Journal: FEBS Open Bio

Article Title: A thermal gradient modulates the oxidative metabolism and growth of human keratinocytes

doi: 10.1002/2211-5463.12303

Figure Lengend Snippet: The effect of the temperature on cellular energy metabolism. Primary keratinocytes ( KER ) and HaCaT cells were incubated for 10 days at 37 and 33 °C, and the levels of mitochondrial ATP were measured by a chemiluminescence‐based assay. (A) Data were expressed as nmol/mg of proteins and (B) as the percentage of their respective counterpart kept at 37 °C (relative ATP ), in order to emphasize the higher induction of ATP synthesis in KER compared to HaCaT. (C) The intracellular LDH activity was assayed in the same conditions. Measurements were taken in triplicate, and data are presented as means ± SD ( n = 3). * P < 0.05 compared to the cells at 37 °C.

Article Snippet: At the first medium change (5 days after the initial plating), the medium was further supplemented with 10 ng·mL −1 epidermal growth factor (Sigma‐Aldrich) and changed every 48 − 72 h. The immortalized human epidermal keratinocyte cell line HaCaT was purchased from American Type Culture Collection (ATCC, Manassas, VA, USA), and the cells were cultured in DMEM that had been supplemented with 10% FBS and 1% antibiotics (penicillin/streptomycin; Sigma‐Aldrich) at 37 and 33 °C in a humidified atmosphere containing 5% CO 2 .

Techniques: Incubation, Chemiluminescence Immunoassay, Activity Assay

Journal: FEBS Open Bio

Article Title: A thermal gradient modulates the oxidative metabolism and growth of human keratinocytes

doi: 10.1002/2211-5463.12303

Figure Lengend Snippet: The influence of the temperature on the expression of a modulator of the mitochondrial respiratory chain. Primary keratinocytes ( KER ) and HaCaT cells were incubated for 10 days at 37 and 33 °C, and the levels of mitochondrial estrogen receptor beta ( ER β) were analyzed by western blotting in mitochondrial extracts. VDAC levels were used as internal controls for protein loading. (A) The blots are representative of a set of three independent experiments. (B) Bands from KER extracts were quantified, normalized for loading as a ratio to VDAC expression, and data plotted on graph as values relative to control. Data represent the mean ± SD of three independent experiments. * P < 0.05 compared to the cells at 37 °C.

Article Snippet: At the first medium change (5 days after the initial plating), the medium was further supplemented with 10 ng·mL −1 epidermal growth factor (Sigma‐Aldrich) and changed every 48 − 72 h. The immortalized human epidermal keratinocyte cell line HaCaT was purchased from American Type Culture Collection (ATCC, Manassas, VA, USA), and the cells were cultured in DMEM that had been supplemented with 10% FBS and 1% antibiotics (penicillin/streptomycin; Sigma‐Aldrich) at 37 and 33 °C in a humidified atmosphere containing 5% CO 2 .

Techniques: Expressing, Incubation, Western Blot, Control

Journal: FEBS Open Bio

Article Title: A thermal gradient modulates the oxidative metabolism and growth of human keratinocytes

doi: 10.1002/2211-5463.12303

Figure Lengend Snippet: The impact of the temperature on a key protein of the uncoupling process. After 10 days at 37 and 33 °C, the mitochondrial extracts from primary keratinocytes ( KER ) and HaCaT cells were analyzed by western blotting to detect the expression of the mitochondrial uncoupling protein 1 ( UCP 1). VDAC levels were used as internal controls for protein loading. The reported molecular weights of the two proteins are indicated. The blots are representative of a set of three independent experiments.

Article Snippet: At the first medium change (5 days after the initial plating), the medium was further supplemented with 10 ng·mL −1 epidermal growth factor (Sigma‐Aldrich) and changed every 48 − 72 h. The immortalized human epidermal keratinocyte cell line HaCaT was purchased from American Type Culture Collection (ATCC, Manassas, VA, USA), and the cells were cultured in DMEM that had been supplemented with 10% FBS and 1% antibiotics (penicillin/streptomycin; Sigma‐Aldrich) at 37 and 33 °C in a humidified atmosphere containing 5% CO 2 .

Techniques: Western Blot, Expressing

Journal: FEBS Open Bio

Article Title: A thermal gradient modulates the oxidative metabolism and growth of human keratinocytes

doi: 10.1002/2211-5463.12303

Figure Lengend Snippet: The effect of the temperature on cell morphology and growth. Primary keratinocytes ( KER ) and HaCaT cells were grown for 10 days at 37 and 33 °C. (A) Representative pictures of the cells at different temperatures. (B) The cells in each dish were counted and the values of the cells kept at 33 °C are expressed as the percentage of their counterpart grown at 37 °C. The data are expressed as the means ± SD of three independent experiments. * P < 0.05 compared to the cells at 37 °C.

Article Snippet: At the first medium change (5 days after the initial plating), the medium was further supplemented with 10 ng·mL −1 epidermal growth factor (Sigma‐Aldrich) and changed every 48 − 72 h. The immortalized human epidermal keratinocyte cell line HaCaT was purchased from American Type Culture Collection (ATCC, Manassas, VA, USA), and the cells were cultured in DMEM that had been supplemented with 10% FBS and 1% antibiotics (penicillin/streptomycin; Sigma‐Aldrich) at 37 and 33 °C in a humidified atmosphere containing 5% CO 2 .

Techniques:

Journal: FEBS Open Bio

Article Title: A thermal gradient modulates the oxidative metabolism and growth of human keratinocytes

doi: 10.1002/2211-5463.12303

Figure Lengend Snippet: A working model of the proposed molecular mechanisms underlying the effect of external temperature on metabolism of human primary keratinocytes and HaCaT cells. (A) At 37 °C, the mitochondrial metabolism supports both the synthesis of ATP and the rerouting of intermediates (acetyl‐CoA) to support proliferation. (B) At 33 °C, the thermal gradient between internal and external temperature is balanced by heat production at the electron transport chain ( ETC ) site, either by uncoupling or by proton leak; the acetyl‐CoA is consumed in the tricarboxylic acid cycle ( TCA cycle), the oxidative metabolism prevails over the biosynthetic process, and the cell reduces proliferation. AS , ATP synthase; UCP , uncoupling protein; Succ, succinate.

Article Snippet: At the first medium change (5 days after the initial plating), the medium was further supplemented with 10 ng·mL −1 epidermal growth factor (Sigma‐Aldrich) and changed every 48 − 72 h. The immortalized human epidermal keratinocyte cell line HaCaT was purchased from American Type Culture Collection (ATCC, Manassas, VA, USA), and the cells were cultured in DMEM that had been supplemented with 10% FBS and 1% antibiotics (penicillin/streptomycin; Sigma‐Aldrich) at 37 and 33 °C in a humidified atmosphere containing 5% CO 2 .

Techniques:

Journal: Purinergic Signalling

Article Title: Upregulation of P2Y2 receptors by retinoids in normal human epidermal keratinocytes

doi: 10.1007/s11302-005-7331-5

Figure Lengend Snippet: Changes in mRNA expression for P2Y receptors induced by ATRA in NHEKs. Diagram shows the percentage of the quantity after amplification by real-time RT-PCR for P2Y1, P2Y2 and P2Y11 receptor mRNAs extracted from NHEKs treated with 1 µM ATRA for 2 h. Asterisks show significant difference from control groups ( white columns , ** P < 0.01). mRNAs of P2Y2 receptors were increased by more than twofold vs. control. Data were obtained from at least three independent experiments.

Article Snippet: Normal human epidermal keratinocytes (NHEKs) were obtained as cyropreserved first-passage cells from neonatal foreskins (Kurabo, Osaka, Japan).

Techniques: Expressing, Amplification, Quantitative RT-PCR, Control

Journal: Purinergic Signalling

Article Title: Upregulation of P2Y2 receptors by retinoids in normal human epidermal keratinocytes

doi: 10.1007/s11302-005-7331-5

Figure Lengend Snippet: Time- and concentration-dependency of three different retinoids-induced changes in mRNAs in NHEKs. Diagram shows the quantity of P2Y2 mRNAs detected by real-time RT-PCR after treatment with 0.001–1 µM ATRA (A), Am80 (B) and 9- cis RA (C) for 2–24 h. The P2Y2 mRNA levels in cells treated with various concentrations of retinoids were normalized by those in retinoids-untreated control cells at each incubation period (2, 6, 12 and 24 h), and expressed as “percentage (%) of control.” All these retinoids, and especially Am80, caused a linear increase in P2Y2 mRNAs in a concentration- and time-dependent fashion. Asterisks show significant difference in the P2Y2 mRNA levels from control groups ( * P < 0.05; ** P < 0.01). Data were obtained from at least three independent experiments.

Article Snippet: Normal human epidermal keratinocytes (NHEKs) were obtained as cyropreserved first-passage cells from neonatal foreskins (Kurabo, Osaka, Japan).

Techniques: Concentration Assay, Quantitative RT-PCR, Control, Incubation

Journal: Purinergic Signalling

Article Title: Upregulation of P2Y2 receptors by retinoids in normal human epidermal keratinocytes

doi: 10.1007/s11302-005-7331-5

Figure Lengend Snippet: Enhancement by ATRA and Am80 of P2Y2 receptor-mediated increase in [Ca 2+ ]i in NHEKs. A. Typical traces of the UTP-evoked changes in [Ca 2+ ]i in NHEKs. NHEKs were incubated with 0.1 µM ATRA ( middle ) or Am80 ( bottom ) for 6 h, incubated with normal culture medium for another 18 h, and then the fura-2 based [Ca 2+ ]i measurement was performed. UTP (100 µM) was applied to cells for 10 s and the increase in the ΔF340/F380 ratio was calculated ( n = 110–125). After the initial UTP-application, the extracellular Ca 2+ was removed (0 Ca 2+ ), and the second UTP was applied to the cells in the absence of extracellular Ca 2+ . Effect of ATRA and Am80 on the UTP-evoked elevation in [Ca 2+ ]i in NHEKs in the presence and absence of extracellular Ca 2+ was summarized in B. Asterisks show significant difference from control (without retinoids) ( * P < 0.05; ** P < 0.01).

Article Snippet: Normal human epidermal keratinocytes (NHEKs) were obtained as cyropreserved first-passage cells from neonatal foreskins (Kurabo, Osaka, Japan).

Techniques: Incubation, Control

Journal: Purinergic Signalling

Article Title: Upregulation of P2Y2 receptors by retinoids in normal human epidermal keratinocytes

doi: 10.1007/s11302-005-7331-5

Figure Lengend Snippet: Visualization of release of ATP from NHEKs. The image panels in A show ATP-derived photons ( white dots ) in a field of ATRA-treated ( right ) and -untreated control NHEKs ( left ). NHEKs were incubated with 0.1 µM ATRA for 6 h. Cells were bathed in luciferin-luciferase reagent and the bioluminescence signals were obtained with a VIM camera (see Materials and methods Section) with an exposure time of 10 s. Sequential images show the ATP-derived photon-signals before (−10 s; b & b′) and 10 (c & c′), 20 (d & d′) and 30 s (e & e′) after mechanical stimulation. The positions of the pipettes are shown in phasecontrast images of NHEKs (a & a′). In B, the accumulative photon intensity in 60 s was converted to the absolute extracellular ATP concentration using a standard ATP-photon intensity relationship curve determined with an ATP standard solution (control, n = 17; ATRA-treated, n = 14). Photons within 50 mms squares around the stimulated site (shown as white squares in a & a′ panels in A) were calculated. Asterisks show significant difference from basal groups ( ** P < 0.01) and dagger shows significant difference from control basal groups († P < 0.05).

Article Snippet: Normal human epidermal keratinocytes (NHEKs) were obtained as cyropreserved first-passage cells from neonatal foreskins (Kurabo, Osaka, Japan).

Techniques: Derivative Assay, Control, Incubation, Luciferase, Concentration Assay

Journal: Purinergic Signalling

Article Title: Upregulation of P2Y2 receptors by retinoids in normal human epidermal keratinocytes

doi: 10.1007/s11302-005-7331-5

Figure Lengend Snippet: ATRA-induced changes in expression pattern of P2 receptors in NHEKs.

Article Snippet: Normal human epidermal keratinocytes (NHEKs) were obtained as cyropreserved first-passage cells from neonatal foreskins (Kurabo, Osaka, Japan).

Techniques: Expressing, Control

Journal: International Journal of Molecular Sciences

Article Title: Antioxidant Artemisia princeps Extract Enhances the Expression of Filaggrin and Loricrin via the AHR/OVOL1 Pathway

doi: 10.3390/ijms18091948

Figure Lengend Snippet: ( A ) Localization of NRF2 is visualized by an immunofluorescence technique. NRF2 is mainly located in the cytoplasm of control keratinocytes. Nuclear staining of NRF2 is enhanced in the keratinocytes treated with APE for 6 h. Nuclei are stained with 4′,6-diamidino-2-phenylindole (DAPI). Scale bar, 20 μm; ( B ) the number of NHEKs with nuclear-predominant staining of NRF2 is significantly increased by APE treatment than that of control; ( C ) APE upregulates the gene expression of antioxidant enzyme NQO1 ; and ( D ) APE also upregulates the gene expression of antioxidant enzyme HO1 . ** p < 0.01.

Article Snippet: The NHEKs were cultured in normal human epidermal keratinocyte proliferation medium, HuMedia-KG2 (KG2; Kurabo, Osaka, Japan), containing insulin, human recombinant epidermal growth factor, hydrocortisone, bovine pituitary extract, and antibacterial substances.

Techniques: Immunofluorescence, Control, Staining, Gene Expression

Journal: International Journal of Molecular Sciences

Article Title: Antioxidant Artemisia princeps Extract Enhances the Expression of Filaggrin and Loricrin via the AHR/OVOL1 Pathway

doi: 10.3390/ijms18091948

Figure Lengend Snippet: ( A ) Localization of AHR is visualized by an immunofluorescence technique. AHR is mainly located in the cytoplasm of control keratinocytes. Nuclear staining of AHR is enhanced in the keratinocytes treated with APE for 6 h. Nuclei are stained with 4′,6-diamidino-2-phenylindole (DAPI). Scale bar, 20 μm; ( B ) the number of NHEKs with nuclear-predominant staining of AHR is significantly increased by APE treatment than that of control; ( C ) APE dose-dependently upregulates the gene expression of CYP1A1 , a specific AHR-responsive metabolizing enzyme. ** p < 0.01; and ( D ) APE-induced CYP1A1 upregulation is canceled in keratinocytes transfected with AHR siRNA. ** p < 0.01.

Article Snippet: The NHEKs were cultured in normal human epidermal keratinocyte proliferation medium, HuMedia-KG2 (KG2; Kurabo, Osaka, Japan), containing insulin, human recombinant epidermal growth factor, hydrocortisone, bovine pituitary extract, and antibacterial substances.

Techniques: Immunofluorescence, Control, Staining, Gene Expression, Transfection

Journal: International Journal of Molecular Sciences

Article Title: Antioxidant Artemisia princeps Extract Enhances the Expression of Filaggrin and Loricrin via the AHR/OVOL1 Pathway

doi: 10.3390/ijms18091948

Figure Lengend Snippet: APE (0.03%)-induced FLG ( A ) and LOR ( B ) upregulation is canceled in keratinocytes with AHR knockdown. APE-induced FLG ( C ) and LOR ( D ) upregulation is also inhibited in keratinocytes with OVOL1 knockdown. * p < 0.05.

Article Snippet: The NHEKs were cultured in normal human epidermal keratinocyte proliferation medium, HuMedia-KG2 (KG2; Kurabo, Osaka, Japan), containing insulin, human recombinant epidermal growth factor, hydrocortisone, bovine pituitary extract, and antibacterial substances.

Techniques: Knockdown