P450 (e.g. CYP17)

A. ZR75-1 (AR+, ER+) and B. MDA MB 231 (AR-, TNBC) cells were treated with either abiraterone or vehicle, then used as targets in a CTL assay using CEA-specific CD8+ T cells as effector cells at an E:T ratio of 30:1. Error bars indicate mean ± S.E.M. for quadruplicate measurements. Statistical analyses were done by Student's t-test, * = P < 0.01 vs. vehicle control. Data are representative of 2 independent experiments.

(A) KMS11 and (B) L363 cells were plated in 5mM glucose medium with ritonavir or DMSO (D) for 17 hours. Glucose consumption rates are normalized to untreated cells (not shown). (C) KMS11 and (D) L363 cells were treated with ritonavir or DMSO for 72 hours. Relative viable cell numbers were determined by MTS assay and normalized to untreated cells (not shown).

Concomitant Western blot analysis of ER and TFF1 assessed after 16 h treatment with E2 or abiraterone in the presence or absence of MG-132 (10 μM).

Comparative mean±SD plasma concentration versus time curves of 40mg/kg oral posaconazole (PSZ) administrated in normolipidemic (NL), intermediate hyperlipidemic (IHL) or extreme hyperlipidemic (HL) rat groups. An insert showing a clear snap of the plasma concentrations for the first 7 h.

Voriconazole increases the osteogenic activity of human osteoblasts in vitro. Osteoblasts were exposed to voriconazole (VCZ) or fluconazole (FCZ) at 15 or 200 μg/ml in osteoinductive medium (OS+) for up to 7 days. As a positive control, cells were also exposed to sodium fluoride (NaF) at 10 μM in nonosteoinductive medium (OS−). Osteoblastic differentiation was evaluated by measuring alkaline phosphatase (ALP) activity (A) and staining for calcium deposition with alazarin red S stain (B and C); calcium deposition was quantified by measuring the absorbance (A415) of extracted alazarin red S (B) and was assessed by the light microscopic appearance (white arrows) of stained osteoblast cultures (C). Values represent the means ± SD. *, P < 0.05, compared to osteoblasts grown under osteogenic conditions without treatment.

a, b, Melanoma-bearing mice were treated with avasimibe (Ava) or DMSO control (5 times) (control, n=9; avasimibe, n=8). c-e, STORM analysis of TCR clustering of tumour-infiltrating CD8+ T cells. c, Representative images. d, Ripley’s K-function analysis of TCR molecules. e, The r value at the maximal L(r)-r value of Ripley’s K-function curves (control, n=100; avasimibe, n=85). f, g, A combined therapy (avasimibe and anti-PD-1) or monotherapies (avasimibe or anti-PD-1) in treating melanoma (n=10). Avasimibe, 5 times; anti-PD-1, 4 times. h, i, Cytokine/granule productions of PD-1hi and PD-1lo tumour-infiltrating CD8+ T cells (control, n=12; avasimibe, n=11).

MCF-7 and SKBR-3 cells were treated as indicated and morphology of autophagosomes was observed with TEM (C). Autophagosomes (Avi, immature autophagic vacuoles, bold arrow), autolysosomes (Avd, degraded/late autophagic vacuoles, sharp arrow).

C. Impact of CYP17A1 inhibitors on genes involved in androgen biosynthesis. H295R cells were treated with control (DMSO) or 1 μM orteronel and galeterone for 72 h and then RNA was isolated and transcribed to cDNA. Results of qRT-PCR validation of HSD3B2, CYP17A1 and AKR1C3 genes relative to the housekeeping gene cyclophilin A. Expression of the genes was analyzed by SYBR Green real-time PCR. Analysis of relative gene expression was determined by the 2−ΔΔCt method. *p < 0.05.

Effects of rifampin alone or in combination with cobicistat on the mean DRV CLint.app. in primary human hepatocytes in vitro. Cryopreserved primary human hepatocytes were incubated with RIF (0.5 to 20 μM) (hatched bars) or with COBI (0.13 to 12.76 μM) and RIF (0.5 to 20 μM) (gray bars) for 72 h. All cells were then incubated with RIF (0.5 to 20 M) or RIF (0.5 to 20 μM) together with COBI (0.13 to 12.76 μM), as described above, together with DRV (5 μM) for 60 min. Control cells were treated with DRV (5 μM) alone for 60 min (black bar). The results shown represent the mean DRV CLint.app. from three biological replicates measured in hepatocytes from three independent donors (donors HU1399, HU1574, and HU1587). Error bars indicate SDs.

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.

SKH-1 mice were subjected to UVB radiation (1000 J/m2 daily, 5 days), or topical apigenin (Api, 5 μmol, in 200 μl vehicle, DMSO: acetone 1:9) was applied 1 h prior to each UVB exposure. Mice were also sham-irradiated and treated with apigenin, and the control group of mice was subjected to sham irradiation and vehicle. 24 h after the final UVB exposure, the mice were euthanized, dorsal skin was harvested, fixed in formalin and paraffin-embedded. (A) Representative Hematoxylin and Eosin (H&E) staining of skin sections (scale bar, 100 μm). (B) Quantitation of epidermal thickness (mean ± SD), *, P < 0.001; **, P< 0.0001. Three sections per mouse and 3 mice per group were evaluated.

NKILA stably overexpressed and control SMMC-7721 and HepG2 cells were treated with a series dose of Baicalein (0, 12.5, 25, 50, 100 μM) for 48 h. Then, cell proliferation was detected by Glo cell viability assays. The control condition was defined as media alone with no NKILA overexpression.

(d) Antiviral activity of SZA on assembly and release. Huh7 cells were electroporated with JFH-1 RNA of HCV, followed by 4 h treatment of the indicated concentrations of SZA. At 48 h after electroporation, cells were subjected to three cycles of freeze and thaw to test the intracellular viral infectivity; supernatants of the electroporated cells were collected for the detection of extracellular viral infectivity. 100 μM naringenin was introduced as a positive control. Results are shown as relative HCV infectivity compared to DMSO treated group. Data shown as mean ± SD of three independent experiments.

Piperine attenuates inflammation in the substantia nigra (SN) of the MPTP-treated brains. (A) Iba-1-immunostaining was performed on brain sections for microglia. Piperine alleviated microglia activating caused by MPTP. (B) Brain tissue was stained with the anti-interleukin-1β (IL-1β) antibody in the SN. MPTP notably increased the expression of IL-1β in the SN of the mice and the increase was partly blocked by piperine. (C) Western blot analysis of IL-1β expression in the SN. (D) Densitometric analysis of western blot analyses of IL-1β. Densitometry was measured by Quantity One 1-D analyze software (Universal Hood II; Bio-Rad Laboratories, Hercules, CA, USA). Values are presented as mean ± standard deviation, *P<0.05, compared to MPTP.

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.

A. ZR75-1 (AR+, ER+) and B. MDA MB 231 (AR-, TNBC) cells were treated with either abiraterone or vehicle, then used as targets in a CTL assay using CEA-specific CD8+ T cells as effector cells at an E:T ratio of 30:1. Error bars indicate mean ± S.E.M. for quadruplicate measurements. Statistical analyses were done by Student's t-test, * = P < 0.01 vs. vehicle control. Data are representative of 2 independent experiments.

(A) KMS11 and (B) L363 cells were plated in 5mM glucose medium with ritonavir or DMSO (D) for 17 hours. Glucose consumption rates are normalized to untreated cells (not shown). (C) KMS11 and (D) L363 cells were treated with ritonavir or DMSO for 72 hours. Relative viable cell numbers were determined by MTS assay and normalized to untreated cells (not shown).

Concomitant Western blot analysis of ER and TFF1 assessed after 16 h treatment with E2 or abiraterone in the presence or absence of MG-132 (10 μM).

Comparative mean±SD plasma concentration versus time curves of 40mg/kg oral posaconazole (PSZ) administrated in normolipidemic (NL), intermediate hyperlipidemic (IHL) or extreme hyperlipidemic (HL) rat groups. An insert showing a clear snap of the plasma concentrations for the first 7 h.

Voriconazole increases the osteogenic activity of human osteoblasts in vitro. Osteoblasts were exposed to voriconazole (VCZ) or fluconazole (FCZ) at 15 or 200 μg/ml in osteoinductive medium (OS+) for up to 7 days. As a positive control, cells were also exposed to sodium fluoride (NaF) at 10 μM in nonosteoinductive medium (OS−). Osteoblastic differentiation was evaluated by measuring alkaline phosphatase (ALP) activity (A) and staining for calcium deposition with alazarin red S stain (B and C); calcium deposition was quantified by measuring the absorbance (A415) of extracted alazarin red S (B) and was assessed by the light microscopic appearance (white arrows) of stained osteoblast cultures (C). Values represent the means ± SD. *, P < 0.05, compared to osteoblasts grown under osteogenic conditions without treatment.

a, b, Melanoma-bearing mice were treated with avasimibe (Ava) or DMSO control (5 times) (control, n=9; avasimibe, n=8). c-e, STORM analysis of TCR clustering of tumour-infiltrating CD8+ T cells. c, Representative images. d, Ripley’s K-function analysis of TCR molecules. e, The r value at the maximal L(r)-r value of Ripley’s K-function curves (control, n=100; avasimibe, n=85). f, g, A combined therapy (avasimibe and anti-PD-1) or monotherapies (avasimibe or anti-PD-1) in treating melanoma (n=10). Avasimibe, 5 times; anti-PD-1, 4 times. h, i, Cytokine/granule productions of PD-1hi and PD-1lo tumour-infiltrating CD8+ T cells (control, n=12; avasimibe, n=11).

MCF-7 and SKBR-3 cells were treated as indicated and morphology of autophagosomes was observed with TEM (C). Autophagosomes (Avi, immature autophagic vacuoles, bold arrow), autolysosomes (Avd, degraded/late autophagic vacuoles, sharp arrow).

C. Impact of CYP17A1 inhibitors on genes involved in androgen biosynthesis. H295R cells were treated with control (DMSO) or 1 μM orteronel and galeterone for 72 h and then RNA was isolated and transcribed to cDNA. Results of qRT-PCR validation of HSD3B2, CYP17A1 and AKR1C3 genes relative to the housekeeping gene cyclophilin A. Expression of the genes was analyzed by SYBR Green real-time PCR. Analysis of relative gene expression was determined by the 2−ΔΔCt method. *p < 0.05.

Effects of rifampin alone or in combination with cobicistat on the mean DRV CLint.app. in primary human hepatocytes in vitro. Cryopreserved primary human hepatocytes were incubated with RIF (0.5 to 20 μM) (hatched bars) or with COBI (0.13 to 12.76 μM) and RIF (0.5 to 20 μM) (gray bars) for 72 h. All cells were then incubated with RIF (0.5 to 20 M) or RIF (0.5 to 20 μM) together with COBI (0.13 to 12.76 μM), as described above, together with DRV (5 μM) for 60 min. Control cells were treated with DRV (5 μM) alone for 60 min (black bar). The results shown represent the mean DRV CLint.app. from three biological replicates measured in hepatocytes from three independent donors (donors HU1399, HU1574, and HU1587). Error bars indicate SDs.

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.

SKH-1 mice were subjected to UVB radiation (1000 J/m2 daily, 5 days), or topical apigenin (Api, 5 μmol, in 200 μl vehicle, DMSO: acetone 1:9) was applied 1 h prior to each UVB exposure. Mice were also sham-irradiated and treated with apigenin, and the control group of mice was subjected to sham irradiation and vehicle. 24 h after the final UVB exposure, the mice were euthanized, dorsal skin was harvested, fixed in formalin and paraffin-embedded. (A) Representative Hematoxylin and Eosin (H&E) staining of skin sections (scale bar, 100 μm). (B) Quantitation of epidermal thickness (mean ± SD), *, P < 0.001; **, P< 0.0001. Three sections per mouse and 3 mice per group were evaluated.

NKILA stably overexpressed and control SMMC-7721 and HepG2 cells were treated with a series dose of Baicalein (0, 12.5, 25, 50, 100 μM) for 48 h. Then, cell proliferation was detected by Glo cell viability assays. The control condition was defined as media alone with no NKILA overexpression.

(d) Antiviral activity of SZA on assembly and release. Huh7 cells were electroporated with JFH-1 RNA of HCV, followed by 4 h treatment of the indicated concentrations of SZA. At 48 h after electroporation, cells were subjected to three cycles of freeze and thaw to test the intracellular viral infectivity; supernatants of the electroporated cells were collected for the detection of extracellular viral infectivity. 100 μM naringenin was introduced as a positive control. Results are shown as relative HCV infectivity compared to DMSO treated group. Data shown as mean ± SD of three independent experiments.

Piperine attenuates inflammation in the substantia nigra (SN) of the MPTP-treated brains. (A) Iba-1-immunostaining was performed on brain sections for microglia. Piperine alleviated microglia activating caused by MPTP. (B) Brain tissue was stained with the anti-interleukin-1β (IL-1β) antibody in the SN. MPTP notably increased the expression of IL-1β in the SN of the mice and the increase was partly blocked by piperine. (C) Western blot analysis of IL-1β expression in the SN. (D) Densitometric analysis of western blot analyses of IL-1β. Densitometry was measured by Quantity One 1-D analyze software (Universal Hood II; Bio-Rad Laboratories, Hercules, CA, USA). Values are presented as mean ± standard deviation, *P<0.05, compared to MPTP.

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.