Ferroptosis

C, Jurkat cells with SB202190 at 1, 5, and 10 μM were tested, and a decreased SRP72 expression was found when using at 10 μM (lanes 8 and 9). D, results were analyzed and RUA illustrated, finding significant results at 10 μM at 240 versus 0 and 120 versus 0 min (p<0.05).

Total and phosphorylated Akt were detected by western blot in protein lysates of liver, inguinal WAT and gastrocnemius muscle from mice that were treated with an insulin bolus after an overnight fast.

Primary hepatocytes were treated with or without 20μM Wy14643/Pioglitazone/Rosiglitazone for 24h followed by stimulation with or without 1μg/ml recombinant human FGF21 for 15min. FGF21 signaling was analyzed by phosphorylation of ERK1/2 and FRS2 examination.

(D) Indicated NRF2 knockdown HCC cells were treated with erastin (10 μM) and sorafenib (5 μM) with or without indicated inhibitors (ferrostatin-1, 1 μM; liproxstatin-1, 100 nM; ZVAD-FMK, 10 μM; necrostatin-1, 10 μM; necrosulfonamide, 0.5 μM) for 24 hours, and cell viability was assayed (n 5 3, *P < 0.05). Abbreviation: GAPDH, glyceraldehyde 3-phosphate dehydrogenase.

(C) Indicated macrophages were treated with LPS (100 ng/mL) in the absence or presence of GKT137831 (1 μmol/L) for 24 h. PKM2 mRNA and lactate levels were assayed (n = 3, *, p < 0.05 versus LPS group).

Inhibition of breast cancer cell growth using sorafenib. MCF-7 breast cancer cells were treated with increasing concentrations of sorafenib for 5 days. Cell number was measured  using a colorimetric growth assay (crystal violet stain) and expressed relative to DMSO treated control cells.

Cell viabilities with increasing concentrations of cisplatin (CP) and doxorubicin (DOXO) under normoxic and hypoxic condition for 48 hours were determined by MTT assay. IC50 values are presented as the means ?SDs (n=4) and * denotes p<0.05.

Aberrantly activated PI3K/AKT pathway mediates lapatinib resistance in SK-BR-3-LR cells. (A and B) After drug treatment, phosphorylation of HER2, EGFR, AKT, and ERK1/2 was determined by Western blotting using specific antibodies.

Statin-Related Inhibition of Dehydroepiandrosterone Sulfate (DHEAS) Uptake by SLCO2B1 in Prostate Cancer (PC) Cells. B, Uptake of DHEAS in PC cells with 2.5 µM DHEAS and different concentrations of statins when incubated for 60 minutes. Statistical analysis was performed by comparing each condition with the DHEAS 2.5 µM and no statin state except when indicated. C, Uptake of DHEAS in PC cells before (scrambled short hairpin RNA) and after (short hairpin RNA 2B1) SLCO2B1 is knocked down when incubated with 2.5 μM DHEAS and 100 μM atorvastatin for 10 and 60 minutes. Statistical analysis was performed by comparing each condition with scrambled short hairpin RNA after 10 minutes with DHEAS except when indicated. P = .02 for the comparison between scrambled short hairpin RNA with 10 vs 60 minutes of DHEAS incubation for LNCaP and .01 for 22RV1. Other P values are indicated in the figure. Bars indicate means and error bars indicate standard deviation.

U2OS cells were cultured with FAC for 24 hours, washed, followed by chelation with 2 chelators (DFO, DFX) -/+ lysosomal protease inhibitors (E-64d and PepstatinA) and analyzed as in panel A. DFX: Deferasirox.

Western blot analysis indicated protein expression in PDAC cells following treatment with erastin (2.5-40 μmol/L) for 24 hours (n=3, * P < 0.05 vs. untreated group)

(D) Representative western blot and (E) densitometry showing protein levels of HO-1 in HO-1+/+ PTCs treated with RSL3 over time. Data shown represent the Mean ± SEM of three independent experiments performed each time in triplicate; * = p<0.05, ** = p<0.01 compared to lower concentration at each time point.

C, Jurkat cells with SB202190 at 1, 5, and 10 μM were tested, and a decreased SRP72 expression was found when using at 10 μM (lanes 8 and 9). D, results were analyzed and RUA illustrated, finding significant results at 10 μM at 240 versus 0 and 120 versus 0 min (p<0.05).

Total and phosphorylated Akt were detected by western blot in protein lysates of liver, inguinal WAT and gastrocnemius muscle from mice that were treated with an insulin bolus after an overnight fast.

Primary hepatocytes were treated with or without 20μM Wy14643/Pioglitazone/Rosiglitazone for 24h followed by stimulation with or without 1μg/ml recombinant human FGF21 for 15min. FGF21 signaling was analyzed by phosphorylation of ERK1/2 and FRS2 examination.

(D) Indicated NRF2 knockdown HCC cells were treated with erastin (10 μM) and sorafenib (5 μM) with or without indicated inhibitors (ferrostatin-1, 1 μM; liproxstatin-1, 100 nM; ZVAD-FMK, 10 μM; necrostatin-1, 10 μM; necrosulfonamide, 0.5 μM) for 24 hours, and cell viability was assayed (n 5 3, *P < 0.05). Abbreviation: GAPDH, glyceraldehyde 3-phosphate dehydrogenase.

(C) Indicated macrophages were treated with LPS (100 ng/mL) in the absence or presence of GKT137831 (1 μmol/L) for 24 h. PKM2 mRNA and lactate levels were assayed (n = 3, *, p < 0.05 versus LPS group).

Inhibition of breast cancer cell growth using sorafenib. MCF-7 breast cancer cells were treated with increasing concentrations of sorafenib for 5 days. Cell number was measured  using a colorimetric growth assay (crystal violet stain) and expressed relative to DMSO treated control cells.

Cell viabilities with increasing concentrations of cisplatin (CP) and doxorubicin (DOXO) under normoxic and hypoxic condition for 48 hours were determined by MTT assay. IC50 values are presented as the means ?SDs (n=4) and * denotes p<0.05.

Aberrantly activated PI3K/AKT pathway mediates lapatinib resistance in SK-BR-3-LR cells. (A and B) After drug treatment, phosphorylation of HER2, EGFR, AKT, and ERK1/2 was determined by Western blotting using specific antibodies.

Statin-Related Inhibition of Dehydroepiandrosterone Sulfate (DHEAS) Uptake by SLCO2B1 in Prostate Cancer (PC) Cells. B, Uptake of DHEAS in PC cells with 2.5 µM DHEAS and different concentrations of statins when incubated for 60 minutes. Statistical analysis was performed by comparing each condition with the DHEAS 2.5 µM and no statin state except when indicated. C, Uptake of DHEAS in PC cells before (scrambled short hairpin RNA) and after (short hairpin RNA 2B1) SLCO2B1 is knocked down when incubated with 2.5 μM DHEAS and 100 μM atorvastatin for 10 and 60 minutes. Statistical analysis was performed by comparing each condition with scrambled short hairpin RNA after 10 minutes with DHEAS except when indicated. P = .02 for the comparison between scrambled short hairpin RNA with 10 vs 60 minutes of DHEAS incubation for LNCaP and .01 for 22RV1. Other P values are indicated in the figure. Bars indicate means and error bars indicate standard deviation.

U2OS cells were cultured with FAC for 24 hours, washed, followed by chelation with 2 chelators (DFO, DFX) -/+ lysosomal protease inhibitors (E-64d and PepstatinA) and analyzed as in panel A. DFX: Deferasirox.

Western blot analysis indicated protein expression in PDAC cells following treatment with erastin (2.5-40 μmol/L) for 24 hours (n=3, * P < 0.05 vs. untreated group)

(D) Representative western blot and (E) densitometry showing protein levels of HO-1 in HO-1+/+ PTCs treated with RSL3 over time. Data shown represent the Mean ± SEM of three independent experiments performed each time in triplicate; * = p<0.05, ** = p<0.01 compared to lower concentration at each time point.