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Fig. 9 The effect of immune-checkpoint inhibitors (ICIs) on cell death of AR-depleted melanoma cells analyzed by LDH release determination. Surface expression of PD-1 (A) and <t>CTLA-4</t> (B) on NK cells, identified as CD56+ CD3−cells. WB analysis for PD-L1 (C), PD-1 (D) and CTLA-4 (E) expression in the indicated melanoma cell lines. Tubulin or GAPDH were revealed as loading controls. AMM16 (F), WM266-4 (L) and MEL-CAL (P) cells were transfected with control siRNA (siRNA ctrl) or AR siRNA (siRNA AR). Lysate proteins were analyzed by WB using the indicated antibodies. Melanoma cells were untreated or treated with 5, 10 or 35 μg/ml of Atezolizumab (G, M, Q), Pembrolizumab (H, N, R) or Ipilimumab (I, O, S) for 26 h. LDH release was determined as indicated in Methods section. In G–I, M–O, Q–S, data are expressed as mean ± SD of 3 different experiments each in triplicate; *p < 0.05; **p < 0.01.
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Fig. 9 The effect of immune-checkpoint inhibitors (ICIs) on cell death of AR-depleted melanoma cells analyzed by LDH release determination. Surface expression of PD-1 (A) and <t>CTLA-4</t> (B) on NK cells, identified as CD56+ CD3−cells. WB analysis for PD-L1 (C), PD-1 (D) and CTLA-4 (E) expression in the indicated melanoma cell lines. Tubulin or GAPDH were revealed as loading controls. AMM16 (F), WM266-4 (L) and MEL-CAL (P) cells were transfected with control siRNA (siRNA ctrl) or AR siRNA (siRNA AR). Lysate proteins were analyzed by WB using the indicated antibodies. Melanoma cells were untreated or treated with 5, 10 or 35 μg/ml of Atezolizumab (G, M, Q), Pembrolizumab (H, N, R) or Ipilimumab (I, O, S) for 26 h. LDH release was determined as indicated in Methods section. In G–I, M–O, Q–S, data are expressed as mean ± SD of 3 different experiments each in triplicate; *p < 0.05; **p < 0.01.
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Fig. 9 The effect of immune-checkpoint inhibitors (ICIs) on cell death of AR-depleted melanoma cells analyzed by LDH release determination. Surface expression of PD-1 (A) and CTLA-4 (B) on NK cells, identified as CD56+ CD3−cells. WB analysis for PD-L1 (C), PD-1 (D) and CTLA-4 (E) expression in the indicated melanoma cell lines. Tubulin or GAPDH were revealed as loading controls. AMM16 (F), WM266-4 (L) and MEL-CAL (P) cells were transfected with control siRNA (siRNA ctrl) or AR siRNA (siRNA AR). Lysate proteins were analyzed by WB using the indicated antibodies. Melanoma cells were untreated or treated with 5, 10 or 35 μg/ml of Atezolizumab (G, M, Q), Pembrolizumab (H, N, R) or Ipilimumab (I, O, S) for 26 h. LDH release was determined as indicated in Methods section. In G–I, M–O, Q–S, data are expressed as mean ± SD of 3 different experiments each in triplicate; *p < 0.05; **p < 0.01.

Journal: Cell death & disease

Article Title: Role of the androgen receptor in melanoma aggressiveness.

doi: 10.1038/s41419-025-07350-4

Figure Lengend Snippet: Fig. 9 The effect of immune-checkpoint inhibitors (ICIs) on cell death of AR-depleted melanoma cells analyzed by LDH release determination. Surface expression of PD-1 (A) and CTLA-4 (B) on NK cells, identified as CD56+ CD3−cells. WB analysis for PD-L1 (C), PD-1 (D) and CTLA-4 (E) expression in the indicated melanoma cell lines. Tubulin or GAPDH were revealed as loading controls. AMM16 (F), WM266-4 (L) and MEL-CAL (P) cells were transfected with control siRNA (siRNA ctrl) or AR siRNA (siRNA AR). Lysate proteins were analyzed by WB using the indicated antibodies. Melanoma cells were untreated or treated with 5, 10 or 35 μg/ml of Atezolizumab (G, M, Q), Pembrolizumab (H, N, R) or Ipilimumab (I, O, S) for 26 h. LDH release was determined as indicated in Methods section. In G–I, M–O, Q–S, data are expressed as mean ± SD of 3 different experiments each in triplicate; *p < 0.05; **p < 0.01.

Article Snippet: The staining of NK cells was done using the following antibodies: APC CD56 (clone REA196)/ IgG1, FITC CD3 (clone BW264/56)/IgG2a, PE CD152 (CTLA-4) (clone BNI3/ IgG2a) from Miltenyi Biotec; BV510 CD3 (clone HIT3a)/IgG2a, BB700 CD56 (clone NCAM16.2)/IgG2b, PE-CF594 CD279 (PD-1) (clone EH12.1)/IgG1 from BD Bioscience.

Techniques: Expressing, Transfection, Control