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. Author manuscript; available in PMC: 2022 Jul 10.
Published in final edited form as: Cancer Lett. 2021 Apr 17;510:79–92. doi: 10.1016/j.canlet.2021.04.004

Fig. 4. Combination treatment induces senescence through ROS generation in HNSCC.

Fig. 4.

a. Representative image of SA-β-gal staining indicates cell senescence in UMSCC1 and Cal27 cells after palbociclib (2.5 μM), afatinib (250 nM) or combination (palbociclib- 2.5 μM and afatinib- 250 nM) for 48 h. Treatment with N-acetyl cysteine (15 mM) for 2–4 h prior to combination treatment (right panel). b. Intracellular levels of reactive oxygen species (ROS) measured with Fluorometric Intracellular Ros Kit after 48 h of treatment [palbociclib (1 & 2.5 μM), afatinib (250 nM) and combination (palbociclib- 1 & 2.5 μM and afatinib- 250 nM)]. c. Immunofluorescence of mitochondrial Tom20 staining in UMSCC1 after treatment [afatinib-250 nm, palbociclib-2.5 μM, and combination (afatinib-250 nm & palbociclib-2.5 μM)] for 48 h. d. Active mitochondria as shown by JC-1 red/green staining. Quantification of ratio of red/green (bottom panel). e. Western blot of ROS scavenging proteins after treatment described (b) in UMSCC1 and Cal27. Data represent mean ± SEM. n=2. ANOVA followed by Tukey’s multiple comparisons test between groups (b & d). *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001, and ns=non-significant.