Fig 1: UNC5A and UNC5C induce caspase-3 activation through DAPK1/PR65b during UPR. (A) Quantification of netrin-1–receptor expression by qRT-PCR (mean + SEM, n = 3). (B–F) Identification of the implicated netrin-1 receptors. HepaRG cells were transfected with siRNAs and treated with DTT or not for 4 hours (mock). (B) Assessment of XBP1 mRNA splicing by RT-PCR. Representative result, n = 3. (C) Assessment of netrin-1 protein knockdown by immunoblotting. Representative result, n = 3. (D and E) Assessment of transcript knockdown efficiencies. Graphs indicate (D) UNC5A and (E) UNC5C mRNA levels in siRNA-treated cells in comparison with control siRNA-treated cells (mean + SEM; n = 3; Mann–Whitney test; P < .05). (F) Caspase-3 activation is reversed by UNC5A or UNC5C knockdown after UPR induction. Graph indicates caspase-3 activity ratio of DTT vs untreated cells for each condition (mean + SEM; n = 3; Mann–Whitney test; P < .05). (G–J) Identification of the downstream signaling pathway. HepaRG cells were transfected with siRNAs and treated with DTT or not for 4 hours (mock). (G) Assessment of XBP1 mRNA splicing by RT-PCR. Representative result, n = 3. (H) Evaluation of netrin-1, DAPK1, and PR65ß depletion by immunoblotting. Representative result, n = 3. (I) Caspase-3 activation is reversed by DAPK1 or PR65ß knockdown. Graph indicates the caspase-3 activity ratio for each condition (mean + SEM; n = 3; Mann–Whitney test; P < .05). (J) PP2A activity is increased by netrin-1 depletion and reversed by reduced expression of PR65ß. Graph indicates PP2A activity ratio for each condition (mean + SEM; n = 3; Mann–Whitney test; P < .05). *,**, or *** refer to statistical analyses.
Fig 2: eIF3a upregulates the expression of PPP2R1B by promoting its translation. (A) A375 cells were transfected with a non-targeting siRNA or an eIF3a siRNA, and levels of the eIF3a and PPP2R1B mRNAs were measured using qRT-PCR. (B) HEK293T cells were transfected with a control plasmid or a Flag-eIF3a plasmid, and the expression of the eIF3a and PPP2R1B mRNAs was measured using qRT-PCR. (C) A375 or SK-28 cells were transfected with a non-targeting siRNA or an eIF3a siRNA, followed by treatment with puromycin for 30 min. Puromycin incorporation was measured using western blotting. (D) RIP assay indicating that the PPP2R1B mRNA bound to the eIF3a protein. (E) Luciferase assay showing significantly lower activity of the PPP2R1B reporter construct after eIF3a knockdown in HEK293T cells. (F) A375 cells were transfected with a non-targeting siRNA or an eIF3a siRNA, followed by treatment with 20 µM HCQ for 24 h. The levels of eIF3a, PPP2R1B and LC3 were measured using western blotting. (G) A375 cells were transfected with a nontargeting siRNA or an eIF3a siRNA, followed by treatment with 20 µM MG132 for 4 h. The levels of eIF3a and PPP2R1B were measured using western blotting.
Fig 3: eIF3a suppresses ERK activity by upregulating the expression of PPP2R1B. (A) Differential expression of three protein phosphatases was observed after silencing eIF3a using mass spectrometry. (B) A375 and SK-28 cells were transfected with a non-targeting siRNA or an eIF3a siRNA, and the levels of eIF3a, PPP2R1B, p-ERK and ERK were measured using western blotting. (C) A375 and SK-28 cells were treated with the indicated concentrations of the eIF3a inhibitor L-mimosine, and the levels of eIF3a, PPP2R1B, p-ERK and ERK were measured using western blotting. (D) HEK293T cells were transfected with a control plasmid or a Flag-eIF3a plasmid, and the levels of Flag, eIF3a, PPP2R1B, p-ERK and ERK were measured using western blotting. (E) A375 cells were transfected with the indicated concentrations of a non-targeting siRNA or an eIF3a siRNA, and the levels of eIF3a, PPP2R1B and p-ERK were measured using western blotting. (F) A375 and SK-28 cells were transfected with a non-targeting siRNA or PPP2R1B siRNA, and the levels of eIF3a, PPP2R1B, p-ERK and ERK were measured using western blotting. (G) A375 and A375R cells were transfected with a control plasmid or a HA-PPP2R1B plasmid, and the levels of PPP2R1B, p-ERK and ERK were measured using western blotting. (H) SK-28 or HEK293T cells were transfected with a non-targeting siRNA or an eIF3a siRNA, followed by transfection with a HA-PPP2R1B plasmid. The levels of eIF3a, HA, PPP2R1B, p-ERK and ERK were measured using western blotting.
Fig 4: Model showing the role of the eIF3a-PPP2R1B-ERK axis in regulating BRAF inhibitor resistance. In normal BRAFV600E mutant cells, the BRAF inhibitor suppresses ERK activity and cell proliferation. When eIF3a is depleted, PPP2R1B translation is inhibited, resulting in the persistence of p-ERK and the subsequent occurrence of drug resistance.
Fig 5: The association of eIF3a and PPP2R1B is validated in samples from patients with melanoma. (A) IHC analyses of eIF3a and PPP2R1B levels in melanoma tissues from the HPA database. (B) Pearson’s correlation analyses of eIF3a and PPP2R1B mRNA levels in melanoma (data from TCGA). (C) Pearson’s correlation analyses of eIF3a and PPP2R1B mRNA levels in lung cancer. (D) Pearson’s correlation analyses of eIF3a and PPP2R1B mRNA levels in breast cancer. (E) Pearson’s correlation analyses of the eIF3a and PPP2R1B mRNA levels in primary, metastatic, and uveal melanoma. TPM represents transcripts per million.
Supplier Page from Abcam for Anti-PPP2R1B antibody [EPR10158]