Journal: BMB Reports

Article Title: Cereblon upregulation overcomes thalidomide resistance in multiple myeloma through mitochondrial functional reprogramming

doi: 10.5483/BMBRep.2024-0130

Figure Lengend Snippet: CRBN overexpression overcomes thalidomide resistance in KMS20 cells through mitochondrial reprogramming mediated by AMPKα1. (A) Schematic diagram illustrating the combination treatment of thalidomide and Ad-CRBN to overcome drug resistance in a thalidomide-resistant xenograft model in BALB/c nude mice. Figure courtesy of Biorender.com. (B) Representative images of BALB/c nude mice bearing KMS20 cells treated with thalidomide and Ad-CRBN injections. Images were captured 7 days post-thalidomide treatment (day 18). (C-D) Measurement of tumor size and monitoring of survival in thalidomide-resistant KMS20 xenograft model in BALB/c nude mice post Ad-CRBN and/or thalidomide treatment. Ad-Vehicle, n = 5; Ad Vehicle ＋ Thalidomide, n = 4; Ad-CRBN, n = 7; Ad-CRBN ＋ thalidomide, n = 7. (E) Dose-dependent effects of thalidomide treatment on KMS20 cell viability during CRBN overexpression. Data represent the average of three independent experiments. (F) Dose-dependent effects of thalidomide, Ad-Vehicle, or Ad-CRBN on mitochondrial function parameters such as ATP (n = average of 4 independent experiments for each group), (G) ∆Ψm (Ad-Vehicle and thalidomide, n = average of 4 independent experiments; Ad-CRBN and thalidomide, n = average of 7 independent experiments), and (H) mtROS (n = average of 7 independent experiments for each group). (I) Representative western blot images demonstrating markers of mitochondrial biogenesis, p-AMPKα1, and t-AMPKα1 during combined CRBN overexpression and dose-dependent thalidomide treatment in KMS20 cells. Data are presented as mean ± SD. C, D Log-rank test. *P < 0.05, **P < 0.01, ****P < 0.0001 vs. respective groups E, F, G, H, Two-Way ANOVA with Sidak’s posthoc analysis. The numbers above graphs indicate significant P-values. ATP, adenosine triphosphate; ∆Ψm, mitochondrial membrane potential; mtROS, mitochondrial ROS; Thal, thalidomide.

Article Snippet: The membranes were blocked with 5% skim milk in 0.01M Tris-buffered saline (pH 7.5) containing 0.5% Tween 20 and incubated with the appropriate primary antibodies CRBN (HPA045910, Sigma-Aldrich); PGC-1α (sc-517380, Santa Cruz Biotechnology, Dallas, TX, USA); NRF1 (46743S, Cell Signalling, Danvers, MA, USA); ERRα (13826S, Cell Signalling); TFAM (sc-376672, Santa Cruz Biotechnology); p-AMPKα1 (2535, Cell Signalling); AMPKα1 (2795, Cell Signalling); α-Tubulin (2144, Cell Signalling).

Techniques: Over Expression, Western Blot, Membrane

Journal: BMB Reports

Article Title: Cereblon upregulation overcomes thalidomide resistance in multiple myeloma through mitochondrial functional reprogramming

doi: 10.5483/BMBRep.2024-0130

Figure Lengend Snippet: CRBN overexpression overcomes thalidomide resistance in KMS20 cells through mitochondrial reprogramming mediated by AMPKα1. (A) Schematic diagram illustrating the combination treatment of thalidomide and Ad-CRBN to overcome drug resistance in a thalidomide-resistant xenograft model in BALB/c nude mice. Figure courtesy of Biorender.com. (B) Representative images of BALB/c nude mice bearing KMS20 cells treated with thalidomide and Ad-CRBN injections. Images were captured 7 days post-thalidomide treatment (day 18). (C-D) Measurement of tumor size and monitoring of survival in thalidomide-resistant KMS20 xenograft model in BALB/c nude mice post Ad-CRBN and/or thalidomide treatment. Ad-Vehicle, n = 5; Ad Vehicle ＋ Thalidomide, n = 4; Ad-CRBN, n = 7; Ad-CRBN ＋ thalidomide, n = 7. (E) Dose-dependent effects of thalidomide treatment on KMS20 cell viability during CRBN overexpression. Data represent the average of three independent experiments. (F) Dose-dependent effects of thalidomide, Ad-Vehicle, or Ad-CRBN on mitochondrial function parameters such as ATP (n = average of 4 independent experiments for each group), (G) ∆Ψm (Ad-Vehicle and thalidomide, n = average of 4 independent experiments; Ad-CRBN and thalidomide, n = average of 7 independent experiments), and (H) mtROS (n = average of 7 independent experiments for each group). (I) Representative western blot images demonstrating markers of mitochondrial biogenesis, p-AMPKα1, and t-AMPKα1 during combined CRBN overexpression and dose-dependent thalidomide treatment in KMS20 cells. Data are presented as mean ± SD. C, D Log-rank test. *P < 0.05, **P < 0.01, ****P < 0.0001 vs. respective groups E, F, G, H, Two-Way ANOVA with Sidak’s posthoc analysis. The numbers above graphs indicate significant P-values. ATP, adenosine triphosphate; ∆Ψm, mitochondrial membrane potential; mtROS, mitochondrial ROS; Thal, thalidomide.

Article Snippet: The membranes were blocked with 5% skim milk in 0.01M Tris-buffered saline (pH 7.5) containing 0.5% Tween 20 and incubated with the appropriate primary antibodies CRBN (HPA045910, Sigma-Aldrich); PGC-1α (sc-517380, Santa Cruz Biotechnology, Dallas, TX, USA); NRF1 (46743S, Cell Signalling, Danvers, MA, USA); ERRα (13826S, Cell Signalling); TFAM (sc-376672, Santa Cruz Biotechnology); p-AMPKα1 (2535, Cell Signalling); AMPKα1 (2795, Cell Signalling); α-Tubulin (2144, Cell Signalling).

Techniques: Over Expression, Western Blot, Membrane