hek293t cells  (ATCC)

Journal: Bioactive Materials

Article Title: Bioengineered extracellular vesicles escape lysosomal degradation and deliver Tet-PKM2 for macrophage immunometabolic reprogramming and periodontitis treatment

doi: 10.1016/j.bioactmat.2026.01.002

Figure Lengend Snippet: Generation of PKM2-overexpressing cells and generation of Tet-PKM2-enriched LEVs. ( A ) Schematic illustration depicting the overexpression of PKM2 in HEK293T cells by lentiviral transduction and subsequent allosteric activation of Tet-PKM2 via TEPP-46 treatment. Cells transfected with ov-NC or ov-PKM2 were named the NC or OV, respectively. ( B ) Relative mRNA expression levels of PKM2 in the NC and OV groups (qRT‒PCR, n = 4). ( C ) Representative immunoblot bands of PKM2 in the NC and OV groups. ( D ) Semiquantitative analysis of the expression levels of PKM2 in the NC and OV groups ( n = 6). ( E ) PK activity in PKM2-overexpressing HEK293T cells in response to treatment with various concentrations of TEPP-46 (0, 10, 20, 40, and 70 μM) ( n = 6). ( F ) Representative immunoblot bands of PKM2 conformational states in PKM2-overexpressing cells after treatment with TEPP-46 (40 μM) for 0, 8, and 24 h (DSS cross-linking assay). ( G ) Semiquantitative analysis of the expression levels of Tet-PKM2 in PKM2-overexpressing cells after treatment with TEPP-46 (40 μM) for 0, 8, and 24 h ( n = 4). ( H ) Schematic illustration of the isolation of LEVs and SEVs by differential velocity centrifugation. ( I ) Representative TEM images showing the morphology of SEVs and LEVs. ( J ) Size distributions of SEVs and LEVs (NTA). ( K ) Particle counts of SEVs and LEVs (NTA). ( L ) Particle-to-protein ratios of SEVs and LEVs. ( M ) Cellular uptake of SEVs and LEVs (labeled with PKH67; green) by macrophages (immunofluorescence assay); cell skeletons were stained with phalloidin (red), and nuclei were stained with DAPI (blue). ( N ) Expression of CD63, HSP70, TSG101, calnexin, and GM130 in whole-cell lysates (Cells), SEVs, and LEVs (Western blot). ( O ) Representative immunoblot bands of PKM2 conformational states in LEVs and SEVs. ( P ) Semiquantitative analysis of the expression levels of Tet-PKM2 in LEVs and SEVs ( n = 3) ( Q ) Conformational states of PKM2 in LEVs Tet−PKM2 in response to TEPP-46 treatment. ( R ) Semiquantitative analysis of the expression levels of Tet-PKM2 in LEVs following TEPP-46 treatment ( n = 3). The data are expressed as the mean ± SEM. Statistical analysis was performed by one-way ANOVA ( E and G ) and Student's t -test ( B , D , K , L , P , and R ). ns indicates no significant difference between the indicated columns; ∗ p < 0.05, ∗∗ p < 0.01, and ∗∗∗ p < 0.001 indicate significant differences between the indicated columns.

Article Snippet: Tet-PKM2 in HEK293T cells was allosterically activated by TEPP-46 (MCE, HY-18657, USA).

Techniques: Over Expression, Transduction, Activation Assay, Transfection, Expressing, Western Blot, Activity Assay, Isolation, Centrifugation, Labeling, Immunofluorescence, Staining

Journal: Bioactive Materials

Article Title: Bioengineered extracellular vesicles escape lysosomal degradation and deliver Tet-PKM2 for macrophage immunometabolic reprogramming and periodontitis treatment

doi: 10.1016/j.bioactmat.2026.01.002

Figure Lengend Snippet: Generation of bioengineered LEVs (LEVs@TA) through in situ TA modifications. ( A ) Schematic illustration showing the interaction of TA with the phospholipid bilayer of LEVs via hydrogen bonding and the uptake of LEVs@TA by macrophages. ( B ) The percentages of CY5-TA-modified cells following incubation with gradient concentrations of CY5-TA (0, 0.1, 1, 5, and 10 μM) for 24 h (flow cytometry assay). ( C ) Colocalization of CY5-TA on HEK293T cells following incubation in 10 μM CY5-TA for 24 h (fluorescence microscopy). The nuclei were stained with DAPI (blue). ( D ) The percentages of CY5-TA-modified LEVs following incubation with gradient concentrations of CY5-TA (0, 10, 20, 50, and 100 μM) for 24 h (flow cytometry assay). ( E ) Colocalization of CY5-TA and PKH67-labeled LEVs (green) following incubation with 100 μM CY5-TA for 24 h (fluorescence microscopy). ( F ) Snapshots of CGMD simulations depicting the uptake of LEVs and LEVs@TA by macrophages at 0, 5, 10, 15, and 20 ns. ( G ) Representative in vivo fluorescence images showing good stability of DIO-labeled-LEVs@CY5-TA in vivo .

Article Snippet: Tet-PKM2 in HEK293T cells was allosterically activated by TEPP-46 (MCE, HY-18657, USA).

Techniques: In Situ, Modification, Incubation, Flow Cytometry, Fluorescence, Microscopy, Staining, Labeling, In Vivo

Journal: iScience

Article Title: Metabolic orchestration of NOD1 signaling by AMPK-mediated phosphorylation of ZDHHC5

doi: 10.1016/j.isci.2026.115245

Figure Lengend Snippet: Cellular energy stress suppresses PGN-induced NOD1 signaling (A) Mouse BMDM cells were treated with or without glucose for 6 h, then stimulated with C12-iE-DAP (1 μg/mL) for 30 min, p65 and p38 phosphorylation were analyzed by immunoblotting. (B) Mouse BMDM cells were treated with 2-DG (25 mM) in glucose-free medium for 6 h, then stimulated with C12-iE-DAP (1 μg/mL) for 30 min, p65 and p38 phosphorylation were analyzed by immunoblotting. (C) Mouse BMDM cells were treated with metformin (2 mM) for 6 h, then stimulated with C12-iE-DAP (1 μg/mL) for 30 min, p65 and p38 phosphorylation were analyzed by immunoblotting. (D) Mouse iBMDM cells were treated with or without glucose and stimulated with C12-iE-DAP (5 μg/mL) for 7 h. The IL-6 release in the medium was measured with ELISA. For each experimental group, three supernatant samples were analyzed. ∗∗ p < 0.01, NS, p > 0.05. mean ± s.d., Student’s t test. (E) Mouse iBMDM cells were treated with 2-DG (25 mM) in glucose-free medium and stimulated with C12-iE-DAP (5 μg/mL) for 7 h. The IL-6 release in the medium was measured with ELISA. For each experimental group, three supernatant samples were analyzed. ∗∗ p < 0.01, NS, p > 0.05. mean ± s.d., Student’s t test. (F) Mouse iBMDM cells were pre-treated with DMSO or Compound C (5 μM) and treated with or without glucose for 6 h, then stimulated with C12-iE-DAP (1 μg/mL) for 30 min, p65 and p38 kinase phosphorylation were analyzed by immunoblotting. (G) Mouse iBMDM cells were pre-treated with DMSO or Compound C (5 μM) and then treated with or without glucose and stimulated with C12-iE-DAP (5 μg/mL) for 7 h. The IL-6 release in the medium was measured with ELISA. For each experimental group, three supernatant samples were analyzed. ∗∗ p < 0.01, NS, p > 0.05. mean ± s.d., Student’s t test. (H) Mouse iBMDM cells were treated with MK-8722 (2 μM) for 8 h, then stimulated with C12-iE-DAP (1 μg/mL) for 30 min, p65 and p38 phosphorylation were analyzed by immunoblotting. (I) HEK293T cells were treated with or without glucose for 6 h. Representative fluorescence images show the localization of GFP-NOD1 were presented. Scale bar = 10 μm for all images. (J) HEK-293T cells expressing FLAG-NOD1 were treated with or without glucose for 6h. Total, cytosolic, and membrane fractions were immunoblotted with the indicated antibodies. (K) HEK293T cells were pre-treated with DMSO or Compound C (5 μM) and then treated with glucose starvation for 6h. Representative fluorescence images show the localization of GFP-NOD1 were presented. Scale bar = 10 μm for all images. (L) HEK-293T cells expressing FLAG-NOD1 were pre-treated with DMSO or Compound C (5 μM) and treated with or without glucose for 6h. Total, cytosolic, and membrane fractions were immunoblotted with the indicated antibodies.

Article Snippet: HEK293T , ATCC , CRL-11268.

Techniques: Phospho-proteomics, Western Blot, Enzyme-linked Immunosorbent Assay, Fluorescence, Expressing, Membrane

Journal: iScience

Article Title: Metabolic orchestration of NOD1 signaling by AMPK-mediated phosphorylation of ZDHHC5

doi: 10.1016/j.isci.2026.115245

Figure Lengend Snippet: AMPK inhibits NOD1 palmitoylation (A) HEK293T cells expressing FLAG-NOD1 were pre-treated with or without glucose and then labeled with alkyne-palmitic acid (alk-C16) for 8 h. The level of NOD1 palmitoylation was detected by click chemistry reaction, followed by FLAG agarose enrichment, and visualized by Western blots. (B) HEK293T cells expressing FLAG-NOD1 were treated with 2-DG (25 mM) in glucose-free medium and labeled with alkyne-palmitic acid (alk-C16) for 8 h. The level of NOD1 palmitoylation was detected by click chemistry reaction. (C) HEK293T cells expressing FLAG-NOD1 were treated with metformin (5 mM) and labeled with alkyne-palmitic acid (alk-C16) for 8 h. The level of NOD1 palmitoylation was detected by click chemistry reaction. (D) Mouse iBMDM cells were treated with metformin (2 mM) for 8 h. The level of NOD1 palmitoylation was detected by the ABE method. (E) HEK293T cells expressing FLAG-NOD1 were pre-treated with DMSO or Compound C (5 μM) and then treated with metformin (5 mM) and labeled with alkyne-palmitic acid (alk-C16) for 8 h. The level of NOD1 palmitoylation was detected by click chemistry reaction. (F) AMPKα WT or DKO MEF cells were treated with metformin (2 mM) for 8 h. The levels of NOD1 palmitoylation were detected by the ABE method.

Article Snippet: HEK293T , ATCC , CRL-11268.

Techniques: Expressing, Labeling, Western Blot

Journal: iScience

Article Title: Metabolic orchestration of NOD1 signaling by AMPK-mediated phosphorylation of ZDHHC5

doi: 10.1016/j.isci.2026.115245

Figure Lengend Snippet: AMPK interacts with and phosphorylates ZDHHC5 (A) Endogenous immunoprecipitation (IP) between ZDHHC5 and AMPKα was performed in HEK293T cells with AMPKα antibody. And the precipitates were blotted with the indicated antibodies. (B) Interactions between AMPKα 1/2 and ZDHHC5 were investigated by GST-pull-down assay. (C) HEK293T cells were transfected with FLAG-ZDHHC5, and immunoprecipitation was performed using FLAG M2 beads. The immunoprecipitates were then analyzed by Western blotting with a Phospho-AMPK Substrate motif antibody. (D) HEK293T cells were transfected with FLAG-ZDHHC5, and immunoprecipitation was performed using FLAG M2 beads. The immunoprecipitates were treated with λ-phosphatase (λppase) at 30°C for 30 min and analyzed by Western blotting with n phospho-AMPK substrate motif antibody. (E) HEK293T cells were transfected with FLAG-ZDHHC5 and treated with metformin (5 mM) for 8 h. Immunoprecipitation was then performed using FLAG M2 beads, and the immunoprecipitates were analyzed by Western blotting with a Phospho-AMPK Substrate motif antibody. (F) HEK293T cells were transfected with FLAG-ZDHHC5, then pre-treated with DMSO or Compound C (5 μM) and treated with metformin (5 mM) for 8 h. Immunoprecipitation was then performed using FLAG M2 beads, and the immunoprecipitates were analyzed by Western blotting with a Phospho-AMPK Substrate motif antibody. (G) AMPKα WT or DKO MEF cells were transfected with FLAG-ZDHHC5 and treated with metformin (2 mM) for 8 h. Immunoprecipitation was then performed using FLAG M2 beads, and the immunoprecipitates were analyzed by Western blotting with a Phospho-AMPK Substrate motif antibody. (H) Identification of AMPK phosphorylation sites on ZDHHC5 by mass spectrometry. (I) HEK293T cells were transfected with the indicated plasmids, and immunoprecipitation was performed using FLAG M2 beads. The immunoprecipitates were then analyzed by Western blotting with a phospho-AMPK substrate motif antibody. (J) In vitro kinase assay for ZDHHC5. Indicated GST protein was incubated with AMPK in kinase buffer supplemented with 125 μM ATP and 150 μM AMP for 30 min at 30 °C, and then analyzed with phospho-AMPK substrate motif antibody.

Article Snippet: HEK293T , ATCC , CRL-11268.

Techniques: Immunoprecipitation, Pull Down Assay, Transfection, Western Blot, Phospho-proteomics, Mass Spectrometry, In Vitro, Kinase Assay, Incubation

Journal: iScience

Article Title: Metabolic orchestration of NOD1 signaling by AMPK-mediated phosphorylation of ZDHHC5

doi: 10.1016/j.isci.2026.115245

Figure Lengend Snippet: AMPK phosphorylation inhibits ZDHHC5 plasma membrane association (A) HEK293T cells expressing FLAG-ZDHHC5 were treated with metformin (5 mM) and labeled with alkyne-palmitic acid (alk-C16) for 8 h. The level of ZDHHC5 palmitoylation was detected by click chemistry reaction. (B) Mouse iBMDM cells were treated with metformin (2 mM) for 8 h. The level of ZDHHC5 palmitoylation was detected by the ABE method. (C) HEK293T cells expressing FLAG-ZDHHC5 were treated with MK-8722 (2 μM) and labeled with alkyne-palmitic acid (alk-C16) for 8 h. The level of ZDHHC5 palmitoylation was detected by click chemistry reaction. (D) HEK293T cells expressing FLAG-ZDHHC5 were pre-treated with DMSO or Compound C (5 μM) and then treated with metformin (5 mM) and labeled with alkyne-palmitic acid (alk-C16) for 8 h. The level of ZDHHC5 palmitoylation was detected by click chemistry reaction. (E) AMPKα WT or DKO MEF cells were treated with metformin (2 mM) for 8 h. The levels of ZDHHC5 palmitoylation were detected by the ABE method. (F) HEK293T cells were treated with metformin (5 mM) or glucose starvation for 6 h. Representative fluorescence images show the localization of endogenous ZDHHC5 were presented. Scale bars = 10 μm for all images. (G) HEK293T cells were treated with metformin (5 mM) or glucose starvation for 6h. Total, cytosolic, membrane, and nuclear fractions of endogenous ZDHHC5 were immunoblotted with indicated antibodies. (H) HEK293T cells were pre-treated with DMSO or Compound C (5 μM) and then treated with metformin (5 mM) for 6h. Representative fluorescence images show the localization of endogenous ZDHHC5 were presented. Scale bars = 10 μm for all images. (I) HEK293T cells were transfected with FLAG-Golga7B and HA-ZDHHC5 and treated with metformin (5 mM) or glucose starvation for 8h. The interaction between ZDHHC5 and Golga7B was investigated by immunoprecipitation and immunoblot with the indicated antibodies. (J) HEK293T cells were transfected with the indicated plasmids and then labeled with alkyne-palmitic acid (alk-C16) for 8 h. The level of ZDHHC5 palmitoylation was detected by click chemistry reaction. (K) HEK293T cells were transfected with the indicated plasmids and treated with metformin (5 mM) and then labeled with alkyne-palmitic acid (alk-C16) for 8 h. The level of ZDHHC5 palmitoylation was detected by click chemistry reaction. (L) HEK293T cells were treated with metformin (5 mM) for 6 h. Representative fluorescence images show the localization of FLAG-ZDHHC5-WT or 2A were presented. Scale bars = 10 μm for all images. (M) HEK-293T cells expressing FLAG-ZDHHC5-WT or 2A were treated with metformin (5 mM) for 6 h. Total, cytosolic, and membrane fractions were immunoblotted with the indicated antibodies.

Article Snippet: HEK293T , ATCC , CRL-11268.

Techniques: Phospho-proteomics, Clinical Proteomics, Membrane, Expressing, Labeling, Fluorescence, Transfection, Immunoprecipitation, Western Blot

Journal: iScience

Article Title: Metabolic orchestration of NOD1 signaling by AMPK-mediated phosphorylation of ZDHHC5

doi: 10.1016/j.isci.2026.115245

Figure Lengend Snippet: AMPK-mediated ZDHHC5 phosphorylation inhibits NOD1 activation (A) ZDHHC5-knockout HEK293T cells reconstituted with ZDHHC5 wild-type (WT) or 2A mutant were transfected to express FLAG-NOD1, then treated with metformin (5 mM) and labeled with alk-C16 for 6 h. NOD1 palmitoylation was detected by click chemistry reaction. (B) ZDHHC5-knockout HEK293T cells reconstituted with ZDHHC5 wild-type (WT) or 2A mutant were treated with metformin (5 mM) for 6 h. Representative fluorescence images show the localization of GFP-NOD1 were presented. Scale bar = 10 μm for all images. (C) ZDHHC5-knockout HEK293T cells reconstituted with ZDHHC5 wild-type (WT) or 2A mutant were treated with or without glucose for 6 h. Representative fluorescence images show the localization of GFP-NOD1 were presented. Scale bar = 10 μm for all images. (D) ZDHHC5-knockout HEK293T cells reconstituted with ZDHHC5 wild-type (WT) or 2A mutant were transfected to express FLAG-NOD1, and treated with metformin (5 mM) for 6 h. Total, cytosolic, and membrane fractions were immunoblotted with the indicated antibodies. (E) BMDMs were generated from Zdhhc5 −/− mice, and were reconstituted with ZDHHC5 wild-type (WT) or 2A mutant using lentiviral transduction. The reconstituted BMDM cells were treated with metformin (2 mM) for 6 h, followed by stimulation with C12-iE-DAP (1 μg/mL) for 30 min. p65 and p38 kinase phosphorylation were analyzed by immunoblotting. (F) ZDHHC5-knockdown iBMDMs were reconstituted with ZDHHC5 wild-type (WT) or 2A mutant using lentiviral transduction. The reconstituted iBMDM cells were treated with metformin (2 mM) for 6 h, followed by stimulation with C12-iE-DAP (1 μg/mL) for 30 min, p65 and p38 kinase phosphorylation were analyzed by immunoblotting. (G) ZDHHC5-knockdown iBMDMs were reconstituted with ZDHHC5 wild-type (WT) or 2A mutant using lentiviral transduction. The reconstituted iBMDMs cells were treated with or without glucose and stimulated with C12-iE-DAP (5 μg/mL) for 7 h. The IL-6 release in the medium was measured with ELISA. For each experimental group, three supernatant samples were analyzed. ∗∗ p < 0.01, NS, p > 0.05. mean ± s.d., Student’s t test.

Article Snippet: HEK293T , ATCC , CRL-11268.

Techniques: Phospho-proteomics, Activation Assay, Knock-Out, Mutagenesis, Transfection, Labeling, Fluorescence, Membrane, Generated, Transduction, Western Blot, Knockdown, Enzyme-linked Immunosorbent Assay

Journal: iScience

Article Title: Metabolic orchestration of NOD1 signaling by AMPK-mediated phosphorylation of ZDHHC5

doi: 10.1016/j.isci.2026.115245

Figure Lengend Snippet: The AMPK-ZDHHC5 axis is inhibited by C12-iE-DAP (A) Mouse iBMDM cells were transfected with FLAG-ZDHHC5 and stimulated with C12-iE-DAP (1 μg/mL) for 30 min. Immunoprecipitation was then performed using FLAG M2 beads, and the immunoprecipitates were analyzed by Western blotting with a phospho-AMPK substrate motif antibody. (B) Mouse iBMDM cells were transfected with FLAG-ZDHHC5 and treated with metformin (2 mM) for 8 h, followed by stimulation with C12-iE-DAP (1 μg/mL) for 30 min. After stimulation, immunoprecipitation was performed using FLAG M2 beads, and the immunoprecipitates were analyzed by Western blotting with a Phospho-AMPK Substrate motif antibody. (C) Mouse iBMDM cells were stimulated with C12-iE-DAP (1 μg/mL) for 30 min. The level of ZDHHC5 palmitoylation was detected by the ABE method. (D) Mouse iBMDM cells were stimulated with C12-iE-DAP (1 μg/mL) for 1 h. Total, cytosolic, and membrane fractions were immunoblotted with the indicated antibodies. (E) Mouse iBMDM cells were treated with metformin (2 mM) for 8 h, then stimulated with C12-iE-DAP (1 μg/mL) for 30 min. The level of ZDHHC5 palmitoylation was detected by the ABE method. (F) Mouse iBMDM cells were stimulated with C12-iE-DAP (1 μg/mL) for 30 min. The level of NOD1 palmitoylation was detected by the ABE method. (G) Mouse iBMDM cells were stimulated with C12-iE-DAP (1 μg/mL) for 1 h. Total, cytosolic, and membrane fractions were immunoblotted with the indicated antibodies. (H) Mouse iBMDM cells were treated with metformin (2 mM) for 8 h, then stimulated with C12-iE-DAP (1 μg/mL) for 30 min. The level of NOD1 palmitoylation was detected by the ABE method. (I) ZDHHC5-knockout iBMDMs were reconstituted with ZDHHC5 wild-type (WT) or 2A mutant using lentiviral transduction. The reconstituted iBMDM cells were stimulated with C12-iE-DAP (1 μg/mL) for 30 min. The level of ZDHHC5 palmitoylation was detected by the ABE method. (J) ZDHHC5-knockout iBMDMs were reconstituted with ZDHHC5 wild-type (WT) or 2A mutant using lentiviral transduction. The reconstituted iBMDM cells were stimulated with C12-iE-DAP (1 μg/mL) for 30 min. The level of NOD1 palmitoylation was detected by the ABE method. (K) HEK293T cells expressing FLAG-NOD1-WT or E266K mutant were treated with or without glucose and then labeled with alk-C16 for 6 h. The NOD1 palmitoylation level was detected using a click chemistry reaction. (L) A working model illustrates the role of energy stress-induced AMPK activation in NOD1 signaling.

Article Snippet: HEK293T , ATCC , CRL-11268.

Techniques: Transfection, Immunoprecipitation, Western Blot, Membrane, Knock-Out, Mutagenesis, Transduction, Expressing, Labeling, Activation Assay