Table 1.
Primers used for real-time PCR.
Fig 1.
The effect of puerarin on the viability, injury and proliferative activity in high-passage NHDFs.
A, B: The effect of puerarin (pue) on cell viability of young (A) and senescent NHDFs (B) was analyzed by CCK-8 assay. n = 5. C: The effect of cellular LDH release in high passage NHDFs. Data was obtained from LDH assay. n = 6. D, E: The effect of puerarin on the cell proliferative activity in senescent NHDFs. Cells were stained for BrdU and nuclei by specific antibody and DAPI, respectively, and photographed at 100×magnification (D). The percentage of BrdU positive cells per total cells was calculated from 15 microscopic fields for each group. (E). Scale bars = 100μm.
Fig 2.
The effect of puerarin on ROS generation in high-passage NHDFs.
ROS generation in senescent NFDFs (sene) with or without 50 μM puerarin (+pue 50) was measured by fluorescent ROS probe CellROX Green (A). CellROX intensity was calculated from 10 images for each group (B). Scale bars = 100μm.
Fig 3.
The effect of puerarin on cellular senescence in high-passage NHDFs.
A, B: The effect of puerarin on SA-β-galactosidase activity. Young and senescent (sene) NHDFs treated with 20 or 50 μM puerarin (pue) were stained by SA-β-galactosidase assay (A). The percentage of SA-β-galactosidase positive cells per total cells was calculated from 15 microscopic fields for each group. Scale bars = 100 μm.
Fig 4.
The effect of puerarin on the expression of SMA in high passage NHDFs.
A-B: Young or senescent (sene) NHDFs with or without 50 μM puerarin (+pue 50) were stained for SMA by the immunocytochemical method. The percentage of SMA positive cells per total cells was calculated from 15 microscope fields for each group. Scale bars = 100μm. C The expression of SMA mRNA in young and senescent NHDFs treated with or without 25–50 μM puerarin (pue) for 72 h. n = 4.
Fig 5.
The effect of puerarin on gene expressions of reticular and papillary markers.
The expressions of PDPN (papillary marker), CNN1 (reticular marker) in young and senescent NHDFs treated with or without 25–50 μM puerarin (pue) for 72 h. n = 3–5.
Fig 6.
The effect of puerarin on CNN1 and PDPN expressions in senescent NHDFs.
(A) Young or senescent (sene) NHDFs with or without 50 μM puerarin (+pue 50) were stained for CNN1 (green) and PDPN (red) by the immunocytochemical method. (B) Fluorescent intensities of CNN1 positive area were calculated from 5 microscpic fields for each group. Scale bars = 100μm.
Fig 7.
The effect of puerarin on the translocation of estrogen receptors in senescent NHDFs.
(A) Senescent (sene) NHDFs with or without 50 μM puerarin (+pue 50) were stained for ERα (upper) or ERβ (lower) by the immunocytochemical method. (B) Fluorescent intensities of ERβ that translocated into nuclei were calculated from more than 100 nuclei (3 microscpic fields) for each group. Scale bars = 50μm.
Fig 8.
Estrogen receptor antagonist blocked puerarin-mediated reduction of SMA and CNN1.
(A) The expressions of SMA and CNN1 mRNAs in senescent NHDFs treated with 50 μM puerarin (+pue50) and estrogen receptor antagonist, 1 μM fulvostrant (+Fulv) for 72 h. n = 4. (B-E) Senescent (sene) NHDFs treated with 50 μM puerarin (+pue50) and estrogen receptor antagonist, 1 μM fulvostrant (+Fulv) were stained for SMA (B) and CCN1 (C) by the immunocytochemical method. (D) The percentage of SMA positive cells per total cells was calculated from 11 microscope fields for each group. (E) Fluorescent intensities of CNN1 positive area were calculated from 11 microscpic fields for each group. Scale bars = 100μm.