Journal: Nature Communications

Article Title: APOA1 binding protein promotes lymphatic cell fate and lymphangiogenesis by relieving caveolae-mediated inhibition of VEGFR3 signaling

doi: 10.1038/s41467-025-60611-w

Figure Lengend Snippet: a TEM analysis of control and recombinant AIBP-treated hLECs. Arrows depict caveolae. b Quantification of caveolae in ( a ). n = 35 (control) and n = 36 (AIBP) cells. Data are Mean ± SE; unpaired two-sided t -test with Welch’s correction. c TEM analysis of caveolae in ECs of the cardinal vein from control and apoa1bp2 −/− zebrafish. Arrows indicate closed caveolae and arrowheads show open caveolae. d Quantification of caveolae in cardinal vein ECs. n = 27 (control) and n = 29 ( apo1bp2 −/− ) cells. Data are Mean ± SE; unpaired two-sided t -test with Welch’s correction. e Scheme illustration of 4 hydroxy-tamoxifen (4OHT)-induced ubiquitous mEos2-APOA1 expression. f Representative images of control and mEos2-APOA1 expressed embryos after 4OHT treatment. Embryos were imaged at 2 dpf. The white dashed line demarcates the control animals. The results are representative of 3 independent repeats. g Photoconversion and vascular circulation of mEos2-APOA1. The head regions of embryos were exposed to UV light for 1 min to induce photoconversion. h Analysis of mEos2-APOA1 secretion. The specified tail region was imaged, and the maximum RFP-to-GFP signal ratio within the ISV lumen was quantified. n = 8 intersegmental vessels from 2 embryos. Data are presented as mean ± SEM, analyzed using one-way ANOVA with Tukey’s post-hoc test. i Maxi-projection confocal images of Prox1 + cells in the apoa1bp −/− ; fli1a:egfp zebrafish with control (mEos2-APOA1) and mEos2-APOA1 overexpression (ubi:Gal4-ERT2+mEos2-APOA1) at 4 dpf and immunostained with GFP (green) and Prox1 (red) antibodies. Arrowheads show the Prox1 + LECs in TD. j Quantitative data of Prox1 + LEC in TD (7 somites). n = 10 ( mEos-APOA1 ) and n = 11 ( ubi:Ert2-Gal4; mEos-APOA1 ) embryos. Data are Mean ± SE; unpaired two-sided t -test with Welch’s correction. CV cardinal vein. Scale bar: 400 nm in a and 500 nm in c ; 100 µm in ( f , g , i ). Source data are prov i ded as a file.

Article Snippet: Primary human dermal lymphatic microvascular endothelial cells (hLECs) were purchased from PromoCell (Cat # C-12216 and C-12217).

Techniques: Control, Recombinant, Expressing, Over Expression

Journal: Nature Communications

Article Title: APOA1 binding protein promotes lymphatic cell fate and lymphangiogenesis by relieving caveolae-mediated inhibition of VEGFR3 signaling

doi: 10.1038/s41467-025-60611-w

Figure Lengend Snippet: a − d Effect of MβCD on VEGFR3 phosphorylation. a hLECs were growth factor-starved, and treated with 10 mM MβCD for 5, 15, and 30 min, and the resulting cells were further stimulated with 100 ng/mL VEGFC. The resulting cells were lysed and blotted using CAV-1, VEGFR3, GAPDH antibodies. b Quantitative analysis of panel a. Mean ± SD, n = 3 repeats; two-way ANOVA with Dunnett’s post-hoc test. c , hLECs were treated as in panel a. and cell lysates were immunoprecipitated using VEGFR3 antibody. Immunoblotting was performed using anti-phosphotyrosine (4G10) and VEGFR3 antibodies. d Quantitative analysis of ( c ). n = 3 repeats. Data are presented as mean ± SD and were analyzed using one-way ANOVA with Tukey’s post-hoc test. e , f Effect of AIBP treatment on VEGFR3 distribution in caveolar fractions. e hLECs were incubated with either recombinant AIBP or vehicle control in EBM2 supplemented with 10% FBS for 2 h, and the cells were subjected to sucrose gradient ultracentrifugation. n = 3 repeats. The resulting fractions were collected for Western blot analysis as indicated. Tx treatment; cav: caveolar fraction; n.c non-caveolar fraction. f Quantitative data of ( e ). Mean ± SD; two-way ANOVA with Sidak’s post-hoc test. n = 3 repeats. g , h Effect of AIBP and HDL co-treatment on VEGFR3 signaling. g hLECs were growth factor-starved and treated with HDL, AIBP, or HDL and AIBP in combination, and further stimulated with VEGFC. The resulting cells were lysed and immunoblotted as indicated. h Quantitative data of ERK and AKT activation. Mean ± SD; two-way ANOVA with Tukey’s post-hoc test. n = 3 repeats. Ctrl: control. i Maxi-projection confocal images of Prox1 + and pErk1/2 + cells in the apoa1bp −/− ; fli1a:egfp zebrafish at 36 hpf following immunostaining using GFP, pErk1/2, and Prox1 antibodies. Dorsal (DA) aorta and cardinal vein (CV) were imaged. Arrows show the Prox1 + LECs with pErk1/2 expression. j Quantitative data of pErk1/2 intensity in Prox1 + LECs. Data are Mean ± SE; unpaired two-sided t -test with Welch’s correction. n = 146 (control) and n = 166 ( apoa1bp −/− ) cells. Scale bar: 50 µm. Source data are provided as a file.

Article Snippet: Primary human dermal lymphatic microvascular endothelial cells (hLECs) were purchased from PromoCell (Cat # C-12216 and C-12217).

Techniques: Phospho-proteomics, Immunoprecipitation, Western Blot, Incubation, Recombinant, Control, Activation Assay, Immunostaining, Expressing

Journal: Nature Communications

Article Title: APOA1 binding protein promotes lymphatic cell fate and lymphangiogenesis by relieving caveolae-mediated inhibition of VEGFR3 signaling

doi: 10.1038/s41467-025-60611-w

Figure Lengend Snippet: a Conserved CAV-1 binding site on VEGFR3 in human (Hu), mouse (Ms), and zebrafish (Zf). The conserved amino acids are shown in blue. b Co-immunoprecipitation of endogenous VEGFR3 and CAV-1 in hLECs. Lysates from two 10 cm confluent plates of hLECs were combined, then equally divided for immunoprecipitation using VEGFR3 antibody or control protein A beads. The samples were subsequently immunoblotted for CAV-1 and VEGFR3. c , d VEGFR3 AAA loses its binding to CAV-1. c hLECs were transfected with control EGFP, VEGFR3-EGFP (R3), or VEGFR3 AAA -EGFP (R3 AAA ) using lentivirus-mediated gene transduction. After 72 hours, the resulting cells were lysed and immunoprecipitated with GFP antibody conjugated to agarose beads and immunoblotted using GFP and CAV-1 antibodies. d The input lysates were immunoblotted using GFP, CAV1, or GAPDH antibody as indicated. e Localization of VEGFR3 and VEGFR3 AAA in caveolae. hLECs were transduced with VEGFR3-APEX2 or VEGFR3 AAA -APEX2 Lenti-viral particles, and after 72 h, cells were fixed with 2.5% glutaraldehyde, stained using DAB substrate kit, and pelleted for TEM analysis. An enlarged view of a single caveola, highlighted with a white box, is shown in the top left corner of each image. f – h hLECs were transduced using lentivirus, and the resulting cells were growth factor starved and treated with 100 ng/mL VEGFC for 20 min, cells were then lysed and immunoblotted as indicated. R3/R3 AAA -EGFP denotes detection using GFP antibody. Quantitative data of VEGFR3 activation ( g ), ERK activation ( i ), and AKT activation ( j ) were shown. Mean ± SD; two-way ANOVA with Tukey’s post-hoc test. n = 3 independent repeats in g , i , j . Endg: endogenous. Scale bar: 400 nm. Source data are provided as a file.

Article Snippet: Primary human dermal lymphatic microvascular endothelial cells (hLECs) were purchased from PromoCell (Cat # C-12216 and C-12217).

Techniques: Binding Assay, Immunoprecipitation, Control, Transfection, Transduction, Staining, Activation Assay

Journal: The FASEB Journal

Article Title: The deubiquitinating enzyme USP48 stabilizes TRAF2 and reduces E-cadherin-mediated adherens junctions

doi: 10.1096/fj.201700415RR

Figure Lengend Snippet: Knockdown of USP48 or inhibition of TNIK and JNK promotes epithelial barrier integrity. A) Scheme showing principle of ECIS system. B) Knockdown of USP48 increases TEER in A549 cells. A549 cells cultured on ECIS gold electrodes were transfected with control siRNA or Usp48 siRNA. After transfection, changes in TEER at 0 to 36 h were measured with ECIS. Data are presented as means ± sd (n = 4). C) Knockdown of USP48 increases TEER in Beas2B cells. Beas2B cells cultured on ECIS gold electrodes were transfected with control siRNA or Usp48 siRNA. After transfection, changes in TEER at 48 h were measured with ECIS. Data are presented as means ± sd (n = 4). P < 0.01 compared to control siRNA–transfected cells. The rest of the cells were analyzed by USP48, TRAF2, and β-actin immunoblotting. D) Inhibition of TNIK or JNK increases TEER. Beas2B cells cultured on ECIS gold electrodes were treated with DMSO (10 μM), KY-05009 (10 μM), or SP600125 (10 μM), and changes in TEER at 0 to 36 h were measured with ECIS. Data are presented as means ± sd, n = 4. E) Knockdown of USP48 promotes LPA-increased TEER. Human bronchial epithelial cells cultured on ECIS gold electrodes were transfected with control siRNA or Usp48 siRNA and incubated for 3 d. Cells were then challenged with LPA (0.5 μM), and changes in TEER were measured with ECIS. F) Scheme showing GSK3β phosphorylates and activates USP48, thus stabilizing TRAF2 and influencing TNIK/JNK-mediated E-cadherin expression and epithelial barrier integrity.

Article Snippet: Human lung epithelial cells [Beas2B and human bronchial epithelial cells; American Type Culture Collection (ATCC), Manassas, VA, USA] and murine lung epithelial 12 (MLE12) cells (ATCC) were cultured with medium supplemented with hydrocortisone, insulin, transferrin, estrogen, selenium, 10% fetal bovine serum, and antibiotics at 37°C in 5% CO 2 incubator.

Techniques: Knockdown, Inhibition, Cell Culture, Transfection, Control, Western Blot, Incubation, Expressing

Journal: Cancer research

Article Title: Cancer-induced muscle wasting requires p38β MAPK-mediated activation of p300

doi: 10.1158/0008-5472.CAN-19-3219

Figure Lengend Snippet: (A) LLC induces p300 phosphorylation on Ser-12 and this reaction is critical to p300-mediated acetylation and activation of C/EBPβ. C2C12 myoblasts were transfected with a plasmid encoding phosphorylation-defective mutant of p300, p300-S12A or p300-S89A, or empty vector as control in 3 independent experiments. After differentiation, myotubes were treated with LLC cell conditioned medium (LCM) or conditioned medium of non-tumorigenic NL20 cells for 2 h. Cell lysates were analyzed by Western blotting. (B) Overexpression of p300-S12A attenuates muscle weight loss in LLC tumor-bearing mice. Tibialis muscle (TA) of LLC tumor-bearing mice was transfected with the p300-S12A-encoding plasmid (n = 5). The contralateral TA was transfected with empty vector as control. After the development of cachexia, TA muscles were collected and weighed on day 21. Overexpression of p300-S12A was confirmed by Western blotting analysis against p300. (C) Overexpression of p300-S12A prevents the loss of myofiber mass in LLC tumor-bearing mice. H&E-stained TA cross sections of (B) were analyzed for myofiber cross-sectional area. (D) Overexpression of p300-S12A in TA attenuates muscle weight loss in KPC tumor-bearing mice (n = 5). (E) Overexpression of p300-S12A in TA prevents the loss of myofiber mass of KPC tumor-bearing mice (n = 5). (F) Overexpression of p300-S12D in TA causes loss of muscle weight in cancer-free mice (n = 5). (G) Overexpression of p300-S12D in TA causes loss of myofiber mass in cancer-free mice (n = 5). * indicates a statistically significant difference (p < 0.05) determined by one-way ANOVA (A), paired Student t-test (B, D and F) or Chi-square test (C, E and G).

Article Snippet: Conditioned medium of non-tumorigenic NL20 cells (human lung epithelial cells, ATCC) was used as control.

Techniques: Activation Assay, Transfection, Plasmid Preparation, Mutagenesis, Western Blot, Over Expression, Staining

Journal: bioRxiv

Article Title: AIBP-CAV1-VEGFR3 axis dictates lymphatic cell fate and controls lymphangiogenesis

doi: 10.1101/2020.10.16.342998

Figure Lengend Snippet: ( A and B ). Cholesterol removal potentiates VEGFR3 signaling. ( A ) hLECs were serum-starved, and treated with 10 mM MβCD for 30 min, and cells were further stimulated with 100 ng/ml VEGFC. The resulting cells were lysed and blotted with Cav-1 or GAPDH antibodies. ( B ) hLECs were treated as in ( A ). and cell lysates were immunoprecipitated using VEGFR3 antibody. Immunoblotting was performed using anti-phosphotyrosine (4G10) and VEGFR3 antibodies. ( C ) AIBP-mediated cholesterol efflux disrupts caveolae and reduces CAV-1 levels in the caveolar fractions. hLECs were treated with recombinant 200 ng/ml AIBP, 100 μg/ml HDL 3 , or both in serum-free EBM2 for 6 hours, and the cells were subjected to sucrose-mediated ultracentrifugation. The resulting fractions were collected for Western blot analysis as indicated. ( D ) AIBP-mediated cholesterol efflux increases VEGFR3 signaling. hLECs were serum-starved and treated as in ( C ), and further stimulated with 100 ng/ml VEGFC. The resulting cells were lyzed and immunoblotted as indicated. ( E and F ), Quantitative data of AKT activation ( E ) and ERK activation ( F ). Mean±SD, n=3 independent repeats. *, p<0.05; **, p<0.01; ns: not significant.

Article Snippet: Human dermal lymphatic microvascular endothelial cells (hLECs) (Angio-Proteomie, Cat # cAP-0003) were cultured in Endothelial Cell Medium (ScienCell, Cat # 1001).

Techniques: Immunoprecipitation, Western Blot, Recombinant, Activation Assay

Journal: bioRxiv

Article Title: AIBP-CAV1-VEGFR3 axis dictates lymphatic cell fate and controls lymphangiogenesis

doi: 10.1101/2020.10.16.342998

Figure Lengend Snippet: ( A ) Conserved CAV-1 binding site on VEGFR3 in human (Hu), mouse (Ms), and zebrafish (Zf). ( B ) VEGFR3 AAA loses its binding to CAV-1. hLECs were transfected with control EGFP (Ctrl), VEGFR3-EGFP (R3), or VEGFR3 AAA -EGFP (R3 AAA ) using lentivirus-mediated gene transfer. After 72 hours, the resulting cells were lyzed and immunoprecipitated with EGFP antibody coupled to the magnetic Dynabeads and immunoblotted using VEGFR3 and CAV-1 antibodies. Immunoblotting of overexpressed VEGFR3-EGFP and VEGFR3 AAA -EGFP (R3/R3 AAA -EGFP) were detected using VEGFR3 antibody. The input lysates were shown on the right. ( C ) VEGFR3 AAA increases VEGFR3 signaling. hLECs were transduced as in ( B ), and the resulting cells were serum starved and treated with 100 ng/ml VEGFC for 20 min, cells were then lysed and immunoblotted as indicated. Quantitative data of VEGFR3 activation ( D ), AKT activation ( E ), and ERK activation ( F ) were shown. n=3 independent repeats. *, p<0.05; **, p<0.01; ***, p<0.001.

Article Snippet: Human dermal lymphatic microvascular endothelial cells (hLECs) (Angio-Proteomie, Cat # cAP-0003) were cultured in Endothelial Cell Medium (ScienCell, Cat # 1001).

Techniques: Binding Assay, Transfection, Control, Immunoprecipitation, Western Blot, Activation Assay

Journal: Bioactive Materials

Article Title: Drug-eluting intraocular lens with sustained bromfenac release for conquering posterior capsular opacification

doi: 10.1016/j.bioactmat.2021.07.015

Figure Lengend Snippet: Effects of bromfenac on TGF-β2-induced EMT and cell migration in HLEC-B3 cells: the FN (A), MMP2 (B), and α-SMA (C) mRNA expression induced by 10 ng/mL TGF-β2 at 2 h, 12 h, and 24 h, measured by qPCR (*p < 0.05, **p < 0.01 vs TGF-β2 free group); effects of bromfenac in different concentrations on the TGF-β2-induced upregulation of FN (D), MMP2 (E), and α-SMA (F) mRNA expression measured by qPCR (*p < 0.05, **p < 0.01 vs TGF-β2 treated alone group); (G) FN, MMP2, and α-SMA protein expression induced by 10 ng/mL TGF-β2 for different time points detected by Western blot; (H) effects of bromfenac in different concentrations on the TGF-β2-induced upregulation of FN, MMP2, and α-SMA protein expression detected by Western blot. The cells were pre-treated, with or without bromfenac, for 24 h before induced by 10 ng/mL TGF-β2 for 24 h. (I) the wound-healing assay showed the effects of bromfenac in different concentrations on TGF-β2-induced cell migration (scale bar, 500 μm; n = 3; error bars represent SEM).

Article Snippet: A human lens epithelial cell line (HLEC-B3) was obtained from the American type culture collection (ATCC, Manassas, VA, US) and grown in Dulbecco's Modified Eagle Medium—Nutrient Mixture F-12 (DMEM/F12; Gibco, Grand Island, NY, US), containing 10% fetal bovine serum (FBS; Gibco) at 37 °C in a 5% CO 2 humidified atmosphere.

Techniques: Migration, Expressing, Western Blot, Wound Healing Assay

Journal: Bioactive Materials

Article Title: Drug-eluting intraocular lens with sustained bromfenac release for conquering posterior capsular opacification

doi: 10.1016/j.bioactmat.2021.07.015

Figure Lengend Snippet: Immunofluorescence staining of the change of cellular EMT markers: 80 μg/mL bromfenac inhibited the upregulation of FN (A), MMP2 (B), and α-SMA (C) induced by 10 ng/mL TGF-β2 in HLEC-B3 cells (scale bar, 100 μm).

Article Snippet: A human lens epithelial cell line (HLEC-B3) was obtained from the American type culture collection (ATCC, Manassas, VA, US) and grown in Dulbecco's Modified Eagle Medium—Nutrient Mixture F-12 (DMEM/F12; Gibco, Grand Island, NY, US), containing 10% fetal bovine serum (FBS; Gibco) at 37 °C in a 5% CO 2 humidified atmosphere.

Techniques: Immunofluorescence, Staining

Journal: Bioactive Materials

Article Title: Drug-eluting intraocular lens with sustained bromfenac release for conquering posterior capsular opacification

doi: 10.1016/j.bioactmat.2021.07.015

Figure Lengend Snippet: Effects of bromfenac on the TGF-β2-activated ERK/GSK-3β pathway in HLEC-B3. U0126 suppressed TGF-β2-induced up-regulation of the EMT markers FN, MMP2, α-SMA, and Snail. (A) TGF-β2 stimulated the phosphorylation of ERK1/2 and GSK-3β, and (B) bromfenac significantly inhibited phosphorylation. The cells were pre-treated with or without bromfenac for 24 h before induced by 10 ng/mL TGF-β2. (C) TGF-β2 stimulated the phosphorylation of Smad2/3, but (D) bromfenac could not inhibit phosphorylation; and pre-treatment with U0126 for 2 h suppressed the TGF-β2-induced upregulation of FN (E), MMP2 (F), α-SMA (G), and Snail (H) mRNA, and protein expression (I) (*p < 0.05, **p < 0.01 vs TGF-β2 treated alone group, n = 3, error bars represent SEM).

Article Snippet: A human lens epithelial cell line (HLEC-B3) was obtained from the American type culture collection (ATCC, Manassas, VA, US) and grown in Dulbecco's Modified Eagle Medium—Nutrient Mixture F-12 (DMEM/F12; Gibco, Grand Island, NY, US), containing 10% fetal bovine serum (FBS; Gibco) at 37 °C in a 5% CO 2 humidified atmosphere.

Techniques: Phospho-proteomics, Expressing

Journal: Bioactive Materials

Article Title: Drug-eluting intraocular lens with sustained bromfenac release for conquering posterior capsular opacification

doi: 10.1016/j.bioactmat.2021.07.015

Figure Lengend Snippet: Effects of U0126 and CHIR-99021 on TGF-β2-induced EMT in HLEC-B3 and the role of Snail in TGF-β2-induced EMT. The cells were treated with 20 nM CHIR-99021 for 24 h and 20 μM U0126 for 2 h before 10 ng/mL TGF-β2 treatment. The mRNA (A, B, C, and D) and protein (E) expression of FN, MMP2, α-SMA, and Snail detected by qPCR and Western blot analysis, respectively (*p < 0.05, **p < 0.01 between groups); (F) the TGF-β2-induced phosphorylation of GSK-3β after treatment with CHIR-99021 and U0126 and evaluated by Western blot analysis. The cells were pre-treated with or without bromfenac for 24 h before induced by 10 ng/mL TGF-β2, and Snail expression (G) was detected by Western blot analysis. The cells were transfected with Snail siRNA or NC siRNA before bromfenac and 10 ng/mL TGF-β2 treatment. The protein (H) and mRNA (I, J, and K) expression of FN, MMP2, and α-SMA detected by qPCR and Western blot analysis, respectively (*p < 0.05, **p < 0.01 between groups). NS represents no significance compared with the Snail siRNA + TGF-β2 treated alone group (n = 3, error bars represent SEM).

Article Snippet: A human lens epithelial cell line (HLEC-B3) was obtained from the American type culture collection (ATCC, Manassas, VA, US) and grown in Dulbecco's Modified Eagle Medium—Nutrient Mixture F-12 (DMEM/F12; Gibco, Grand Island, NY, US), containing 10% fetal bovine serum (FBS; Gibco) at 37 °C in a 5% CO 2 humidified atmosphere.

Techniques: Expressing, Western Blot, Phospho-proteomics, Transfection

Journal: PLoS ONE

Article Title: Transcriptional Protein Sp1 Regulates LEDGF Transcription by Directly Interacting with Its Cis -Elements in GC-Rich Region of TATA-Less Gene Promoter

doi: 10.1371/journal.pone.0037012

Figure Lengend Snippet: A and B, mRNA expression levels of LEDGF/p75 ( black bars ) and Sp1 ( gray bars ) were analyzed by real time PCR. Total RNA was isolated from LECs separated from lenses of human subjects of different age groups and reverse transcribed cDNA was subjected to real time PCR analysis with specific primers as detailed in . Age group 1 (n = 4, 16–26 years); Age group 2 (n = 3, 34–42 years); Age group 3 (n = 7, 52–75 years). n; denotes number of subjects. The data represent the mean ± S.D. from three independent experiments (** p<0.001 ). C, Western analysis of LEDGF/p75 and Sp1 protein using their corresponding specific antibodies. hLECs isolated from eye lenses of 24- and 64-year-old human subjects were cultured as described in . Cellular proteins from confluent cells were extracted, and equivalent amounts were loaded onto SDS-PAGE, transferred to a PVDF membrane and processed for immunoblotting. Western analysis showed the expression levels of LEDGF/p75 ( upper panel) and Sp1 ( middle panel). Lower panel, membrane probed with β-actin antibody as loading/internal control. The same membrane was probed and reprobed with antibodies following stripping and restriping to obtain relative expression of Sp1, LEDGF/p75 or β-actin. Each band of blot was quantified using densitometer shown at the right . Images are representatives from three independent experiments. D and E, Sp1 upregulated expression of LEDGF/p75 protein and mRNA in hLECs in dose dependent fashion. hLECs were transfected with either pCMV-vector or increasing amounts of pCMV-Sp1 (2, 4 and 8 µg) as indicated and described in section. Total Protein and RNA were extracted after 48 h of transfection and were used for Western analysis (D) and real time PCR (E) respectively, using specific probes. D, left , Western analysis data showing the expression levels of LEDGF/p75 ( upper panel) in cells transfected with plasmid encoding Sp1 at different concentrations ( middle panel). Lower panel, membrane probed with β-actin antibody. The same membrane was probed and reprobed with antibodies following stripping and restriping to obtain relative expression of Sp1, LEDGF/p75 or β-actin. Right , Histogram displaying relative protein band density indicated as values ± S.D. of three independent experiments. E, Histogram showing the values (mean ± S.D.) of Sp1 concentration-dependent expression of LEDGF/p75 mRNA (black bars vs gray bars) obtained from three independent experiments (** p <0.001). F, A Sp1 inhibitor, artemisinin, reduced expression of LEDGF/p75 in LECs in dose-dependent manner. Cultured cells were treated with either increasing concentrations of artemisinin (50, 150 and 300 µM) or with vehicle control. Cell lysates were resolved onto SDS-PAGE and analyzed by Western blot for the effects of artemisinin on expression of LEDGF/p75 and Sp1 protein. Relative band density in pixels is shown below the Western blot images (* p <0.01, ** p <.001). β-actin was used as internal control. G and H, Representative immunoblots showing depletion of Sp1 using Sp1 Knockdown assay. Sp1-specific shRNA constructs were transiently (G) and stably (H) transfected as described in section. Protein lysate was prepared and Western analysis was carried out. The same membrane was probed and reprobed with antibodies following stripping and restriping to obtain relative expression of Sp1 or LEDGF/p75 or β-actin. Relative band density in pixels is shown below the Western blot images (** p <.001).

Article Snippet: Human lens epithelial cells (hLECs) (a gift of Dr. V. N. Reddy, Eye Research Institute, Oakland University, Rochester, MI) and Cos7 cells (ATCC; CRL-1651) were maintained routinely in our laboratory following the method described elsewhere .

Techniques: Expressing, Real-time Polymerase Chain Reaction, Isolation, Reverse Transcription, Western Blot, Cell Culture, SDS Page, Membrane, Control, Stripping Membranes, Transfection, Plasmid Preparation, Concentration Assay, Knockdown, shRNA, Construct, Stable Transfection

Journal: PLoS ONE

Article Title: Transcriptional Protein Sp1 Regulates LEDGF Transcription by Directly Interacting with Its Cis -Elements in GC-Rich Region of TATA-Less Gene Promoter

doi: 10.1371/journal.pone.0037012

Figure Lengend Snippet: A, Left half, diagrams showing the 5′-deletion constructs of LEDGF/p75 promoter linked to CAT reporter gene used for transient transfections. Right half, CAT activity of the LEDGF/p75 promoter deletion constructs and empty CAT vector in hLECs. 5′-deletion mutant constructs and pGFP were cotransfected into hLECs. 48 h later, protein was extracted and CAT activity was measured. CAT activity ( right ) was normalized to GFP readings (O.D.). The data represent the mean ± S.D. from three independent experiments. B, Point mutation analysis showing Sp1 site-dependent transcriptional activity of the LEDGF/p75 gene promoter in hLECs. Left half, schematic representation of Sp1-site-directed mutants of LEDGF/p75 promoter linked to CAT. Right half, CAT activity of the wild-type (WT) and its mutant constructs (Mut-3, Mut-2, Mut-1 and Mut- 1+2+3) and empty CAT vector in hLECs. All data are presented as the mean ± S.D. derived from three independent experiments (* p <0.01, ** p <0.001).

Article Snippet: Human lens epithelial cells (hLECs) (a gift of Dr. V. N. Reddy, Eye Research Institute, Oakland University, Rochester, MI) and Cos7 cells (ATCC; CRL-1651) were maintained routinely in our laboratory following the method described elsewhere .

Techniques: Construct, Transfection, Activity Assay, Plasmid Preparation, Mutagenesis, Derivative Assay

Journal: PLoS ONE

Article Title: Transcriptional Protein Sp1 Regulates LEDGF Transcription by Directly Interacting with Its Cis -Elements in GC-Rich Region of TATA-Less Gene Promoter

doi: 10.1371/journal.pone.0037012

Figure Lengend Snippet: A, Representative gel-shift mobility assays showing Sp1 binding to radiolabeled oligonucleotide probes containing consensus Sp1 sites as indicated. Nuclear extracts isolated from hLECs were incubated with 32 p-labeled probes containing Sp1 binding sites (WT-probes) or their corresponding mutants (Mut probes). Nuclear extracts bound to oligos containing Sp1 sites and yielded to complex, Sp1/DNA (Cm1) (A, lanes 1, 3 and 5). No complex occurred with mutant probes (A, lanes 2, 4, and 6). The oligonucleotide probes of both wild-type and mutated sequence used in assay are shown adjacent to image. B, Gel-shift assay showing the binding of Sp1 in nuclear extract of Sp1 overexpressed with hLECs to 32 p-labeled probes with its site. Nuclear extract isolated from cells transfected with plasmid encoding Sp1 or its corresponding vector was incubated with WT-probe1 or standard control probe (sc-2502; Santa Cruz Biotech). The DNA-protein complex was resolved on a 5% acrylamide gel. A discrete Sp1 expression-dependent DNA-protein complex was observed (B; lanes 1 vs 3) in comparison to vector transfected cells (lane 1), while the mutated probe failed to generate the complex (B, lanes 2 and 4). B, Right (lanes 5 and 6) , depletion of endogenous Sp1 with its specific antibody. Nuclear extracts were incubated with either anti-Sp1 antibody (lane 6) or normal rabbit IgG (lane 5), and recovered nuclear extracts were incubated with the same probes (lanes 5 and 6). Lanes 7 and 8, standard control containing • Sp1 site (sc-2502, Santa Cruz Biotech) or its @ mutant (sc-2503) processed for gel-shift assay using the same nuclear extracts. Extreme right , Depletion assay using anti-Sp3 antibody with nuclear extract showing no change in Sp1/DNA complex (lane 10) and the complex was indistinguishable from Lane 9. Images are representatives from three independent consistent observations.

Article Snippet: Human lens epithelial cells (hLECs) (a gift of Dr. V. N. Reddy, Eye Research Institute, Oakland University, Rochester, MI) and Cos7 cells (ATCC; CRL-1651) were maintained routinely in our laboratory following the method described elsewhere .

Techniques: Gel Shift, Binding Assay, Isolation, Incubation, Labeling, Mutagenesis, Sequencing, Transfection, Plasmid Preparation, Control, Acrylamide Gel Assay, Expressing, Comparison, Depletion Assay

Journal: PLoS ONE

Article Title: Transcriptional Protein Sp1 Regulates LEDGF Transcription by Directly Interacting with Its Cis -Elements in GC-Rich Region of TATA-Less Gene Promoter

doi: 10.1371/journal.pone.0037012

Figure Lengend Snippet: A, Interrupting Sp1 activity by artemisinin interrupted LEDGF/p75 promoter activity in a concentration-dependent manner. Upper panel, a diagram of the LEDGF/p75 promoter representing three Sp1-binding sites (−170/+35) used for CAT activity. A selective Sp1 inhibitor , artemisinin, reduced the activity of LEDGF/p75 promoter in LECs in dose-dependent fashion. Artemisinin or its diluents (control) were added to culture medium of LEDGF/p75 promoter constructs or empty vector transfected cells monolayer. Cells were disrupted and CAT activities were measured as described in the section. Data are the mean of three experiments, and error bars indicate standard deviation (** p<0.001 ). B and C, Influence of Sp1 overexpression on transcriptional activity of LEDGF/p75 promoter. Plasmid encoding pCAT- LEDGF/p75 (−170/+35) or pCAT-V was cotransfected into hLECs ( B ) and Cos7 cells ( C ) with indicated amounts of pCMV-Sp1. Following CAT assay, CAT values were analyzed and represented as histograms, with Sp1 (black bar) or without Sp1 (open bar) overexpression. Empty CAT vector shows insignificant CAT activity (gray bar). Transfections were carried out as described in and level of Sp1 protein was evaluated using Western analysis (B and C, Upper panel). The data are representative of at least three independent experiments. Each value represents the mean ± S.D. (** p<0.001 ).

Article Snippet: Human lens epithelial cells (hLECs) (a gift of Dr. V. N. Reddy, Eye Research Institute, Oakland University, Rochester, MI) and Cos7 cells (ATCC; CRL-1651) were maintained routinely in our laboratory following the method described elsewhere .

Techniques: Activity Assay, Concentration Assay, Binding Assay, Control, Construct, Plasmid Preparation, Transfection, Standard Deviation, Over Expression, Western Blot

Journal: PLoS ONE

Article Title: Transcriptional Protein Sp1 Regulates LEDGF Transcription by Directly Interacting with Its Cis -Elements in GC-Rich Region of TATA-Less Gene Promoter

doi: 10.1371/journal.pone.0037012

Figure Lengend Snippet: A, Repression of LEDGF/p75 transcription by Sp1 Sumoylation. Cells were transfected or cotransfected with pCAT- LEDGF/p75 (pCAT-LED) or pCAT vector (pCAT-V) and/or with increasing amounts of a plasmid encoding Sumo1 (pEGFP-Sumo1) as indicated. Cells were disrupted at predefined times and processed for CAT assay. Data indicate CAT activity in cells overexpressing different amounts of Sumo1 (A, gray bars) and without Sumo1 (black bars). Experiments were performed three times, and data are presented as mean ± S.D. B, ChIP assay coupled with desumoylation and DNA-protein complex dissociation experiments showed that the effect of Sumo1 on the abundance of Sp1 was concentration-dependent. hLECs were transfected with either pCMV-Sp1 alone or cotransfected with pEGFP-Sumo1 or pFLAG-Senp1. ChIP assay was performed in duplicates from each sample with anti-Sp1 or anti-Sp3 antibody or control IgG. Following processing, one set of precipitated samples was submitted for PCR analysis of Sp1 responsive region of LEDGF/p75 promoter (B) as described in . In another set of experiments, DNA bound proteins were eluted with high salt solution, and Western analysis was performed on elutes to measure Sp1 prevalence by anti-Sp1-antibody (C: lane 1, pCMV-Sp1; lane 2, pEGFP-Sumo1; lane 3, pFLAG-Senp1; lane 4, pCMV-Sp1 plus pEGFP-Sumo1; lane 5, pCMV-Sp1 plus pFLAG-Senp1; p-vector). Images shown in the panel (B) are of representatives of Sp1-3 (site 3) region. Similar results were obtained with Sp1-1 (site 1) and Sp1-2 (site 2) in the LEDGF/p75 promoter when ChIP-PCR analysis was done (data not shown). Following stripping of Sp1 immunoblotted membrane, the same membrane was reprobed with Sp3 specific antibody, and no bands were observed (D).

Article Snippet: Human lens epithelial cells (hLECs) (a gift of Dr. V. N. Reddy, Eye Research Institute, Oakland University, Rochester, MI) and Cos7 cells (ATCC; CRL-1651) were maintained routinely in our laboratory following the method described elsewhere .

Techniques: Transfection, Plasmid Preparation, Activity Assay, Concentration Assay, Control, Western Blot, Stripping Membranes, Membrane

Journal: PLoS ONE

Article Title: Transcriptional Protein Sp1 Regulates LEDGF Transcription by Directly Interacting with Its Cis -Elements in GC-Rich Region of TATA-Less Gene Promoter

doi: 10.1371/journal.pone.0037012

Figure Lengend Snippet: A, hLECs were transfected with either mock, negative control siRNA or LEDGF/p75 siRNA. Following transfection, cell extracts were prepared and expression was examined by Western analysis using Anti-LEDGF/p75 antibody (A). Relative density in pixels is shown on the right . B, Control siRNA (siControl) or LEDGF/p75 siRNA (siLEDGF/p75) transfected cells were seeded in 12-well plates and submitted to UVB exposure as described in . A survival assay-MTS assay was conducted, and data shown are mean ± S.D. values of three independent experiments. ** p<0.001 compared with control siRNA. C, Sp1 overexpression in cells with siRNA LEDGF/p75 conferred resistance against UVB stress. LEDGF/p75 siRNA transfected hLECs were transiently re-transfected with pCMV-Sp1 and then exposed to UVB stress. MTS assay was performed to evaluate vulnerability. * p<0.01 compared with respective controls. D, Sp1 or LEDGF/p75 overexpression in hLECs provided cytoprotection against stress induced by UVB. Cells were cultured and exposed to different doses of UV stress as indicated. Cell viability was analyzed using MTS assay as described in . * * p<0.001 compared with respective controls. Data represent mean ± S.D. from three independent experiments.

Article Snippet: Human lens epithelial cells (hLECs) (a gift of Dr. V. N. Reddy, Eye Research Institute, Oakland University, Rochester, MI) and Cos7 cells (ATCC; CRL-1651) were maintained routinely in our laboratory following the method described elsewhere .

Techniques: Transfection, Negative Control, Expressing, Western Blot, Control, Clonogenic Cell Survival Assay, MTS Assay, Over Expression, Cell Culture