Journal: bioRxiv

Article Title: Translational control of AMPK activity in melanoma

doi: 10.64898/2025.12.30.697000

Figure Lengend Snippet: (A) Western blot analysis of A375 and MelJuso (NRAS-mutant) melanoma cells treated with increasing concentrations of the eIF4A inhibitor Rocaglamide A (RocA) for 20 h. Both cell lines exhibited dose-dependent increases in phosphorylated AMPK, accompanied by decreased levels of LKB1 and its cofactor MO25. (B) Western blot analysis of G361 (BRAF V600E -mutant, LKB1 -null) melanoma cells treated with increasing concentrations of RocA for 20 h. AMPK activation occurred in a dose-dependent manner despite the absence of functional LKB1, confirming LKB1-independent AMPK activation. (C) Western blot analysis of A375, MelJuso, and G361 cells treated with increasing concentrations of the eIF4E-eIF4G disruptor 4E1RCat for 20 h. All three cell lines exhibited increased AMPK activity regardless of LKB1 status or driver mutation. (D) Western blot analysis of A375 and MelJuso cells transfected with two different LKB1-targeting siRNAs for 48 h, combined with 100 nM RocA treatment for the last 20 h. LKB1 knockdown did not prevent RocA-induced AMPK activation, confirming the LKB1-independent mechanism. Non-targeting control siRNA (si-NT) was used as a control. (D) Western blot analysis of A375, G361, and MelJuso cells transfected with CaMKKβ-targeting siRNAs for 48 h, combined with 100 nM RocA treatment for the last 20 h. CaMKKβ depletion did not impair RocA-induced AMPK activation, indicating that CaMKKβ is not required for AMPK activation by eIF4F inhibition. Non-targeting control siRNAs (si-NT) were used as a control. (E) Western blot analysis of A375 and MelJuso cells transfected with MLK3-targeting siRNAs for 48 h, combined with 100 nM RocA treatment for the last 20 h. MLK3 protein levels decreased following both RocA treatment and siRNA-mediated knockdown, but MLK3 depletion did not prevent RocA-induced AMPK activation. Non-targeting siRNAs (si-NT) were used as a control. Vinculin, GAPDH, or total AMPK levels served as loading controls. The control samples (CTRL) were treated with an equivalent volume of the vehicle (DMSO). The upper index letters refer to the corresponding loading control detected on the same membrane.

Article Snippet: Small-molecule inhibitor stocks of Rocaglamide A, 4E1RCat, CR-1-31-B, Rotenone (MedChemExpress), PD184352, YM201636 (Selleckchem) and Okadaic acid (Merck) were prepared in dimethylsulfoxide (DMSO, Merck).

Techniques: Western Blot, Mutagenesis, Activation Assay, Functional Assay, Activity Assay, Transfection, Knockdown, Control, Inhibition, Membrane

Journal: Science Advances

Article Title: Identification of an anaplastic subtype of prostate cancer amenable to therapies targeting SP1 or translation elongation

doi: 10.1126/sciadv.adm7098

Figure Lengend Snippet: ( A ) Cell viability assay in indicated cells treated with 4E1RCat (top) or HHT (bottom). Data represent means ± SD from a representative experiment with four technical repeats and the experiment was replicated three times with similar results. ( B to D ) Targeting translation by 4E1RCat or HHT inhibits CRPC migration (B), sphere formation (C), and total protein synthesis (D) in PC3 cells in vitro. ( E to G ) Inhibitory effects of HHT on the growth of PC3 xenografts (E and F) and a syngeneic murine RM1 model (G) in vivo. In (E) and (G), shown are the tumor growth curves (left; insets present tumor randomizations), endpoint tumor images (middle), and tumor weight (right) of indicated models treated with vehicle or HHT. In (F), shown are the effects of HHT treatment on body weight change of immunodeficient mice in the PC3 model. n.s., not significant. ( H ) HHT inhibits TRAMP (the transgenic adenocarcinoma of the mouse prostate) PCa in vivo. Shown is a schematic of in vivo HHT treatment (top) and endpoint images and weight of GU and prostate tissues (bottom) in TRAMP mice treated with vehicle or HHT. ( I and J ) Clinical effect of HHT on patients with CRPC. Three patients who failed prior ADT were enrolled and, except for patient 1 (I), both patients 2 (J) and 3 (fig. S7G) received radical prostatectomy with bone metastasis detected afterward. For pain assessment, the Brief Pain Inventory–Short Form (BPI-SF) is used, in which the “least,” “worst,” and “average” describe the “pain as its worst,” “pain as its least,” and “pain on average” in the past 24 hours. BPI relief percentage denotes how much relief a patient has received after treatment in the past 24 hours. The red arrow marks the pelvic lymph node areas in (J). All P values were determined by a two-tailed unpaired Student’s t test. ** P < 0.01 and *** P < 0.001.

Article Snippet: For all in vitro experiments, HHT (TopScience, catalog no. T3380), 4E1RCat (TopScience, catalog no. T1742), and plicamycin (or Mithramycin A; MCE, catalog no. HY-A0122) were first dissolved in PBS or dimethyl sulfoxide (DMSO) as instructed.

Techniques: Viability Assay, Migration, In Vitro, In Vivo, Transgenic Assay, Two Tailed Test