Journal: Cell Cycle

Article Title: Degraded melanocores are incompetent to protect epidermal keratinocytes against UV damage

doi: 10.1080/15384101.2018.1456601

Figure Lengend Snippet: Effects of siRNA-mediated knockdown of CTSV in KCs on melanocore degradation. (A) HaCaT cells were transfected with CTSV siRNA or the scrambled control for 72 h. The efficiency of CTSV knockdown by these siRNAs was tested using western blotting and RT-PCR. The histogram (on the right) shows the densitometric quantification of data with means ± SD of 3 independent experiments. (B) Cell lysates were prepared from siRNA-transfected HaCaT as noted, and individual melanocores were isolated from human MCs in culture, as described in the Materials and Methods. The melanocore specimens were incubated with the cell lysates at 37°C for 8 h. Representative TEM images show that lysates of CTSV-transfected HaCaT cells have less of a destructive effect on melanocores than lysates of the scrambled control, as indicated by the arrows (shown on the left). The melanocore specimens were also incubated with 1 μg/mL rhCTSV or a heat-inactivated control; TEM images are shown on the right. (C and D) Comparison of percentages of damaged melanocores from 3 independent experiments. *P <0.05. (E) CTSV enzyme activity of cell lysates was estimated using the fluorogenic dipeptide substrate Z-Leu-Arg-AMC. The histogram shows the means ± SD of fluorescence intensities from 3 independent experiments. *P < 0.05, **P < 0.01.

Article Snippet: The percentage of damaged melanocores was calculated to represent the efficacy of melanocore degradation. siRNA-induced CTSV mRNA knockdown Small interfering RNA (siRNA) duplexes targeting CTSV (Cat#: sc-44526) and a non-targeting scrambled control (Cat#: sc-37007) were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA).

Techniques: Transfection, Western Blot, Reverse Transcription Polymerase Chain Reaction, Isolation, Incubation, Activity Assay, Fluorescence

Journal: Cell Cycle

Article Title: Degraded melanocores are incompetent to protect epidermal keratinocytes against UV damage

doi: 10.1080/15384101.2018.1456601

Figure Lengend Snippet: Melanocore degradation is sensitive to CTSV up-expression in Ca2+-induced differentiated KCs. (A) Human KCs were cultured with EpiLife medium supplemented with 0.06 mM or 1.3 mM calcium for 48 h. The total RNA or protein was extracted from cultured cells, the mRNA and protein level were detected using qPCR and western blotting, respectively. Representative blot is shown on the left, the densitometric quantification of the intensity of the bands is shown on the middle. The levels of CTSV mRNA are shown on the right. *P < 0.05. (B) KCs were transfected with CTSV siRNA or the scrambled control for 72 h in the medium containing 0.06 mM or 1.3 mM calcium, as described for Figure 5. The cells were incubated with equal amount of melanocores for 72 h. The melanocore-incorporated KCs were harvested for the determination of Pmel 17/gp100 protein. Representative blot is shown on the left, the histogram (on the right) shows the densitometric quantification of data with means ± SD of 3 independent experiments. *P < 0.05. (C) The melanocore-incorporated KCs were cultured in the medium containing 0.06 mM or 1.3 mM calcium for varying time points (0, 24, 48, and 72h). The cells were harvested for chase analysis of Pmel 17/gp100 protein. The histogram (on the left) shows the densitometric quantification of data with means ± SD of 3 independent experiments. Human dermal fibroblasts (HDFBs) as a control are shown on the right. *P < 0.05.

Article Snippet: The percentage of damaged melanocores was calculated to represent the efficacy of melanocore degradation. siRNA-induced CTSV mRNA knockdown Small interfering RNA (siRNA) duplexes targeting CTSV (Cat#: sc-44526) and a non-targeting scrambled control (Cat#: sc-37007) were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA).

Techniques: Expressing, Cell Culture, Western Blot, Transfection, Incubation

Journal: Cell Cycle

Article Title: Degraded melanocores are incompetent to protect epidermal keratinocytes against UV damage

doi: 10.1080/15384101.2018.1456601

Figure Lengend Snippet: Suppression of CTSV mRNA and enzyme activity in KCs exposed to UVA or UVB. (A) HaCaT cells were seeded into 6-well cell culture plates and were irradiated with UVA (0, 3, 10 and 30 J/cm2) or UVB (0, 10, 30 and 100 mJ/cm2). The cells were harvested at 24 h post-irradiation. Protein and mRNA transcript levels of CTSV were determined by western-blotting and semi-quantitative RT-PCR, respectively. Representative images of 3 experiments are shown. (B) qPCR was performed to measure CTSV mRNA levels. The intensities of western-blot bands were quantified using Image J densitometry software. The levels of CTSV mRNA and protein are reported from 3 independent experiments. *P < 0.05, **P < 0.01. (C) Cell lysates were prepared from KCs exposed to 30 mJ/cm2 UVB and then incubated with isolated melanocore specimens at 37°C for 8 h. Representative TEM images show that UVB-exposed KCs have less of a destructive effect on melanocores than the unexposed control, as indicated by the arrows. (D) Comparison of percentages of damaged melanocores from 3 independent experiments. *P < 0.05. (E) Enzyme activity of CTSV in UVB-exposed keratinocyte lysates was estimated using the fluorogenic dipeptide substrate Z-Leu-Arg-AMC. Results are averages of 3 independent experiments. **P < 0.01.

Article Snippet: The percentage of damaged melanocores was calculated to represent the efficacy of melanocore degradation. siRNA-induced CTSV mRNA knockdown Small interfering RNA (siRNA) duplexes targeting CTSV (Cat#: sc-44526) and a non-targeting scrambled control (Cat#: sc-37007) were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA).

Techniques: Activity Assay, Cell Culture, Irradiation, Western Blot, Quantitative RT-PCR, Software, Incubation, Isolation

Journal: Cell Cycle

Article Title: Degraded melanocores are incompetent to protect epidermal keratinocytes against UV damage

doi: 10.1080/15384101.2018.1456601

Figure Lengend Snippet: Effects of siRNA-mediated knockdown of CTSV in KCs on melanocore degradation. (A) HaCaT cells were transfected with CTSV siRNA or the scrambled control for 72 h. The efficiency of CTSV knockdown by these siRNAs was tested using western blotting and RT-PCR. The histogram (on the right) shows the densitometric quantification of data with means ± SD of 3 independent experiments. (B) Cell lysates were prepared from siRNA-transfected HaCaT as noted, and individual melanocores were isolated from human MCs in culture, as described in the Materials and Methods. The melanocore specimens were incubated with the cell lysates at 37°C for 8 h. Representative TEM images show that lysates of CTSV-transfected HaCaT cells have less of a destructive effect on melanocores than lysates of the scrambled control, as indicated by the arrows (shown on the left). The melanocore specimens were also incubated with 1 μg/mL rhCTSV or a heat-inactivated control; TEM images are shown on the right. (C and D) Comparison of percentages of damaged melanocores from 3 independent experiments. *P <0.05. (E) CTSV enzyme activity of cell lysates was estimated using the fluorogenic dipeptide substrate Z-Leu-Arg-AMC. The histogram shows the means ± SD of fluorescence intensities from 3 independent experiments. *P < 0.05, **P < 0.01.

Article Snippet: Small interfering RNA (siRNA) duplexes targeting CTSV (Cat#: sc-44526) and a non-targeting scrambled control (Cat#: sc-37007) were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA).

Techniques: Transfection, Western Blot, Reverse Transcription Polymerase Chain Reaction, Isolation, Incubation, Activity Assay, Fluorescence

Journal: Cell Cycle

Article Title: Degraded melanocores are incompetent to protect epidermal keratinocytes against UV damage

doi: 10.1080/15384101.2018.1456601

Figure Lengend Snippet: Melanocore degradation is sensitive to CTSV up-expression in Ca2+-induced differentiated KCs. (A) Human KCs were cultured with EpiLife medium supplemented with 0.06 mM or 1.3 mM calcium for 48 h. The total RNA or protein was extracted from cultured cells, the mRNA and protein level were detected using qPCR and western blotting, respectively. Representative blot is shown on the left, the densitometric quantification of the intensity of the bands is shown on the middle. The levels of CTSV mRNA are shown on the right. *P < 0.05. (B) KCs were transfected with CTSV siRNA or the scrambled control for 72 h in the medium containing 0.06 mM or 1.3 mM calcium, as described for Figure 5. The cells were incubated with equal amount of melanocores for 72 h. The melanocore-incorporated KCs were harvested for the determination of Pmel 17/gp100 protein. Representative blot is shown on the left, the histogram (on the right) shows the densitometric quantification of data with means ± SD of 3 independent experiments. *P < 0.05. (C) The melanocore-incorporated KCs were cultured in the medium containing 0.06 mM or 1.3 mM calcium for varying time points (0, 24, 48, and 72h). The cells were harvested for chase analysis of Pmel 17/gp100 protein. The histogram (on the left) shows the densitometric quantification of data with means ± SD of 3 independent experiments. Human dermal fibroblasts (HDFBs) as a control are shown on the right. *P < 0.05.

Article Snippet: Small interfering RNA (siRNA) duplexes targeting CTSV (Cat#: sc-44526) and a non-targeting scrambled control (Cat#: sc-37007) were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA).

Techniques: Expressing, Cell Culture, Western Blot, Transfection, Incubation