Journal: Physiological Reports

Article Title: DDAH1 regulates apoptosis and angiogenesis in human fetal pulmonary microvascular endothelial cells

doi: 10.14814/phy2.14150

Figure Lengend Snippet: siRNA‐mediated knockdown of DDAH1 in hfPMVEC resulted in less NO production with no significant change in eNOS protein levels. (A) DDAH1 siRNA transfection of hfPMVECs resulted in effective knockdown of DDAH1 protein levels. hfPMVECs were transfected with either scramble siRNA or DDAH1 siRNA for 24 h, recovered for 24 h, and protein isolated. Representative western blot and bar graph of densitometry data for DDAH1 protein levels normalized to β ‐actin ( n = 6 in each group). * P < 0.05, DDAH1 siRNA different from scramble. (B) DDAH1 knockdown with siRNA resulted in lower NO production than scramble transfected cells. hfPMVECs were transfected with either scramble siRNA or DDAH1 siRNA for 24 h, washed and allowed to recover for 24 h. Cell were incubated for an additional 24 h and cell media harvested for determination of nitrites using a chemiluminescence NO analyzer. Nitrite levels were normalized to protein concentration in each plate ( n = 3 in each group). * P < 0.05, DDAH1 siRNA different from scramble. (C) DDAH1 siRNA transfection of hfPMVECs resulted in no significant change of eNOS protein levels. The protein isolated under B above was used in western blotting for eNOS protein levels. Representative western blot and bar graph of densitometry data for eNOS protein level normalized to β ‐actin ( n = 6 in each group).

Article Snippet: The following primary antibodies were used: DDAH1 (1:1000, Cat#: PA5‐35306, Lot#: RG2217435E, Thermo Scientific, Waltham, MA), eNOS (1:1000, Cat#:610296, Lot#:9, BD Transduction Laboratories, San Diego, CA), Cleaved caspase‐3 (1:1000, Cat#:9664, Lot#:20, Cell Signaling Danvers, MA), Total caspase‐3 (1:1000 Cat#:9662, Lot#:18, Cell Signaling), Cleaved caspase‐8 (1:1000, Cat#:9749, Lot#7, Cell Signaling), Total caspase‐8 (1:1000, Cat#: 4790, Lot#:2, Cell Signaling), Cleaved caspase‐9 (1:1000, Cat#:9505, Lot#:3, Cell Signaling), Total caspase‐9 (1:1000, Cat#:9502, Lot#:2, Cell Signaling), p21 (1:500, Cat#:471, Lot#: C196, Santa Cruz Biotechnology, Dallas, TX), and PCNA (1:5000, Cat#: P8825, Lot#: O37K4777, Sigma‐Aldrich, St. Louis, MO).

Techniques: Knockdown, Transfection, Isolation, Western Blot, Incubation, Protein Concentration

Journal: Physiological Reports

Article Title: DDAH1 regulates apoptosis and angiogenesis in human fetal pulmonary microvascular endothelial cells

doi: 10.14814/phy2.14150

Figure Lengend Snippet: siRNA‐mediated knockdown of DDAH1 in hfPMVEC resulted in lower cleaved caspase‐3 and cleaved caspase‐8 protein levels. hfPMVECs were transfected with either scramble siRNA control or DDAH1 siRNA for 24 h, allowed to recover for 24 h, and then incubated for 24 h and protein isolated for western blot analysis. (A) Transfection with DDAH1 siRNA resulted in lower cleaved caspase‐3 protein levels. Representative western blot and bar graphs for cumulative densitometry data of cleaved caspase‐3 protein levels normalized to total caspase‐3 protein levels ( n = 6 in each group). * P < 0.05, DDAH1 siRNA different from scramble. (B) Transfection with DDAH1 siRNA resulted in lower cleaved caspase‐8 protein levels. Representative western blot and bar graphs for cumulative densitometry data of cleaved caspase‐8 protein levels normalized to caspase‐8 ( n = 6 in each group). * P < 0.05, DDAH1 siRNA different from scramble. (C) Transfection with DDAH1 siRNA resulted in no significant change in cleaved caspase‐9 protein levels. Representative western blot and bar graphs for cumulative densitometry data of cleaved caspase‐9 protein levels normalized to caspase‐9 ( n = 6 in each group). (D) Transfection with DDAH1 siRNA resulted in no significant change in p21 protein levels. Representative western blot and bar graphs for cumulative densitometry data of p21 protein levels normalized to β ‐actin ( n = 6 in each group). (E) Transfection with DDAH1 siRNA resulted in no significant change in PCNA protein levels. Representative western blot and bar graphs for cumulative densitometry data of PCNA protein levels normalized to β ‐actin ( n = 6 in each group).

Article Snippet: The following primary antibodies were used: DDAH1 (1:1000, Cat#: PA5‐35306, Lot#: RG2217435E, Thermo Scientific, Waltham, MA), eNOS (1:1000, Cat#:610296, Lot#:9, BD Transduction Laboratories, San Diego, CA), Cleaved caspase‐3 (1:1000, Cat#:9664, Lot#:20, Cell Signaling Danvers, MA), Total caspase‐3 (1:1000 Cat#:9662, Lot#:18, Cell Signaling), Cleaved caspase‐8 (1:1000, Cat#:9749, Lot#7, Cell Signaling), Total caspase‐8 (1:1000, Cat#: 4790, Lot#:2, Cell Signaling), Cleaved caspase‐9 (1:1000, Cat#:9505, Lot#:3, Cell Signaling), Total caspase‐9 (1:1000, Cat#:9502, Lot#:2, Cell Signaling), p21 (1:500, Cat#:471, Lot#: C196, Santa Cruz Biotechnology, Dallas, TX), and PCNA (1:5000, Cat#: P8825, Lot#: O37K4777, Sigma‐Aldrich, St. Louis, MO).

Techniques: Knockdown, Transfection, Control, Incubation, Isolation, Western Blot

Journal: Physiological Reports

Article Title: DDAH1 regulates apoptosis and angiogenesis in human fetal pulmonary microvascular endothelial cells

doi: 10.14814/phy2.14150

Figure Lengend Snippet: DDAH1 transfection of hfPMVEC resulted in greater viable cell numbers. (A) hfPMVEC transfected with DDAH1 siRNA had greater viable cell numbers at 24, 48, and 72 h than scramble control. hfPMVEC were transfected with DDAH1 siRNA or scramble. After 24 h, the cells were washed, trypsinized, and 5 × 10 4 cells loaded in each well of a six‐well plate. After 24, 48, or 72 h, viable cell numbers were determined by trypan blue exclusion ( n = 3 in each group). * P < 0.05, DDAH1 siRNA different from scramble. (B) Non‐transfected hfPMVEC were seeded at 1 × 10 4 in each well of a six‐well plate and treated with 300 μ mol/L ADMA for 24 h or no treatment controls. ADMA‐treated cells had greater viable cell numbers than no treatment controls ( n = 5 in each group). * P < 0.05, different from control. Dotted line represents seeded cell number.

Article Snippet: The following primary antibodies were used: DDAH1 (1:1000, Cat#: PA5‐35306, Lot#: RG2217435E, Thermo Scientific, Waltham, MA), eNOS (1:1000, Cat#:610296, Lot#:9, BD Transduction Laboratories, San Diego, CA), Cleaved caspase‐3 (1:1000, Cat#:9664, Lot#:20, Cell Signaling Danvers, MA), Total caspase‐3 (1:1000 Cat#:9662, Lot#:18, Cell Signaling), Cleaved caspase‐8 (1:1000, Cat#:9749, Lot#7, Cell Signaling), Total caspase‐8 (1:1000, Cat#: 4790, Lot#:2, Cell Signaling), Cleaved caspase‐9 (1:1000, Cat#:9505, Lot#:3, Cell Signaling), Total caspase‐9 (1:1000, Cat#:9502, Lot#:2, Cell Signaling), p21 (1:500, Cat#:471, Lot#: C196, Santa Cruz Biotechnology, Dallas, TX), and PCNA (1:5000, Cat#: P8825, Lot#: O37K4777, Sigma‐Aldrich, St. Louis, MO).

Techniques: Transfection, Control

Journal: Physiological Reports

Article Title: DDAH1 regulates apoptosis and angiogenesis in human fetal pulmonary microvascular endothelial cells

doi: 10.14814/phy2.14150

Figure Lengend Snippet: hfPMVEC tube formation was lower in cells transfected with DDAH1 siRNA. (A) hfPMVECs were transfected with either scramble siRNA control or DDAH1 siRNA for 24 h. hfPMVECs were washed with DPBS, trypsinized, and resuspended in ECM + 1% FBS. 1.5 × 10 4 hfPMVEC were seeded into Matrigel‐coated well on a 96‐well cell culture plate. The plates were incubated at 37°C 21% O 2 ‐5% CO 2 ‐balance N 2 for 4–6 h at 37°C. Quantification was performed by counting tube branches per view field ( n = 6 in each group) using NIH ImageJ software (ImageJ, 1.47 Version, National Institutes of Health, Bethesda, MD). hfPMVEC transfected with DDAH1 siRNA had less endothelial tube formation as measured by tube branches per view field than scramble control. * P < 0.05, DDAH1 siRNA different from scramble. (B) Non‐transfected hfPMVECs were treated with either vehicle or 300 μ mol/L ADMA for 24 h and tube branches determined as in A above. hfPMVEC treated with 300 μ mol/L ADMA had fewer endothelial tubes as measured by tube branches per field of view than did vehicle treated controls. * P < 0.05, different from scramble control.

Article Snippet: The following primary antibodies were used: DDAH1 (1:1000, Cat#: PA5‐35306, Lot#: RG2217435E, Thermo Scientific, Waltham, MA), eNOS (1:1000, Cat#:610296, Lot#:9, BD Transduction Laboratories, San Diego, CA), Cleaved caspase‐3 (1:1000, Cat#:9664, Lot#:20, Cell Signaling Danvers, MA), Total caspase‐3 (1:1000 Cat#:9662, Lot#:18, Cell Signaling), Cleaved caspase‐8 (1:1000, Cat#:9749, Lot#7, Cell Signaling), Total caspase‐8 (1:1000, Cat#: 4790, Lot#:2, Cell Signaling), Cleaved caspase‐9 (1:1000, Cat#:9505, Lot#:3, Cell Signaling), Total caspase‐9 (1:1000, Cat#:9502, Lot#:2, Cell Signaling), p21 (1:500, Cat#:471, Lot#: C196, Santa Cruz Biotechnology, Dallas, TX), and PCNA (1:5000, Cat#: P8825, Lot#: O37K4777, Sigma‐Aldrich, St. Louis, MO).

Techniques: Transfection, Control, Cell Culture, Incubation, Software

Journal: Physiological Reports

Article Title: DDAH1 regulates apoptosis and angiogenesis in human fetal pulmonary microvascular endothelial cells

doi: 10.14814/phy2.14150

Figure Lengend Snippet: Treatment with an NO donor restored cleaved caspase‐3 and cleaved caspase‐8 protein levels in DDAH1 transfected hfPMVEC. hfPMVEC were transfected with DDAH1 siRNA or scramble for 24 h and then recovered for 24 h. The DDAH1 siRNA transfected hfPMVECs were then treated with either vehicle or 0.1 μ mol/L DETA NONOate, while untreated scramble transfected cells were used as a control. After a 24‐h incubation period, protein was isolated and western blotting done for cleaved and total caspase‐3 and ‐8. (A) Addition of the NO donor to DDAH1 siRNA transfected cells resulted in protein levels of cleaved caspase‐3 similar to those seen in the scramble control and significantly greater than DDAH1 siRNA transfected hfPMVEC treated with vehicle. Representative western blot and bar graphs for cumulative densitometry data for cleaved caspase‐3 protein levels normalized to total caspase‐3 ( n = 3 in each group). * P < 0.05, DDAH1 siRNA different from scramble; # P < 0.05, DDAH1 siRNA + NO donor different from DDAH1 siRNA. (B) Addition of the NO donor to the DDAH1 siRNA transfected hfPMVEC resulted in protein levels of cleaved caspase‐8 similar to those seen in scramble control and significantly greater than DDAH1 siRNA transfected hfPMVEC. Representative western blot and bar graphs for cumulative densitometry data for cleaved caspase‐8 protein levels normalized to caspase‐8 ( n = 3 in each group). * P < 0.05, DDAH1 siRNA different from scramble; # P < 0.05, DDAH1 siRNA + NO donor different from DDAH1 siRNA.

Article Snippet: The following primary antibodies were used: DDAH1 (1:1000, Cat#: PA5‐35306, Lot#: RG2217435E, Thermo Scientific, Waltham, MA), eNOS (1:1000, Cat#:610296, Lot#:9, BD Transduction Laboratories, San Diego, CA), Cleaved caspase‐3 (1:1000, Cat#:9664, Lot#:20, Cell Signaling Danvers, MA), Total caspase‐3 (1:1000 Cat#:9662, Lot#:18, Cell Signaling), Cleaved caspase‐8 (1:1000, Cat#:9749, Lot#7, Cell Signaling), Total caspase‐8 (1:1000, Cat#: 4790, Lot#:2, Cell Signaling), Cleaved caspase‐9 (1:1000, Cat#:9505, Lot#:3, Cell Signaling), Total caspase‐9 (1:1000, Cat#:9502, Lot#:2, Cell Signaling), p21 (1:500, Cat#:471, Lot#: C196, Santa Cruz Biotechnology, Dallas, TX), and PCNA (1:5000, Cat#: P8825, Lot#: O37K4777, Sigma‐Aldrich, St. Louis, MO).

Techniques: Transfection, Control, Incubation, Isolation, Western Blot

Journal: Physiological Reports

Article Title: DDAH1 regulates apoptosis and angiogenesis in human fetal pulmonary microvascular endothelial cells

doi: 10.14814/phy2.14150

Figure Lengend Snippet: Treatment with an NO donor decreased viable cell numbers in DDAH1 siRNA transfected cells. hfPMVEC were transfected with either DDAH1 siRNA or scramble for 24 h, the cells were washed and allowed to recover for 24 h. The cells were then trypsinized and equal numbers seeded in each well of a six‐well plate. Some DDAH1 siRNA transfected cells were treated with 0.1 μ mol/L DETA NONOate and some were treated with both 0.1 μ mol/L DETA NONOate and 100 μ mol/L Z‐DEVD‐FMK. After 72 h, viable cell numbers were counted using trypan blue exclusion ( n = 6, each group) and presented as fold change compared to scramble. Addition of the NO donor to hfPMVEC transfected with DDAH1 siRNA resulted in significantly lower viable cell numbers than vehicle treated DDAH1 siRNA‐transfected hfPMVEC. Addition of the caspase‐3 inhibitor to NO donor treated DDAH1 transfected hfPMVEC resulted in three‐fold greater viable cell numbers than in either scramble control or the DDAH1 siRNA transfected NO donor‐treated hfPMVEC. * P < 0.05, DDAH1 siRNA different than scramble; # P < 0.05, DDAH1 siRNA + NO donor different than DDAH1 siRNA; ¶ DDAH1 siRNA + NO donor + caspase‐3 inhibitor different from all other conditions, P < 0.05.

Article Snippet: The following primary antibodies were used: DDAH1 (1:1000, Cat#: PA5‐35306, Lot#: RG2217435E, Thermo Scientific, Waltham, MA), eNOS (1:1000, Cat#:610296, Lot#:9, BD Transduction Laboratories, San Diego, CA), Cleaved caspase‐3 (1:1000, Cat#:9664, Lot#:20, Cell Signaling Danvers, MA), Total caspase‐3 (1:1000 Cat#:9662, Lot#:18, Cell Signaling), Cleaved caspase‐8 (1:1000, Cat#:9749, Lot#7, Cell Signaling), Total caspase‐8 (1:1000, Cat#: 4790, Lot#:2, Cell Signaling), Cleaved caspase‐9 (1:1000, Cat#:9505, Lot#:3, Cell Signaling), Total caspase‐9 (1:1000, Cat#:9502, Lot#:2, Cell Signaling), p21 (1:500, Cat#:471, Lot#: C196, Santa Cruz Biotechnology, Dallas, TX), and PCNA (1:5000, Cat#: P8825, Lot#: O37K4777, Sigma‐Aldrich, St. Louis, MO).

Techniques: Transfection, Control

Journal: Physiological Reports

Article Title: DDAH1 regulates apoptosis and angiogenesis in human fetal pulmonary microvascular endothelial cells

doi: 10.14814/phy2.14150

Figure Lengend Snippet: Treatment with the NO donor increased tube branches in DDAH1 siRNA transfected hfPMVEC. hfPMVEC were transfected with either DDAH1 siRNA or scramble for 24 h, and the cells were washed and allowed to recover for 24 h. hfPMVEC were then trypsinized and equal numbers seeded in matrigel in each well of a 96‐well plate. Some of the DDAH1 siRNA transfected cells had 0.1 μ mol/L DETA NONOate added and some of the DDAH1 transfected cells had both 0.1 μ mol/L DETA NONOate and 100 μ mol/L Z‐DEVD‐FMK added. Treatments were given to cell media both prior to putting the cells in matrigel and again during the incubation in matrigel. After 4 h, the number of branches were determined. Quantification was performed by counting tube branches per view field ( n = 6 in each group) and presented as fold change compared to scramble. hfPMVEC transfected with DDAH1 siRNA and treated with the NO donor had more tube branches per field of view than did vehicle treated DDAH1 siRNA‐transfected hfPMVEC. hfPMVEC transfected with DDAH1 siRNA and treated with both the NO donor and the caspase‐3 inhibitor had fewer tube branches per field of view than any of the other groups. * P < 0.05, DDAH1 siRNA different from scramble; # P < 0.05, DDAH1 siRNA + NO donor different from DDAH1 siRNA; ¶ DDAH1 siRNA + NO donor + caspase‐3 inhibitor different from all other conditions, P < 0.05.

Article Snippet: The following primary antibodies were used: DDAH1 (1:1000, Cat#: PA5‐35306, Lot#: RG2217435E, Thermo Scientific, Waltham, MA), eNOS (1:1000, Cat#:610296, Lot#:9, BD Transduction Laboratories, San Diego, CA), Cleaved caspase‐3 (1:1000, Cat#:9664, Lot#:20, Cell Signaling Danvers, MA), Total caspase‐3 (1:1000 Cat#:9662, Lot#:18, Cell Signaling), Cleaved caspase‐8 (1:1000, Cat#:9749, Lot#7, Cell Signaling), Total caspase‐8 (1:1000, Cat#: 4790, Lot#:2, Cell Signaling), Cleaved caspase‐9 (1:1000, Cat#:9505, Lot#:3, Cell Signaling), Total caspase‐9 (1:1000, Cat#:9502, Lot#:2, Cell Signaling), p21 (1:500, Cat#:471, Lot#: C196, Santa Cruz Biotechnology, Dallas, TX), and PCNA (1:5000, Cat#: P8825, Lot#: O37K4777, Sigma‐Aldrich, St. Louis, MO).

Techniques: Transfection, Incubation

Journal: Physiological Reports

Article Title: DDAH1 regulates apoptosis and angiogenesis in human fetal pulmonary microvascular endothelial cells

doi: 10.14814/phy2.14150

Figure Lengend Snippet: Proposed model of DDAH1 inhibition of the NO‐mediated activation of cleaved caspase‐3 and resultant effects on viable cell numbers and angiogenesis. DDAH1 inhibition leads to an accumulation of ADMA, which inhibits eNOS function to produce less NO, this leads to lower levels of activated caspase‐3. Less activated caspase‐3 leads to more viable cells and decreased endothelial tube formation.

Article Snippet: The following primary antibodies were used: DDAH1 (1:1000, Cat#: PA5‐35306, Lot#: RG2217435E, Thermo Scientific, Waltham, MA), eNOS (1:1000, Cat#:610296, Lot#:9, BD Transduction Laboratories, San Diego, CA), Cleaved caspase‐3 (1:1000, Cat#:9664, Lot#:20, Cell Signaling Danvers, MA), Total caspase‐3 (1:1000 Cat#:9662, Lot#:18, Cell Signaling), Cleaved caspase‐8 (1:1000, Cat#:9749, Lot#7, Cell Signaling), Total caspase‐8 (1:1000, Cat#: 4790, Lot#:2, Cell Signaling), Cleaved caspase‐9 (1:1000, Cat#:9505, Lot#:3, Cell Signaling), Total caspase‐9 (1:1000, Cat#:9502, Lot#:2, Cell Signaling), p21 (1:500, Cat#:471, Lot#: C196, Santa Cruz Biotechnology, Dallas, TX), and PCNA (1:5000, Cat#: P8825, Lot#: O37K4777, Sigma‐Aldrich, St. Louis, MO).

Techniques: Inhibition, Activation Assay