Figure 5

Identification of Flcn as an AMPK-regulated gene and TFEB as a transcription factor that mediates this response. A) Relative Flcn mRNA levels were assessed with a Biomark gene expression 192.24 IFC δ gene assay. AMPKα1α2^+/+ or AMPKα1α2^−/− MEFs were treated with vehicle (DMSO), 10 µM 991 or 2 mM AICAR, for 0, 0.5, 1, 2, 4, 8, 12, and 24 h. Two-way ANOVA with interaction was fit to log-transformed data. *P < 0.05, **P < 0.01, ***P < 0.001. Each data point represents the mean ± sem (n = 12). B) AMPKα1α2^+/+ or AMPKα1α2^−/− MEFs were treated with 10 µM 991 or 2 mM AICAR for the indicated time points. FLCN was immunoprecipitated (IP) from cell lysates and subjected to Western blot analysis with the FLCN antibody. A representative blot of 3 independent experiments is shown. C) Schematic representation of Flcn-NanoLuc luciferase reporter plasmid and the correspondent luciferase activity profile. Different lengths of Flcn’s promoter region (−8000, −1200, and −100 bp) were inserted upstream of the NanoLuc luciferase reporter gene. The constructs, along with a firefly luciferase plasmid, which served as internal control, were transiently transfected into AMPKα1α2^+/+ or AMPKα1α2^−/− MEFs. Twelve hours post-transfection, cells were treated with vehicle (DMSO) or 30 µM 991 for 12 h, then harvested and luciferase assay was performed. Values were first normalized by transfection efficiency, and then represented as log₂ fold-change ±sd, relative to control (vehicle-treated cells) (n = 3). The dotted lines indicate the log₂ fold-change threshold of ±0.37. Data were analyzed by 2-way ANOVA with the factors of genetic background and promoter length, plus the interaction between the 2 factors. Significance of the genetic background is indicated. ***P < 0.001, **P < 0.01. TSS, transcription start site. D) AMPKα1α2^+/+ or AMPKα1α2^−/− MEFs were transiently transfected with Flcn-NanoLuc luciferase (−1200 or −100 bp) either control or with a mutation on TFEB-binding site (position −40 bp from the TSS), together with a firefly luciferase plasmid for control, and treated as above. Values were first normalized by transfection efficiency, and then represented as log₂ fold-change ± sd, relative to control (vehicle-treated cells) (n = 3). Data were analyzed by a 2-way ANOVA with the factors of promoter length and TFEB-binding site status (control or TFEB-mutated), plus the interaction between these 2 factors. Significance of the TFEB-binding site status is indicated. **P < 0.01. E) TFEB/TFE3 control (TFEB/3^+/+) and double KO (TFEB/3^−/−) MEFs were transiently transfected with Flcn-NanoLuc luciferase (−1200 or −200 bp) control or with a mutation on TFEB-binding site (position −40 bp), together with a firefly luciferase plasmid. Cells were then treated with vehicle (DMSO) or 30 µM 991. Values were normalized and represented as described in D (n = 4). Data were analyzed by 2-way ANOVA with the factors of genetic background, TFEB-binding site status (control or mutated) and interaction. Significance of the genetic background is indicated, ****P < 0.0001. F) mRNA level of Flcn in TFEB/3^+/+ or TFEB/3^−/− MEFs treated with 10 µM 991 or 2 mM AICAR for 4, 12, and 24 h. Data are presented as a box-and-whisker plots (minimum to maximum) of values normalized to control (vehicle-treated cells, 4 h). Data were analyzed by a 2-way ANOVA with the factors of time and treatment, plus the interaction between these 2 factors (n = 9). Significance of the treatment factor is indicated. ****P < 0.0001. G) TFEB/3^+/+ and TFEB/3^−/− MEFs were lysed after 0, 24, and 36 h of treatment with 10 µM 991 or 2 mM AICAR. Cell lysates (20 μg) were subjected to Western blot analysis using the indicated antibodies. FLCN was IP from 500 μg of cell lysate with 1 μg of anti-FLCN antibody. Images are representative of n = 2.