Journal: eLife

Article Title: Pak2 is required for actin cytoskeleton remodeling, TCR signaling, and normal thymocyte development and maturation

doi: 10.7554/eLife.02270

Figure Lengend Snippet: ( A ) Western blot analysis using anti-Pak2 antiserum and cell lysates from the thymus of Pak2 F/F (WT), and Pak2 F/F ;Cd4 -Cre (KO) mice. Anti-Erk1/2 antibody was used as loading control. Shown are representative of two independent experiments. ( B ) Quantitative PCR analysis of Pak2 mRNA expression in DP, semi-mature and mature CD4SP thymocytes. Shown are Pak2 mRNA levels from DP, semi-mature and mature CD4SP thymocytes relative to Pak2 F/F DP thymocytes (error bars; SD). Data are representative of two independent experiments. ( C ) Western blot analysis using anti-PAK2 and cell lysates from the thymi of Pak2 F/F (WT), Pak2 F/+ ;Lck- Cre (HET) and Pak2 F/F ;Lck- Cre (KO) mice. ( D ) Representative flow cytometry analyses of CD4 and CD8 expression on lymphocytes from spleens (n = 5), peripheral lymph nodes (pLn; axillary, brachial and inguinal lymph nodes, n = 4), and mesenteric lymph nodes (mLn, n = 4) from Pak2 F/F (WT) and Pak2 F/F ;Lck -Cre (KO) mice. Numbers in each quadrant represent the percentage of cells in the indicated quadrant. ( E ) Flow cytometry analyses of CD62L and CD44 on CD4+ T cells from Pak2 F/F (WT) and Pak2 F/F ;Lck -Cre (KO) mice. ( F ) Quantification of cell numbers of different lymphocyte subsets from Pak2 F/F and Pak2 F/F ;Lck- Cre mice. Error bars: SD (spleen [ n = 5 mice per genotype], pLn [ n = 4 mice per genotype], and mLn [n = 4 mice per genotype]; blood [n = 2 mice per genotype]). *, 0.01

Article Snippet: Pak2 (TA306346) antibody is from Origene (Rockville, MD).

Techniques: Western Blot, Real-time Polymerase Chain Reaction, Expressing, Flow Cytometry, Two Tailed Test, Generated

Journal: eLife

Article Title: Pak2 is required for actin cytoskeleton remodeling, TCR signaling, and normal thymocyte development and maturation

doi: 10.7554/eLife.02270

Figure Lengend Snippet: ( A ) Quantification of cell numbers of different lymphocyte subsets from Pak2 F/F and Pak2 F/F ;Lck- Cre mice. Error bars: SD (spleen [ n = 5 mice per genotype], pLn [ n = 4 mice per genotype], and mLn [n = 4 mice per genotype]; blood [n = 2 mice per genotype]). *, 0.01

Article Snippet: Pak2 (TA306346) antibody is from Origene (Rockville, MD).

Techniques: Two Tailed Test

Journal: eLife

Article Title: Pak2 is required for actin cytoskeleton remodeling, TCR signaling, and normal thymocyte development and maturation

doi: 10.7554/eLife.02270

Figure Lengend Snippet: ( A ) Flow cytometry analysis of 1:1 mixed bone marrow chimeras. Chimeras were generated by transferring 1:1 mixed WT Pak2 +/+ (CD45.1 + CD45.2 + ) and KO Pak2 F/F ;Cd4 -Cre (CD45.2 + ) donor bone marrows into lethally irradiated C57BL6 hosts that express CD45.1. Data shown are representative of five bone marrow chimeras. CD4-CD8-double negative (DN) thymocytes generated from donor hematopoietic stem cells were gated using CD45.2 markers, and plotted with CD45.1 expression to distinguish T cells produced from WT (CD45.1 positive) and Pak2 F/F ;Cd4- Cre (CD45.1 negative) donors (first panel). Reconstitution of CD19+ B cells from spleen or pLN by WT (CD45.1 positive) and Pak2 F/F ;Cd4 -Cre (CD45.1 negative) donors showed similar contribution of donor BM cells from WT and Pak2 F/F ;Cd4 -Cre mice (second panel). In contrast, CD4 and CD8 T cells from Pak2 F/F ;Cd4 -Cre (CD45.1 negative) donors were markedly underrepresented compared to those from WT (CD45.1 positive) donors (third and fourth panels). ( B ) Total lymphocytes generated either from Pak2 +/+ or Pak2 F/F ;Cd4 -Cre donor bone marrow cells were gated and plotted as CD19 vs CD3 expression. ( C ) Expression of CD4 and CD8 is shown in CD3 positive cells generated either from Pak2 +/+ or Pak2 F/F ;Cd4 -Cre donor bone marrow cells. DOI: http://dx.doi.org/10.7554/eLife.02270.005

Article Snippet: Pak2 (TA306346) antibody is from Origene (Rockville, MD).

Techniques: Flow Cytometry, Generated, Transferring, Irradiation, Expressing, Produced

Journal: eLife

Article Title: Pak2 is required for actin cytoskeleton remodeling, TCR signaling, and normal thymocyte development and maturation

doi: 10.7554/eLife.02270

Figure Lengend Snippet: ( A ) Flow cytometry of lymphocytes from thymi of Pak2 F/F or Pak2 F/F ;Lck -Cre mice (n = 5). ( B ) Quantification of cell numbers of different thymic subsets from Pak2 F/F and Pak2 F/F ;Lck -Cre mice. Error bars: SEM (n = 5 mice per genotype). **, 0.001

Article Snippet: Pak2 (TA306346) antibody is from Origene (Rockville, MD).

Techniques: Flow Cytometry, Two Tailed Test, Expressing

Journal: eLife

Article Title: Pak2 is required for actin cytoskeleton remodeling, TCR signaling, and normal thymocyte development and maturation

doi: 10.7554/eLife.02270

Figure Lengend Snippet: ( A ) Flow cytometry analyses of CD4 SP thymocytes of Pak2 F/F (WT), Pak2 +/+ ;Cd4- Cre (WT), Pak2 F/+ ;Cd4- Cre (Het), or Pak2 F/F ;Cd4- Cre (KO) mice. ( B ) Quantification of cell numbers of different thymic subsets. Error bars: SEM (n = 10 mice). ( C ) CD4 and CD8 FACS analyses showing decreased percentage of CD4SP thymocytes in OTII + ; Pak2 F/F ;Cd4- Cre mice. Shown are representative data from three mice from each genotype. ( D ) Cell numbers of different thymic subsets from OTII + ; Pak2 F/F or OTII + ; Pak2 F/F ;Cd4- Cre mice. Graphs in this figure show mean ± SEM (n = 3). *, p=0.014. ( E ) Expression of TCR transgene assessed by anti-Vα2 antibody in total thymocytes from OTII + ; Pak2 F/F or OTII + ; Pak2 F/F ;Cd4- Cre mice. Shown are representative data from three mice from each genotype. DOI: http://dx.doi.org/10.7554/eLife.02270.007

Article Snippet: Pak2 (TA306346) antibody is from Origene (Rockville, MD).

Techniques: Flow Cytometry, Expressing

Journal: eLife

Article Title: Pak2 is required for actin cytoskeleton remodeling, TCR signaling, and normal thymocyte development and maturation

doi: 10.7554/eLife.02270

Figure Lengend Snippet: ( A ) CD4SP TCR hi thymocytes were gated and analyzed by expression of CD69 and CD62L. Fraction A (CD69 hi CD62L low ), semi-mature stage; Fraction B (CD69 low CD62L low ); Fraction C (CD69 low CD62L hi ), mature stage. Shown are representative data of ten mice per genotype. ( B ) Quantification of cell numbers of A, B, and C fractions in CD4SP thymocytes. Error bars: SEM (n = 10). *, 0.01

Article Snippet: Pak2 (TA306346) antibody is from Origene (Rockville, MD).

Techniques: Expressing, Two Tailed Test

Journal: eLife

Article Title: Pak2 is required for actin cytoskeleton remodeling, TCR signaling, and normal thymocyte development and maturation

doi: 10.7554/eLife.02270

Figure Lengend Snippet: ( A ) CD4SP TCR hi thymocytes were gated and analyzed by expression of CD69 and CD62L. Semi-mature stage (CD69 hi CD62L low ) and mature stage (CD69 low CD62L hi ) are shown. Note lack of the cells in the mature stage. ( B ) Upper panels: expression of TCR transgene assessed by anti-Vα2 antibody in CD4SP thymocytes from OTII + ; Pak2 F/F or OTII + ; Pak2 F/F ;Lck- Cre mice. Lower panels: Vα2 hi thymocytes in CD4SP thymocytes were gated and analyzed by expression of CD69 and CD62L. Shown are representative data from three mice from each genotype. DOI: http://dx.doi.org/10.7554/eLife.02270.009

Article Snippet: Pak2 (TA306346) antibody is from Origene (Rockville, MD).

Techniques: Expressing

Journal: eLife

Article Title: Pak2 is required for actin cytoskeleton remodeling, TCR signaling, and normal thymocyte development and maturation

doi: 10.7554/eLife.02270

Figure Lengend Snippet: ( A ) Apoptosis detected using 7AAD/Annexin V staining in freshly isolated Pak2 F/F or Pak2 F/F ;Cd4 -Cre CD4SP thymocytes. Semi-mature (CD69 hi CD62L low , Fraction A in ), mature (CD69 low CD62L hi , Fraction C in ) and CD69 low CD62L low (Fraction B in ) CD4SP thymocytes were gated and analyzed. Data are representative of two independent experiments. ( B ) Intracellular staining of active caspase 3 following 0, 1, and 2 hr of incubation in 10% FBS serum containing media. Data are representative of two independent experiments. ( C ) Apoptosis detected using 7AAD/Annexin V staining following 24 hr of incubation in 10% FBS serum containing media. Semi-mature and mature CD4SP thymocytes were gated and analyzed. Data are representative of two independent experiments. DOI: http://dx.doi.org/10.7554/eLife.02270.010

Article Snippet: Pak2 (TA306346) antibody is from Origene (Rockville, MD).

Techniques: Staining, Isolation, Incubation

Journal: eLife

Article Title: Pak2 is required for actin cytoskeleton remodeling, TCR signaling, and normal thymocyte development and maturation

doi: 10.7554/eLife.02270

Figure Lengend Snippet: ( A ) Flow cytometry analysis of 1:1 mixed bone marrow chimeras generated by transferring WT ( Pak2 +/+ , CD45.1 + CD45.2 + ) and Pak2 F/F ;Cd4- Cre mice (CD45.2 + ) donor bone marrow cells that contain hematopoietic stem cells (HSCs) into lethally irradiated C57BL6 hosts that express CD45.1+. Thymocytes generated either from Pak2 +/+ or Pak2 F/F ;Cd4- Cre donor bone marrow cells were identified using CD45 congenic markers and CD4 vs CD8 expression was shown. TCR hi CD4SP thymocytes were gated and expression of CD69 and CD62L was examined. Data shown are representative of five bone marrow chimeras. ( B ) Aberrant expression of trafficking molecules and maturation markers in CD4 SP thymocytes generated from Pak2 F/F ;Cd4- Cre donor. ( C ) Impaired expression of IL-7Rα in CD4SP T cells generated from Pak2 F/F ;Cd4- Cre donor bone marrow cells. ( D ) Block in the semi-mature to mature transition of Pak2-deficient CD4 SP thymocytes in fetal thymic organ culture. CD4 vs CD8 (top panels) expression of total culture and CD69 vs CD62L expression within TCR hi CD4SP population (bottom panels). Shown are data representative FTOC experiments of six embryos per each genotype. ( E ) Altered expression of maturation markers in TCR hi CD4SP thymocytes from FTOC. ( F ) IL-7Rα expression in DP, CD4SP, and semi-mature (CD69 hi CD62L low ) CD4SP thymocytes from FTOC. DOI: http://dx.doi.org/10.7554/eLife.02270.011

Article Snippet: Pak2 (TA306346) antibody is from Origene (Rockville, MD).

Techniques: Flow Cytometry, Generated, Transferring, Irradiation, Expressing, Blocking Assay, Organ Culture

Journal: eLife

Article Title: Pak2 is required for actin cytoskeleton remodeling, TCR signaling, and normal thymocyte development and maturation

doi: 10.7554/eLife.02270

Figure Lengend Snippet: ( A ) Defects in mRNA expression of KLF2 in mature CD4SP thymocytes from Pak2 F/F ;Cd4 -Cre mice. KLF2 mRNA levels in semi-mature and mature CD4SP thymocytes relative to Pak2 F/F DP thymocytes (left panel, mean ± SD of triplicates, results are representative of three independent experiment); KLF2 mRNA levels in mature Pak2 F/F ;Cd4 -Cre CD4SP thymocytes relative to mature Pak2 F/F CD4SP thymocytes (middle panel, mean ± SEM; each dot represents one mouse, n = 3); KLF2 mRNA levels in semi-mature Pak2 F/F ;Cd4 -Cre CD4SP thymocytes relative to semi-mature Pak2 F/F CD4SP thymocytes (right panel, mean ± SEM; each dot represents one mouse, n = 3). ***, p=0.0001; **, 0.001

Article Snippet: Pak2 (TA306346) antibody is from Origene (Rockville, MD).

Techniques: Expressing, Western Blot

Journal: eLife

Article Title: Pak2 is required for actin cytoskeleton remodeling, TCR signaling, and normal thymocyte development and maturation

doi: 10.7554/eLife.02270

Figure Lengend Snippet: ( A ) Increased apoptosis in the Qa2 hi CD4SP thymocyte subset from Pak2 F/F ;Cd4 -Cre mice following 24 hr of incubation in media or in plate-bound CD3 or CD3/CD28 stimulation. Data are representative of two independent experiments. ( B ) Increased apoptosis in CD4SP thymocytes from Pak2 F/F ;Cd4 -Cre mice following 24 hr of incubation in media or in plate-bound CD3 or CD3/CD28 stimulation. Apoptosis detected using 7AAD/Annexin V staining. Data are representative of three independent experiments. ( C ) Increased apoptosis in resting semi-mature (CD62L low ) CD4SP thymocytes from Pak2 F/F ;Cd4 -Cre mice detected using Annexin V staining or active caspase 3 intracellular staining following 24 hr of incubation in 10% FBS serum containing media. Data are representative of three (annexin V) and two (active caspase 3) independent experiments. ( D ) Increased apoptosis in semi-mature (CD62L low ) CD4SP thymocytes from Pak2 F/F ;Cd4 -Cre mice detected using Annexin V staining following 24 hr of incubation in media or in plate-bound CD3 or CD3/CD28 stimulation. Data are representative of four independent experiments. DOI: http://dx.doi.org/10.7554/eLife.02270.013

Article Snippet: Pak2 (TA306346) antibody is from Origene (Rockville, MD).

Techniques: Incubation, Staining

Journal: eLife

Article Title: Pak2 is required for actin cytoskeleton remodeling, TCR signaling, and normal thymocyte development and maturation

doi: 10.7554/eLife.02270

Figure Lengend Snippet: ( A ) Inhibition of MAPK pathway by UO126, a MEK inhibitor. Effects of UO126 (a MEK inhibitor) and rapamycin (an inhibitor of mTOR) on phosphorylation of Erk1/2 and RSK were demonstrated by immunoblotting analysis of phosphorylation status of Erk1/2 (T202/Y204) and RSK (T359/S363) in total thymocytes from Pak2 F/F or Pak2 F/F ;Cd4 -Cre mice. Cells were preincubated in the presence of DMSO, rapamycin (25 nM), and UO126 (10 μM) at the stated concentration for 30 min and treated with plate-bound anti-CD3 stimulation for 30 and 60 min. UO126 completely abrogated activation of MAPK and RSK, but rapamycin did not substantially inhibit activation of MAPK and RSK. ( B ) Inhibition of mTORC1-mediated pathway by rapamycin. Effect of rapamycin was examined by immunoblotting analysis of phosphorylation status of p70S6K (T389). Phosphorylation of p70S6K at T389 was completely inhibited by rapamycin (25 nM). ( C ) Phosphorylation of S6 at S235/S236 and p70S6K at T389 was completely dependent upon mTOR-mediated pathway. Cells were preincubated in the presence of DMSO, rapamycin (25 nM), and UO126 (10 μM) at the stated concentration for 30 min. Cells were rested in media or treated with plate-bound anti-CD3 stimulation for 30 and 60 min. Immunoblotting analysis of phosphorylation status of S6 (S235/S236) and p70S6K (T389) in total thymocytes from Pak2 F/F or Pak2 F/F ;Cd4 -Cre mice in media (resting) or following plate-bound anti-CD3 stimulation for 30 and 60 min are shown. ( D ) Intensity of each band shown in C was measured, normalized by intensity of GAPDH (as loading control), and shown in the graph as fold increase. Shown are data representative of three independent experiments. ( E ) Phosphorylation of S6 at S235/S236 and p70S6K at T389 were dependent upon activation of mTOR. Relative intensity of inhibitor-treated samples compared to DMSO-treated samples at 30 min or 60 min after stimulation are shown. Intensity of each band was normalized by DMSO-treated sample at each time point and displayed as relative intensity. Graphs in this figure show mean ± SEM (n = 3). DOI: http://dx.doi.org/10.7554/eLife.02270.014

Article Snippet: Pak2 (TA306346) antibody is from Origene (Rockville, MD).

Techniques: Inhibition, Western Blot, Concentration Assay, Activation Assay

Journal: eLife

Article Title: Pak2 is required for actin cytoskeleton remodeling, TCR signaling, and normal thymocyte development and maturation

doi: 10.7554/eLife.02270

Figure Lengend Snippet: ( A ) Increased actin polymerization in resting DP, CD4 and CD8 SP thymocytes from Pak2 F/F Cd4 -Cre mice. The mean fluorescence intensity of phalloidin-Alexa488 in the DP, CD4SP and CD8SP thymocyte populations was normalized to the value of WT DP thymocytes at rest. (error bars; SEM, n = 3). ****, p<0.0001 ( B ) Increased activation of Rac1 at resting from Pak2 F/F ;Cd4 -Cre thymocytes using Rac1 pull-down assay. WCL; whole cell lysates. Shown are data representative of three independent experiments. ( C ) Spreading areas of CD4SP thymocytes on the cover slips. Cells were allowed to spread for 60 min on coverslips coated with anti-CD3. Z-stack images of F-actin staining of the cells were collected and spreading areas where cells contact the cover slips were analyzed. Shown are data representative of three independent experiments. (error bars; SEM, Pak2 F/F [n = 21], Pak2 F/F ;Cd4 -Cre [n = 25]). ****, p<0.0001. ( D ) T cell spreading and actin polymerization triggered by plate-bound TCR stimulation. Shown are z-stack images of the cells from the contact sites where T cells touch the cover slides to the top of the cells. Left panels; maximal projection of z-stack images of F-actin staining (F-actin staining at the contact site [green] and F-actin staining of the rest of the z-stacks [red]). Scale bar: 5 μm. Middle panels: F-actin staining on the contact site to visualize cell spreading. Right panels: z stack images of F-actin were complied and reconstructed as a 3D image. Shown is the orthogonal image viewed from the z-axis. Shown are data representative of three independent experiments. DOI: http://dx.doi.org/10.7554/eLife.02270.015

Article Snippet: Pak2 (TA306346) antibody is from Origene (Rockville, MD).

Techniques: Fluorescence, Activation Assay, Pull Down Assay, Staining