Low-dose AMPK activity and cellular energy ratio
Cell-based study · Human HCT-116 colon cancer cells [1]
Berberine increased AMPK activity in total-cell and isolated lysosomal fractions without a detected increase in the cellular AMP/ATP ratio under these conditions.
Literature-Reported Treatment Conditions
- Treatment concentration
- 0.1, 0.5 and 2.5 μM
- Exposure duration
- 20 h
- Method
- Cell-culture exposure; assay-specific genetic perturbations where indicated.
- Vehicle
- DMSO; final percentage not reported in the selected methods.
- Co-treatment
- Metformin (250 μM) was a separate comparator, not co-treatment. DMSO was the vehicle control.
- Reported limits
- Final DMSO percentage and per-endpoint replicate allocation are not specified in the selected text. Fraction-purity validation was not established by the reviewed figure.
Methods and source locations
Compound reported in the paper: Berberine chloride; Compound label in the paper: BBR (MedChemExpress HY-18258)
Literature experiments using berberine chloride in HCT-116 cells; not Adooq product validation.
Results: BBR activated lysosomal AMPK moderately at low doses; Figure 1A–C; Methods: Reagents and kits, Lysosome isolation, Determination of intracellular AMP and ATP, AMPK activity assay.
AMPK activity in total-cell lysates: ELISA of substrate phosphorylation in lysates; inhibitor-control signal subtracted; vehicle normalized to 1. — Results first subsection; Figure 1B
AMPK activity in isolated lysosomal fractions: Same lysate AMPK activity assay in isolated lysosomal fractions. — Results first subsection; Figure 1B
Cellular AMP/ATP ratio: Bioluminescence assays for AMP and ATP. — Results first subsection; Figure 1C
AXIN1 and PEN2 perturbation of AMPK signaling
Cell-based study · Human HCT-116 colon cancer cells [1]
AXIN1 knockdown or knockout diminished the AMPK response to berberine. PEN2 knockdown did not suppress the berberine-induced phosphorylation response, although it reduced the response to metformin.
Literature-Reported Treatment Conditions
- Treatment concentration
- 2.5 μM
- Exposure duration
- 20 h after genetic perturbation
- Method
- Cell-culture exposure; assay-specific genetic perturbations where indicated.
- Vehicle
- DMSO; final percentage not reported in the selected methods.
- Co-treatment
- Separate metformin comparator (250 μM). Controls: non-sense control siRNA or wild-type cells, with matched DMSO treatment.
- Reported limits
- Final DMSO percentage and exact numerical effect sizes are not given in the selected text; bar heights were not estimated.
Methods and source locations
Compound reported in the paper: Berberine chloride; Compound label in the paper: BBR (MedChemExpress HY-18258)
Literature experiments using berberine chloride in HCT-116 cells; not Adooq product validation.
Results: AXIN1 but not PEN2 was crucial for BBR-induced AMPK activation; Figure 2A–E; Methods: Cell culture, transfection and drug treatment; RNA interference; Western blot; AMPK activity assay.
AMPKα, Raptor and ULK1 phosphorylation after PEN2 knockdown: Western blot with non-sense siRNA and vehicle controls. — Figure 2A and corresponding Results
AMPK signaling and activity after AXIN1 knockdown or knockout: Western blot and lysate AMPK activity ELISA; non-sense siRNA and wild-type comparators. — Figure 2B–E and corresponding Results
UHRF1 abundance and AMPK response
Cell-based study · Human HCT-116 colon cancer cells [1]
Berberine reduced UHRF1 protein abundance, and UHRF1 overexpression attenuated the AMPK response. Short exposure also reduced the co-immunoprecipitation of AMPKα1 with UHRF1. These cellular observations support a role for UHRF1 regulation without establishing direct binding in this experiment.
Literature-Reported Treatment Conditions
- Treatment concentration
- 0.1, 0.5 and 2.5 μM for the abundance series; 2.5 μM for overexpression and interaction experiments
- Exposure duration
- 20 h for abundance and overexpression experiments; 1 h for co-immunoprecipitation
- Method
- Cell-culture exposure; assay-specific genetic perturbations where indicated.
- Vehicle
- DMSO; final percentage not reported in the selected methods.
- Co-treatment
- UHRF1 or PPP2CA overexpression with corresponding empty-vector controls. Metformin, MK-8722 and AICAR were separate comparator treatments in the abundance experiments.
- Reported limits
- Final DMSO percentage; Figure 5 replicate count and error-bar definition are not stated in its legend. Exact effect sizes are not inferred from graphs.
Methods and source locations
Compound reported in the paper: Berberine chloride; Compound label in the paper: BBR (MedChemExpress HY-18258)
Literature experiments using berberine chloride in HCT-116 cells; not Adooq product validation.
Results: BBR was an AMPK activator reducing UHRF1 expression; Overexpression of UHRF1 abolished BBR’s effect on AMPK activation; Figures 3A–C and 5A–D; Methods: Reagents and kits, Cell culture, transfection and drug treatment, Western blot, AMPK activity assay, IP.
UHRF1 protein abundance: Western blot; β-actin normalization; DMSO control. — Figure 3A–C and corresponding Results
AMPK phosphorylation and activity after UHRF1 overexpression: Western blot and lysate AMPK activity ELISA with empty-vector controls. — Figure 5A–B and corresponding Results
AMPK pathway phosphorylation after PPP2CA overexpression: Western blot with empty-vector controls. — Figure 5C and corresponding Results
AMPKα1 recovered with FLAG-UHRF1: Co-expression and anti-FLAG immunoprecipitation of tagged proteins; corresponding empty-vector and vehicle controls. — Figure 5D; Methods: IP; corresponding Results
