Fig. 2.
Fig. 2. KLF6 and endoglin expression and localization after endothelial denudation in HUVECs. / (A) RT-PCR after HUVEC endothelial denudation. HUVECs were grown and wounded as described in “Materials and methods.” At different times (1-24 hours) after wounding, cells were lysed and RNA was extracted and processed for RT-PCR with KLF6 (K), endoglin (E), Sp1 (S), and actin (A) primers. After 20 cycles, PCR reactions were separated on a 3% Nu-Sieve agarose gel, and bands were quantified by densitometry, then plotted relative to actin cDNA as shown in the bar graph on the right. Shown is 1 of 4 representative experiments that gave similar results. (B) Flow cytometric analysis of endoglin and KLF6. HUVECs were wounded extensively, leaving approximately 20% of the total monolayer remaining intact. After different intervals (0-36 hours), cells were processed for flow cytometry. To detect endoglin on the cell surface, incubation with monoclonal antibody P4A4 was used as described in “Materials and methods.” To detect total KLF6, cells were permeabilized before antibody incubation as described in “Materials and methods.” Cytometry profiles for endoglin and KLF6 are shown on the left and, for comparative purposes, contain a vertical dotted line that indicates the fluorescence intensity of unwounded HUVECs. On the right, a plot summarizing the protein levels during the denudation process is included. Shown is 1 of 5 representative experiments that gave similar results. (C) Immunostaining for KLF6 and endoglin in HUVECs after endothelial denudation. HUVECs, grown as monolayers on gelatinized coverslips, were wounded with a tip of pipette in the middle of the monolayer. For KLF6 immunofluorescence microscopy, cells were incubated with a rabbit polyclonal anti-KLF6 antibody, then washed and incubated with an FITC goat-antirabbit antibody (green fluorescence). For endoglin staining, cells were incubated with P4A4 mouse antibody, followed by a secondary anti–mouse IgG coupled to Alexa 546 (red fluorescence). Representative micrographs from 50 different fields with similar results are presented. Single (top and middle rows) and double (bottom row) immunostaining shows that KLF6 translocates from the cytoplasm to the nucleus, whereas endoglin always localizes to the plasma membrane. Original magnification top panels, × 100; middle and bottom panels, × 60.

KLF6 and endoglin expression and localization after endothelial denudation in HUVECs.

(A) RT-PCR after HUVEC endothelial denudation. HUVECs were grown and wounded as described in “Materials and methods.” At different times (1-24 hours) after wounding, cells were lysed and RNA was extracted and processed for RT-PCR with KLF6 (K), endoglin (E), Sp1 (S), and actin (A) primers. After 20 cycles, PCR reactions were separated on a 3% Nu-Sieve agarose gel, and bands were quantified by densitometry, then plotted relative to actin cDNA as shown in the bar graph on the right. Shown is 1 of 4 representative experiments that gave similar results. (B) Flow cytometric analysis of endoglin and KLF6. HUVECs were wounded extensively, leaving approximately 20% of the total monolayer remaining intact. After different intervals (0-36 hours), cells were processed for flow cytometry. To detect endoglin on the cell surface, incubation with monoclonal antibody P4A4 was used as described in “Materials and methods.” To detect total KLF6, cells were permeabilized before antibody incubation as described in “Materials and methods.” Cytometry profiles for endoglin and KLF6 are shown on the left and, for comparative purposes, contain a vertical dotted line that indicates the fluorescence intensity of unwounded HUVECs. On the right, a plot summarizing the protein levels during the denudation process is included. Shown is 1 of 5 representative experiments that gave similar results. (C) Immunostaining for KLF6 and endoglin in HUVECs after endothelial denudation. HUVECs, grown as monolayers on gelatinized coverslips, were wounded with a tip of pipette in the middle of the monolayer. For KLF6 immunofluorescence microscopy, cells were incubated with a rabbit polyclonal anti-KLF6 antibody, then washed and incubated with an FITC goat-antirabbit antibody (green fluorescence). For endoglin staining, cells were incubated with P4A4 mouse antibody, followed by a secondary anti–mouse IgG coupled to Alexa 546 (red fluorescence). Representative micrographs from 50 different fields with similar results are presented. Single (top and middle rows) and double (bottom row) immunostaining shows that KLF6 translocates from the cytoplasm to the nucleus, whereas endoglin always localizes to the plasma membrane. Original magnification top panels, × 100; middle and bottom panels, × 60.

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