1.5 µg/ml) and cell status was observed after 24-48 h. Maintenance was performed in 0.5 µg/ml puromycin. Viable cells were cultured into 12-well plates and sequentially passaged to the number of cells that met the requirements of subsequent experiments. The time interval from transduction to subsequent experiments was ~2 weeks. During this period, the cell status was observed daily and medium was renewed once every 2 days. A number of cells were used to obtain cell lysate for SDS-PAGE gel electrophoresis and determine whether stable transfection was successful. Cells stably transfected with the plenti-CMV-EGFP plasmid were named EGFP-U251 cells and cells stably transfected with the AMPK-TurboID plasmid were named AMPK-U251 cells.

AMPK-U251 cells were grown into 6-well culture plates and experiments were performed when their density reached ~80%. Cell medium was replaced with complete medium containing biotin (500 µmol/l), magnesium chloride (MgCl₂; 1 µmol/l) and ATP (200 µmol/l) and incubated at 37˚C, and seven labeling time gradients were set, namely 0 and 10 min, 1, 3, 6, 12 and 24 h. Cells were collected, cell lysis was performed and cell lysates were subjected to western blotting using a streptavidin-peroxidase antibody.

To confirm successful stable transfection, whole-cell lysates were prepared from the generated EGFP-U251 and AMPK-U251 cells, as well as control U251 cells. Cells were washed twice with ice-cold PBS and lysed in RIPA lysis buffer (cat. no. P0013B; Beyotime Biotechnology) supplemented with a protease inhibitor cocktail on ice for 30 min. The lysates were clarified by centrifugation at 12,000 x g for 20 min at 4˚C. Protein concentrations were determined using an Enhanced BCA Protein Assay Kit (cat. no. P0009; Beyotime Biotechnology). Equal amounts of protein (10 µg per lane) were denatured in 5X SDS-PAGE loading buffer at 95˚C for 10 min. The samples were then separated by SDS-PAGE on a 10% polyacrylamide gel at 120 V for 1.5 h. Following electrophoresis, the proteins were transferred onto a PVDF membrane (cat. no. IPVH00010; MilliporeSigma) using a wet transfer system at 250 mA for 150 min at 4˚C. The membrane was blocked with Quick Block^™ Blocking Buffer (Beyotime Biotechnology) for 15-30 min at room temperature. Subsequently, the membrane was incubated overnight at 4˚C with primary antibodies diluted in blocking buffer. The following primary antibodies were used: Rabbit anti-AMPK polyclonal antibody (cat. no. 10929-2-AP; Proteintech Group, Inc.) and rabbit anti-FLAG polyclonal antibody (cat. no. 80801-2-RR; Proteintech Group, Inc.), both diluted at 1:1,000. GAPDH was used as a loading control. After washing three times with TBST (10 min each), the membrane was incubated with the HRP-conjugated goat anti-rabbit IgG secondary antibody (cat. no. SA00001-2; diluted at 1:5,000; Proteintech Group, Inc.) for 1 h at room temperature. Protein bands were visualized using an enhanced chemiluminescence detection reagent (cat. no. BL523A; Biosharp Life Sciences). The signals were captured using a chemiluminescence imaging system (ChemiDoc^™ MP Imaging System; Bio-Rad Laboratories, Inc.). Densitometric analysis of the bands was performed using ImageJ software (Version 1.53, National Institutes of Health, Bethesda, MD, USA).

AMPK-U251 cells were cultured into 6-well culture plates and left to reach a density of ~70-80% for subsequent experiments. The original culture medium was discarded and replaced with biotin-labeling buffer containing five varying concentrations (0, 50, 100, 200 and 500 µmol/l; 37˚C) after two washes with PBS, in order to collect sufficient labeled protein and prevent oversaturation of labeling efficiency. The biotin labeling buffer was composed of complete medium containing biotin (500 µmol/l), MgCl₂ (1 µmol/l) and ATP (200 µmol/l) and biotin labeling buffer without ATP had only the ATP component removed. Cells were collected, cell lysis was performed and lysates were subjected to western blotting using a streptavidin-peroxidase antibody.

This method was performed with reference to that previously reported by Branon et al (9). The wild-type U251 cell line and AMPK-U251 cell line were grown in T75 cell culture flasks and the experiment was set up in three groups: i) Blank group, two T75 culture flasks for the wild-type U251 cell line; ii) experimental group I, two T75 culture flasks for the AMPK-U251 cell line; and iii) experimental group II, two T75 culture flasks for the AMPK-U251 cell line treated with 15 µmol/l CCCP. All three groups of cells were cultured using DMEM medium containing biotin (500 µmol/l) and MgCl2 (1 µmol/l) at 37˚C, and the cells were collected after 6 h for sample preparation.

Cells in the T75 cell culture flasks were collected by centrifugation (11,200 x g for 15 min at 4˚C) into 1.5-ml centrifuge tubes, resuspended by adding 1 ml of RIPA buffer (containing PMSF), and placed on ice for ~20 min for lysis. In parallel, the Dynabeads were centrifuged at 11,200 x g for 15 min at 4˚C. The supernatant was transferred to a 1.5-ml centrifuge tube. Subsequently, the streptavidin-coated magnetic beads were washed with 1 ml RIPA buffer for ~2 min at 37˚C, the supernatant was clarified by adsorption on a magnetic stand, RIPA buffer was discarded and washing was repeated 5 times at 37˚C. After the last wash, the beads were divided into three equal parts (200 µl per aliquot), and after precipitation on a magnetic stand the RIPA buffer was discarded. The protein lysate (1 ml) was added to the beads separately, placed at 4˚C and shaken slowly overnight using a 360˚ shaker. The next day, the supernatant was discarded and the samples were washed once with 1 ml potassium chloride, three times with Buffer 1 [3M NaCl 33.5 ml, 1M Tris-HCl (pH 7.4) 0.5 and 16 ml Ultrapure Water] configured in advance, once with 1 ml sodium carbonate buffer and then 2 min (maximum 3 min) with 1 ml 10% SDS, after which 1 ml RIPA buffer was added and soaked continuously for 1 min at 50˚C in a metal bath.

Subsequently, one-third of each sample was removed and processed for silver staining, while the rest of the samples were washed twice with RIPA buffer and PBS, then stored at -20˚C. Cells were counted and lysed at equal cell densities. Protein samples (10 µg per lane) were separated by 10% SDS-PAGE. Subsequent experiments were performed using a silver staining kit (fixative, sensitizing, silver staining, development and termination solutions) following the manufacturer's protocol. the gel was removed and placed in 100 ml fixative on a 60-70 rpm shaker for 20 min. The fixative was discarded and placed in 100 ml 30% ethanol on a slow shaker for 10 min. The fixative was discarded and 100 ml 30% ethanol was added with the samples shaken slowly for 10 min. The ethanol was then discarded, 200 ml deionized water was added and shaken slowly for 10 min. The water was replaced with 100 ml 1X sensitizing solution and shaken for 4-5 min. The sensitizing solution was replaced with 200 ml deionized water and shaken for 1 min, the process was repeated and the water discarded. A total of 100 ml 1X silver staining solution was added and shaken for 30-40 min. Then, 100 ml deionized water was added, shaken for 30 sec, repeated once and then the water was discarded. Subsequently, 100 ml color development solution was added and shaken until bands appeared (3-10 min). The color development solution was replaced with 100 ml termination solution and shaken for 10 min. The termination solution was discarded, 100 ml deionized water was added and shaken for 30 sec, repeated once and the water discarded. Images were captured with a camera. All incubations and washes were performed at room temperature.

The remaining two-thirds of the protein samples were sent to Spectrum Zhonghe (Wuhan) Life Science Technology Co., Ltd., for label-free quantitative MS (32). The LC-MS/MS analysis was performed using an Orbitrap Exploris 480 mass spectrometer coupled with an EASY-nLC 1200 liquid chromatography system (both Thermo Fisher Scientific, Inc.). Ionization was carried out in positive electrospray ionization (ESI+) mode with a spray voltage of 2,000 V. The mass spectrometer was operated in data-dependent acquisition mode, acquiring full MS scans over a mass range of 350-1,250 m/z at a resolution of 15,000, followed by higher-energy collisional dissociation (HCD) fragmentation with a normalized collision energy of 28. The ion transfer tube temperature was set to 320˚C, and the nebulizer (sheath gas) pressure and auxiliary gas flow rate were both set to 3 arbitrary units (these values are not directly provided in psi or l/min, as they are controlled by the instrument software). No multiple reaction monitoring transitions were assessed in this study, as the analysis was performed using label-free quantification. Enrichment analysis was performed on the data obtained. The online analysis tool Sangerbox (version 3.0; http://vip.sangerbox.com/home.html) was used for Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis. Significantly changed proteins were identified using a threshold of FDR <0.05 and |log2FC|>1. A total of 1,045 interacting proteins in the AMPK overexpression group were selected for analysis.

Protein interactions were initially verified using a co-IP assay. Wild-type U251 cells and AMPK-U251 cells were collected into EP tubes and labeled as A1 and B1, respectively. To tubes A1 and B1, 1 ml of IP lysis buffer and 10 µl of PMSF were added. The cells were then gently pipetted to disperse and placed on a rotator at 4˚C for 30 min for lysis. The lysed cells were centrifuged in a refrigerated centrifuge at 4˚C and 11,200 x g for 10 min, and protein concentrations were determined using a BCA assay. Two additional EP tubes were labeled as A2 and B2, and 50 µl of Anti-DYKDDDDK magnetic beads were added to each tube. Subsequently, 1 ml of IP lysis buffer was added to tubes A2 and B2, and the tubes were placed on a rotator at 4˚C for 10 min to wash away impurities; this washing step was repeated twice. After discarding the IP lysis buffer from tubes A2 and B2, 900 µl of the protein lysate from tube A1 was transferred to tube A2, and the same procedure was performed for tube B1 using lysate from tube B1. Tubes A2 and B2 containing the lysate-bead mixtures were placed on a rotator at 4˚C and incubated overnight. The next day, tubes A2 and B2 were removed, the original solution was discarded, and 1 ml of PBS was added. The tubes were placed on a rotator at 4˚C for 10 min to wash; this washing step was repeated twice. After discarding the PBS from tubes A2 and B2, an appropriate amount of 1X loading buffer was added. An appropriate amount of loading buffer was also added to tubes A1 and B1. All four EP tubes were placed in a thermo mixer at 100˚C for 10 min to denature the proteins. Finally, SDS-PAGE gel electrophoresis was performed.

In a second step, AMPK-TurboID cells (5x10⁴ cells/cm²) were seeded in a 3.5-cm cell confocal dish, the medium was discarded when the density reached 80%, PBS added to wash the cells for 3 min and washing was repeated twice. Subsequently, 4% paraformaldehyde was added (37˚C) for 15 min to fix the cells in the dish and then discarded. PBS was added for 3 min and discarded, and washing was repeated twice. Next, 0.5% Triton X-100 was added for 20 min at room temperature and then PBS was added for 3 min and repeated twice. PBS was absorbed from the dish with absorbent paper and 200 µl of normal goat serum (cat. no. AR0009; Wuhan Boster Biological Technology, Ltd.) was added dropwise and incubated at room temperature for 30 min for blocking. The solution was removed with absorbent paper, and a primary antibody solution (2.5 µl DNAJA1 primary antibody; 1:200 dilution; 1 µl Flag; 1:500 dilution primary antibody and 500 µl primary antibody dilution) was added and incubated at 4˚C overnight. The cells were washed for 3 min with PBS twice and PBS was removed with absorbent paper. The secondary antibodies, Cy3-conjugated goat anti-mouse IgG (1:250 dilution; cat. no. A0516; Beyotime Biotechnology) and FITC-conjugated goat anti-mouse IgG (1:250 dilution; cat. no. A0568; Beyotime Biotechnology), were diluted in 250 µl of PBS and added to the cells. After incubation at room temperature for 1 h in the dark, the cells were incubated with DAPI (1 µg/ml; 37˚C) for 5 min and washed three times with PBS for 5 min. After absorbing the PBS with blotting paper, the slides were sealed with anti-fluorescent quenching agent and cells were observed under a confocal Olympus FV3000 microscope (Olympus Corporation).

Data analysis and graphing were conducted with GraphPad Prism (version 10.1.2; Dotmatics). Each group of data as the mean ± SD from three experimental replicates. Comparisons among groups were performed using one-way ANOVA followed by Tukey's honestly significant difference test. P<0.05 was considered to indicate a statistically significant difference.

Fig. 1A shows the composition of the AMPK-TurboID overexpression plasmid, including the target gene AMPK, the biotin labeling enzyme TurboID and the tag protein Flag, as well as two different resistance screening markers, ampicillin and puromycin, whereby ampicillin was used for the screening of positive clones in Esherichia coli and puromycin was used for the screening of stably transfected cells. Fig. 1B illustrates the biotin labeling experiment. First, AMPK-U251 cells that were successfully and stably transfected were placed in a biotin environment with a suitable concentration, so that free biotin could fully enter the cells. Adjacent proteins were covalently labeled with biotin by TurboID. After the protein affinity purification experiment, other proteins that were not labeled with biotin were excluded and the proteins that had been labeled with biotin were enriched. Lastly, the labeled proteins were analyzed by label-free quantitative MS.

As shown in Fig. 2A, a large amount of fluorescence was observed under the microscope in plenti-CMV-EGFP-transfected 293T cells after 48 h, determining that 293T cells had been successfully transfected and the viral fluid supernatant could be collected to infect cells. Subsequently, as demonstrated in Fig. 2B, U251 cells infected with the lentivirus carrying the plenti-CMV-EGFP construct for 48 h exhibited a large amount of fluorescence, implying that the concurrent AMPK-TurboID gene was also effectively infected into U251 cells with high probability.

Comparative analysis of stably transduced AMPK-TurboID and TurboID-U251 cell lines was conducted through western blotting. Western blotting was performed with both an AMPK antibody and a Flag antibody (Fig. 2C). The results revealed that the AMPK-TurboID fusion protein showed a uniform and clear band at a molecular weight of ~97 kDa (molecular weight of AMPK, 62.3 kDa; molecular weight of TurboID, 35 kDa; Flag size was neglected), while the corresponding protein region in the TurboID-U251 cell line was blank, indicating that the stable cell line was successfully constructed.

The results of the biotin labeling assay showed that interacting proteins of AMPK appeared after 10 min and more markedly after 1 h of labeling (Fig. 3A). To prevent oversaturation of biotin labeling, 6 h was used as the biotin labeling time of AMPK-U251 cells in the subsequent experiment. The results showed that there was some improvement in biotin labeling efficiency with the addition of ATP, however the effect was limited (groups 4-6; Fig. 3B). Considering the possible effect of the addition of ATP on the activity of AMPK, ATP was not added to the subsequent biotin-labeling buffers in the present study.

Subsequently, the U251 and AMPK-U251 cell lines underwent affinity purification and silver staining. As shown in Fig. 3C, U251 as a negative control pulled only a limited number of proteins. Since U251 cells do not express TurboID, they theoretically should not enrich biotin-labeled proteins; therefore, the few protein bands observed likely represent non-specific binding or experimental background noise. Therefore these data were excluded. The remaining two sets of data indicated that AMPK overexpressing cell lines pulled a higher number of biotin-interacting proteins.

Analysis of the MS data revealed a total of 1,808 non-redundant interacting proteins identified by proximity labeling across all groups after removing duplicates, with 329 proteins identified in the U251 blank group and 1,045 proteins identified in the AMPK overexpression group and 1,705 proteins in the CCCP-treated group. Furthermore, after excluding 129 proteins that were also identified in the blank group, 916 interacting proteins remained (Table SI). In addition, 1,705 proteins were identified in the CCCP-treated group and after excluding 296 proteins that were also identified in the blank group, 1,409 interacting proteins remained (Table SII).

The online analysis tool Sangerbox 3.0 was used for GO and KEGG analysis. A total of 1,045 interacting proteins in the AMPK overexpression group were selected for analysis. In the analysis of cellular components, the terms ‘cytosol’, ‘nuclear part’, ‘protein-containing complex’, ‘nuclear lumen’ and ‘non-membrane-bounded organelle’ were enriched (Fig. 4A). In the analysis of molecular functions, the search revealed enrichment of ‘RNA binding’, ‘enzyme binding’, ‘ribonucleotide binding’ and ‘ATP binding’ (Fig. 4B). The enrichment of ‘ATP binding’ reflects AMPK's function as a cellular energy sensor, while ‘RNA binding’ suggests a potential role in post-transcriptional regulation. In addition, a number of significantly enriched terms were identified in the analysis of biological processes, including ‘organelle organization’, ‘positive regulation of metabolic process’, ‘cellular localization’, ‘positive regulation of macromolecule metabolic process’ and ‘cellular macromolecule localization’ (Fig. 4C). These terms directly reflect AMPK's central role in coordinating metabolism and organelle dynamics. In the KEGG analysis, pathways that were found to be enriched included ‘endocytosis’, ‘spliceosome’, ‘RNA transport’, ‘regulation of actin cytoskeleton’, ‘Hippo signaling pathway’, ‘insulin signaling pathway’, ‘insulin resistance’ and ‘ferroptosis’ (Fig. 4D).

MS data revealed that DNAJA1 is an interaction partner of AMPK. DNAJA1, a key protein in the protection against apoptosis, acts as a co-chaperone protein for heat shock protein family A member 1B and negatively regulates the transport of Bax from the cytoplasm to the mitochondria under conditions of cellular stress (33). Co-IP assays revealed that DNAJA1 interacts with AMPK. As shown in Fig. 5A, AMPK may specifically precipitate DNAJA1.

Immunofluorescence detection of the localization of AMPK and DNAJA1 in U251 cells further provided a basis for the interaction between AMPK and DNAJA1. Fig. 5B shows that both AMPK and DNAJA1 were localized in the cytoplasm, providing a spatial and temporal basis to further determine the interactions between AMPK and DNAJA1.

It has previously been established (33) that DNAJA1 is involved in the negative regulation of Bax and thus participates in the anti-apoptotic process. The present study demonstrated the existence of an interaction between AMPK and DNAJA1 and therefore hypothesized that AMPK may interact with DNAJA1 to participate in the anti-apoptotic pathway of cells. As shown in Fig. 6, the expression levels of DNAJA1 were showed a slight non-significant increase and the expression level of Bax was decreased in the AMPK overexpressing cell line compared with those in the U251 cell line, while Bcl2 expression showed no significant change, indicating that the apoptotic process may be attenuated in AMPK overexpressing cells. CCCP is a mitochondrial uncoupler that disrupts mitochondrial membrane potential and depletes cellular ATP levels, thereby inhibiting AMPK activity (34). The CCCP-treated AMPK-TurboID overexpressing cell line exhibited significantly weakened DNAJA1 and Bcl2 levels but enhanced Bax levels compared with those in the AMPK overexpressing cells without CCCP treatment.