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. Author manuscript; available in PMC: 2021 Mar 13.
Published in final edited form as: Cell Rep. 2021 Feb 23;34(8):108768. doi: 10.1016/j.celrep.2021.108768

Figure 4. C1/M2 induces expression of the PGC-1α4 alternative splice variant, leading to IGF-1 upregulation.

Figure 4.

(A) Quantitative real-time PCR analysis of IGF-1 mRNA levels in C1/M2-inducible cells (doxycycline-regulated HEK293-CMVTetRTetOC1/M2 cells) treated with and without 1 μg/mL of doxycycline. Relative fold expression is shown normalized to RPL23 mRNA levels. Data are presented as the mean ± SEM (n = 3 biologic replicates, 4 technical replicates each; Student’s t test; ***p < 0.001).

(B) AlphaLISA-based analysis of secreted IGF-1 in media from C1/M2-inducible cells (HEK293-CMVTetRTetOC1/M2) with and without 1 μg/mL doxycycline treatment. Data are presented as the mean ± SEM (n = 3 biologic replicates; Student’s t test; **p = 0.003).

(C) Chromatin immunoprecipitation analysis of FLAG-C1/M2 promoter occupancy in C1/M2-inducible cells (HEK293-CMVTetRTetOC1/M2). Data are expressed as promoter occupancy in doxycycline-treated cells normalized to promoter occupancy in vehicle-treated cells. GAPDH and NR4A2 promoters were used as negative and positive controls, respectively. Data are presented as the mean ± SEM (n = 3 biologic replicates; Student’s t test; ***p < 0.001, **p < 0.01).

(D) Luciferase assay measuring activation of the distal and proximal PGC-1α promoters in C1/M2-inducible cells (HEK293-CMVTetRTetOC1/M2) with and without 1 μg/mL doxycycline treatment. Data are presented as the mean ± SEM (n = 3 biologic replicates, 4 technical replicates each; Student’s t test; ***p < 0.001).

(E) Quantitative real-time PCR analysis of PGC-1α4 mRNA expression in HMC3A cells with and without shRNA-mediated C1/M2 knockdown. Relative fold expression is shown normalized to RPL23 mRNA levels and to the mock transduced condition. shNS, non-specific shRNA; shMAML2_#1 and shMAML2_#3, shRNAs targeting C1/M2 and MAML2. Data are presented as the mean ± SEM (n = 3 biologic replicates, 4 technical replicates each; Student’s t test; **p < 0.01).

(F) Temporal analysis of C1/M2, PGC-1α4, and IGF-1 expression kinetics in C1/M2-inducible cells (HEK293-CMVTetRTetOC1/M2) treated with 1 μg/mL of doxycycline for 0–48 h. C1/M2 and PGC-1α4 expression is indicated on the left y axis, whereas IGF-1 expression is indicated on the right y axis. Relative fold expression is shown normalized to RPL23 mRNA levels. Data are presented as the mean ± SEM (n = 3 biologic replicates; Student’s t test; ***p < 0.001).

(G) Quantitative real-time PCR analysis of PGC-1α4 expression in C1/M2-positive and C1/M2-negative cell lines. Cell lines are listed along the x axis, with C1/M2 status indicated below in gray (negative) or red (positive). Relative fold expression is shown normalized to RPL23 mRNA levels. Data are presented as the mean ± SEM (n = 3 biologic replicates, 4 technical replicates each).

(H and I) Quantitative real-time PCR analysis of IGF-1 mRNA levels in HMC3A cells transduced with unique gRNAs targeting the PGC-1α1 isoform only (PGC-1α1 sgRNA) or all PGC-1α isoforms (Pan-PGC-1α sgRNA) (H). Quantitative real-time PCR analysis of PGC-1α4 and IGF-1 mRNA expression in PGC-1α4-inducible cells (doxycycline-regulated HEK293-PGKTetOn3GTRE3GSPGC−1α4 cells) treated with 1 μg/mL of doxycycline for 0–48 h (I). Relative fold expression is shown normalized to RPL23 mRNA levels. Data are presented as the mean ± SEM (n = 3 biologic replicates, 4 technical replicates each; Student’s t test; **p < 0.01, ***p < 0.001, ****p < 0.0001).

See also Figure S5.