Journal: American Journal of Respiratory Cell and Molecular Biology

Article Title: Age-Specific Effects on Rat Lung Glutathione and Antioxidant Enzymes after Inhaling Ultrafine Soot

doi: 10.1165/rcmb.2012-0108OC

Figure Lengend Snippet: TAQMAN GENE EXPRESSION ASSAY CATALOG NUMBERS

Article Snippet: Results are expressed as fold changes relative to filtered animals of the same age unless otherwise stated. table ft1 table-wrap mode="anchored" t5 TABLE 1. caption a7 Symbol Assay ID Gene Name NCBI RefSeq GCLc Rn00563101_m1 Glutamate-cysteine ligase catalytic subunit {"type":"entrez-nucleotide","attrs":{"text":"NM_012815.2","term_id":"52138588"}} NM_012815.2 GCLm Rn00568900_m1 Glutamate-cysteine ligase regulatory subunit {"type":"entrez-nucleotide","attrs":{"text":"NM_017305.2","term_id":"51036644"}} NM_017305.2 GPX1 Rn00577994_g1 Glutathione peroxidase NM_030826.3 GSR Rn01482159_m1 Glutathione reductase {"type":"entrez-nucleotide","attrs":{"text":"NM_053906.2","term_id":"309319800"}} NM_053906.2 GSTM1 Rn00755117_m1 Glutathione S-transferase Mu-1 {"type":"entrez-nucleotide","attrs":{"text":"NM_017014.1","term_id":"8393501"}} NM_017014.1 GSTP1 Rn00561378_gH Glutathione S-transferase Pi-1 {"type":"entrez-nucleotide","attrs":{"text":"NM_012577.2","term_id":"169646324"}} NM_012577.2 GSTT1 Rn00583932_m1 Glutathione S-transferase Theta-1 {"type":"entrez-nucleotide","attrs":{"text":"NM_053293.2","term_id":"55926212"}} NM_053293.2 HPRT Rn01527840_m1 Hypoxanthine-guanine phosphoribosyltransferase {"type":"entrez-nucleotide","attrs":{"text":"NM_012583.2","term_id":"70778838"}} NM_012583.2 Open in a separate window TAQMAN GENE EXPRESSION ASSAY CATALOG NUMBERS

Techniques: Gene Expression

Journal: American Journal of Respiratory Cell and Molecular Biology

Article Title: Age-Specific Effects on Rat Lung Glutathione and Antioxidant Enzymes after Inhaling Ultrafine Soot

doi: 10.1165/rcmb.2012-0108OC

Figure Lengend Snippet: GCLc and GCLm gene expression. RT-PCR expression in airway and parenchyma compartments in neonates and adult rats exposed to PFPs. Basal GCLc was consistently expressed in higher abundance than GCLm, and its highest expression was seen in adult airways (A). After PFP exposure, a transient drop in neonatal airway GCLc expression was observed in PFP24 compared with PFP2 (B). No treatment effects were detected in adult animals (C). Data are plotted as means ± SEM (n = 5–7 rats per group, per compartment, per gene). P < 0.05 are denoted as follows: *significantly different from neonates in the same compartment, and †significantly different from airways in the same age. PFP2, PFP24, and PFP48 refer to PFP exposure for 4, 24, and 48 hours, respectively.

Article Snippet: Results are expressed as fold changes relative to filtered animals of the same age unless otherwise stated. table ft1 table-wrap mode="anchored" t5 TABLE 1. caption a7 Symbol Assay ID Gene Name NCBI RefSeq GCLc Rn00563101_m1 Glutamate-cysteine ligase catalytic subunit {"type":"entrez-nucleotide","attrs":{"text":"NM_012815.2","term_id":"52138588"}} NM_012815.2 GCLm Rn00568900_m1 Glutamate-cysteine ligase regulatory subunit {"type":"entrez-nucleotide","attrs":{"text":"NM_017305.2","term_id":"51036644"}} NM_017305.2 GPX1 Rn00577994_g1 Glutathione peroxidase NM_030826.3 GSR Rn01482159_m1 Glutathione reductase {"type":"entrez-nucleotide","attrs":{"text":"NM_053906.2","term_id":"309319800"}} NM_053906.2 GSTM1 Rn00755117_m1 Glutathione S-transferase Mu-1 {"type":"entrez-nucleotide","attrs":{"text":"NM_017014.1","term_id":"8393501"}} NM_017014.1 GSTP1 Rn00561378_gH Glutathione S-transferase Pi-1 {"type":"entrez-nucleotide","attrs":{"text":"NM_012577.2","term_id":"169646324"}} NM_012577.2 GSTT1 Rn00583932_m1 Glutathione S-transferase Theta-1 {"type":"entrez-nucleotide","attrs":{"text":"NM_053293.2","term_id":"55926212"}} NM_053293.2 HPRT Rn01527840_m1 Hypoxanthine-guanine phosphoribosyltransferase {"type":"entrez-nucleotide","attrs":{"text":"NM_012583.2","term_id":"70778838"}} NM_012583.2 Open in a separate window TAQMAN GENE EXPRESSION ASSAY CATALOG NUMBERS

Techniques: Gene Expression, Reverse Transcription Polymerase Chain Reaction, Expressing

Journal: American Journal of Respiratory Cell and Molecular Biology

Article Title: Age-Specific Effects on Rat Lung Glutathione and Antioxidant Enzymes after Inhaling Ultrafine Soot

doi: 10.1165/rcmb.2012-0108OC

Figure Lengend Snippet: GCLc and GCLm protein analysis through Western blotting and immunohistochemistry. Representative GCLc and GCLm Western blots with actin loading control (A). GCLc/m Western blots were quantified: while neonatal GCLc/m expression remained unchanged after exposure (B), a significant up-regulation in GCLc was detected in PFP2 adult rats compared against FA controls (C). Data are plotted as means ± SEM (n = 6 rats per group). P < 0.05 is denoted as follows: ‡significantly different from FA in the same age. GCL immunohistochemical images in neonatal (D–G) and adult (H–K) rats reared in FA (D and H) and exposed to PFP: PFP2 (E and I), PFP24 (F and J), and PFP48 (G and K). Intense GCL staining was observed in adult and neonatale PFP2. In contrast to neonates, staining in adult PFP48 was continually up-regulated. High magnification inserts highlight GCL-positive cells in treated groups. Scale bars for D–K (shown in K) are 50 μm.

Article Snippet: Results are expressed as fold changes relative to filtered animals of the same age unless otherwise stated. table ft1 table-wrap mode="anchored" t5 TABLE 1. caption a7 Symbol Assay ID Gene Name NCBI RefSeq GCLc Rn00563101_m1 Glutamate-cysteine ligase catalytic subunit {"type":"entrez-nucleotide","attrs":{"text":"NM_012815.2","term_id":"52138588"}} NM_012815.2 GCLm Rn00568900_m1 Glutamate-cysteine ligase regulatory subunit {"type":"entrez-nucleotide","attrs":{"text":"NM_017305.2","term_id":"51036644"}} NM_017305.2 GPX1 Rn00577994_g1 Glutathione peroxidase NM_030826.3 GSR Rn01482159_m1 Glutathione reductase {"type":"entrez-nucleotide","attrs":{"text":"NM_053906.2","term_id":"309319800"}} NM_053906.2 GSTM1 Rn00755117_m1 Glutathione S-transferase Mu-1 {"type":"entrez-nucleotide","attrs":{"text":"NM_017014.1","term_id":"8393501"}} NM_017014.1 GSTP1 Rn00561378_gH Glutathione S-transferase Pi-1 {"type":"entrez-nucleotide","attrs":{"text":"NM_012577.2","term_id":"169646324"}} NM_012577.2 GSTT1 Rn00583932_m1 Glutathione S-transferase Theta-1 {"type":"entrez-nucleotide","attrs":{"text":"NM_053293.2","term_id":"55926212"}} NM_053293.2 HPRT Rn01527840_m1 Hypoxanthine-guanine phosphoribosyltransferase {"type":"entrez-nucleotide","attrs":{"text":"NM_012583.2","term_id":"70778838"}} NM_012583.2 Open in a separate window TAQMAN GENE EXPRESSION ASSAY CATALOG NUMBERS

Techniques: Western Blot, Immunohistochemistry, Control, Expressing, Immunohistochemical staining, Staining

Journal: Translational Lung Cancer Research

Article Title: PRRX1 isoform PRRX1A regulates the stemness phenotype and epithelial-mesenchymal transition (EMT) of cancer stem-like cells (CSCs) derived from non-small cell lung cancer (NSCLC)

doi: 10.21037/tlcr-20-633

Figure Lengend Snippet: Primers used to reverse transcriptional quantitative PCR

Article Snippet: Construction of expression plasmid and transfection The full-length complementary DNA (cDNA) of PRRX1A (human PRRX1A, transcript variant pmx-1a; NCBI reference sequence: {"type":"entrez-nucleotide","attrs":{"text":"NM_006902.5","term_id":"1674986205","term_text":"NM_006902.5"}} NM_006902.5 ) and PRRX1B (human PRRX1B, transcript variant pmx-1b; NCBI reference sequence: {"type":"entrez-nucleotide","attrs":{"text":"NM_022716.4","term_id":"1519312415","term_text":"NM_022716.4"}} NM_022716.4 ) were obtained from RiboBio (Guangzhou, China) and ligated into the Hind III-Bam HI site of the p3×FLAG-CMV-10 vector (Sigma–Aldrich, St. Louis, MO, USA).

Techniques:

Journal: Translational Lung Cancer Research

Article Title: PRRX1 isoform PRRX1A regulates the stemness phenotype and epithelial-mesenchymal transition (EMT) of cancer stem-like cells (CSCs) derived from non-small cell lung cancer (NSCLC)

doi: 10.21037/tlcr-20-633

Figure Lengend Snippet: The effect of PRRX1A on proliferation, invasive capacity, and stemness in A549- and SPC-A1-CSCs. After overexpression of Flag-PRRX1A, Flag-PRRX1B (A), and knockdown of PRRX1A and PRRX1 (B) in A549- or SPC-A1-CSCs, the efficacy was detected by performing semi-quantitative Western blot. (C) After PI staining, flow cytometric analyses was performed to detect cell cycle phases. *, P<0.05, vs. vector group. (D) Cell viability was measured after CCK-8 staining. *, P<0.05, vs. vector group. Sphere formation of A549-CSCs (E) and SPC-A1 CSCs (F) after overexpression or knockdown of PRRX1A/B was measured. *, P<0.05, vs. pENTR/U6 vector group; #, P<0.05, vs. pENTR/U6 group. (G) Transwell assay was performed to detect the effect of PRRX1A/B on invasive capacity. *, P<0.05, vs. vector group; #, P<0.05, vs. pENTR/U6 group. PRRX1, paired-related homeobox 1; CSCs, cancer stem-like cells.

Article Snippet: Construction of expression plasmid and transfection The full-length complementary DNA (cDNA) of PRRX1A (human PRRX1A, transcript variant pmx-1a; NCBI reference sequence: {"type":"entrez-nucleotide","attrs":{"text":"NM_006902.5","term_id":"1674986205","term_text":"NM_006902.5"}} NM_006902.5 ) and PRRX1B (human PRRX1B, transcript variant pmx-1b; NCBI reference sequence: {"type":"entrez-nucleotide","attrs":{"text":"NM_022716.4","term_id":"1519312415","term_text":"NM_022716.4"}} NM_022716.4 ) were obtained from RiboBio (Guangzhou, China) and ligated into the Hind III-Bam HI site of the p3×FLAG-CMV-10 vector (Sigma–Aldrich, St. Louis, MO, USA).

Techniques: Over Expression, Knockdown, Western Blot, Staining, Plasmid Preparation, CCK-8 Assay, Transwell Assay

Journal: Translational Lung Cancer Research

Article Title: PRRX1 isoform PRRX1A regulates the stemness phenotype and epithelial-mesenchymal transition (EMT) of cancer stem-like cells (CSCs) derived from non-small cell lung cancer (NSCLC)

doi: 10.21037/tlcr-20-633

Figure Lengend Snippet: The expression patterns of PRRX1A, PRRX1B, and TGF-β in lung cancer tissues. (A) RT-qPCR was performed to assess the expression patterns of stemness factors, PRRX1A, PRRX1B, and EMT hallmarkers. *, P<0.05, vs. adjacent tissues. (B) Expression levels of PRRX1A and TGF-β in lung cancer tissue samples were positively correlated. The correlation between PRRX1 and TGF-β1 (C), E-cadherin (D), N-cadherin (E), and Vimentin (F) were analyzed by using the GEPIA server. PRRX1, paired-related homeobox 1.

Article Snippet: Construction of expression plasmid and transfection The full-length complementary DNA (cDNA) of PRRX1A (human PRRX1A, transcript variant pmx-1a; NCBI reference sequence: {"type":"entrez-nucleotide","attrs":{"text":"NM_006902.5","term_id":"1674986205","term_text":"NM_006902.5"}} NM_006902.5 ) and PRRX1B (human PRRX1B, transcript variant pmx-1b; NCBI reference sequence: {"type":"entrez-nucleotide","attrs":{"text":"NM_022716.4","term_id":"1519312415","term_text":"NM_022716.4"}} NM_022716.4 ) were obtained from RiboBio (Guangzhou, China) and ligated into the Hind III-Bam HI site of the p3×FLAG-CMV-10 vector (Sigma–Aldrich, St. Louis, MO, USA).

Techniques: Expressing, Quantitative RT-PCR

Journal: Translational Lung Cancer Research

Article Title: PRRX1 isoform PRRX1A regulates the stemness phenotype and epithelial-mesenchymal transition (EMT) of cancer stem-like cells (CSCs) derived from non-small cell lung cancer (NSCLC)

doi: 10.21037/tlcr-20-633

Figure Lengend Snippet: PRRX1A regulates EMT processes via TGF-β/TGF-βR signaling pathway. (A) After overexpression of PRRX1A or PRRX1B, the expression levels of TGF-β1, β2, and β3 were determined by performing RT-qPCR analysis. *, P<0.05, vs. vector group. RT-qPCR assay (B) and semi-quantitative Western blot (C) were performed to detect the mRNA levels of E-cadherin, N-cadherin, and Vimentin after blocking TGF-β/TGF-βR signaling pathway. *, P<0.05, vs. vector group. #, P<0.05, vs. PRRX1A group. (D) Transwell assay was performed to detect the invasive capacity. *, P<0.05, vs. vector group; #, P<0.05, vs. vector + TGF-β1 group; &, P<0.05, vs. PRRX1A group. PRRX1, paired-related homeobox 1; EMT, epithelial-mesenchymal transition.

Article Snippet: Construction of expression plasmid and transfection The full-length complementary DNA (cDNA) of PRRX1A (human PRRX1A, transcript variant pmx-1a; NCBI reference sequence: {"type":"entrez-nucleotide","attrs":{"text":"NM_006902.5","term_id":"1674986205","term_text":"NM_006902.5"}} NM_006902.5 ) and PRRX1B (human PRRX1B, transcript variant pmx-1b; NCBI reference sequence: {"type":"entrez-nucleotide","attrs":{"text":"NM_022716.4","term_id":"1519312415","term_text":"NM_022716.4"}} NM_022716.4 ) were obtained from RiboBio (Guangzhou, China) and ligated into the Hind III-Bam HI site of the p3×FLAG-CMV-10 vector (Sigma–Aldrich, St. Louis, MO, USA).

Techniques: Over Expression, Expressing, Quantitative RT-PCR, Plasmid Preparation, Western Blot, Blocking Assay, Transwell Assay

Journal: Translational Lung Cancer Research

Article Title: PRRX1 isoform PRRX1A regulates the stemness phenotype and epithelial-mesenchymal transition (EMT) of cancer stem-like cells (CSCs) derived from non-small cell lung cancer (NSCLC)

doi: 10.21037/tlcr-20-633

Figure Lengend Snippet: PRRX1A potentially binds to SOX2 and regulates stemness factors. (A) RT-qPCR was performed to detect the different expression patterns between CSCs and parental cells. *, P<0.05, vs. A549 group; #, P<0.05, vs. SPC-A1 group. (B) After overexpression or knockdown of PRRX1A/B, the stemness factors were quantitatively analyzed. *, P<0.05, vs. pENTR/U6 vector group. (C) Co-immunoprecipitation was performed to detect the binding of Flag-PRRX1A or Flag-PRRX1B to SOX2. d Detection of SOX2 protein level after overexpression of PRRX1A/B. PRRX1, paired-related homeobox 1; CSCs, cancer stem-like cells.

Article Snippet: Construction of expression plasmid and transfection The full-length complementary DNA (cDNA) of PRRX1A (human PRRX1A, transcript variant pmx-1a; NCBI reference sequence: {"type":"entrez-nucleotide","attrs":{"text":"NM_006902.5","term_id":"1674986205","term_text":"NM_006902.5"}} NM_006902.5 ) and PRRX1B (human PRRX1B, transcript variant pmx-1b; NCBI reference sequence: {"type":"entrez-nucleotide","attrs":{"text":"NM_022716.4","term_id":"1519312415","term_text":"NM_022716.4"}} NM_022716.4 ) were obtained from RiboBio (Guangzhou, China) and ligated into the Hind III-Bam HI site of the p3×FLAG-CMV-10 vector (Sigma–Aldrich, St. Louis, MO, USA).

Techniques: Quantitative RT-PCR, Expressing, Over Expression, Knockdown, Plasmid Preparation, Immunoprecipitation, Binding Assay

Journal: Nature

Article Title: Structural basis of catalytic activation in human splicing

doi: 10.1038/s41586-023-06049-w

Figure Lengend Snippet: a , Overview of the B AQR cryo-EM structure. Key subunits are colour coded. b , Compositional remodelling of the human spliceosome during catalytic activation. Subunits recruited or destabilized by the ATPase activities of PRP2 and Aquarius are indicated. Destabilized subunits can remain flexibly attached to the spliceosomes, often at lower stoichiometry. NTR, NTC-related proteins.

Article Snippet: Human PRP2 (NCBI Reference Sequence identifier: NM_003587 , transcript variant 1) and human PPIL4 (NCBI Sequence identifiers BC020986 and NM_139126.4 ) open reading frame clones were obtained from OriGene (RC202912) and Applied Biological Materials (373710120000), whereas human GPKOW and SKIP were codon-optimized for expression in insect cells and synthesized by GeneArt (ThermoFisher Scientific).

Techniques: Cryo-EM Sample Prep, Activation Assay

Journal: Nature

Article Title: Structural basis of catalytic activation in human splicing

doi: 10.1038/s41586-023-06049-w

Figure Lengend Snippet: a , Overall maps of state A and B complexes depicted together with the final model. b , Local resolution of the state A maps, estimated in RELION and visualized in ChimeraX. c , Cryo-EM density snapshots. Various subunits are colored and labeled. d , Selected snapshots of modeled B AQR subunits are depicted together with the unsharpened map of the core region of the complex (map M2). e , Structural comparison between B act , B AQR , and C complexes. PRP2, Aquarius, SYF1, the U2 snRNA, and the pre-mRNA substrate are colored and labeled. Note the large-scale repositioning of the PRP2 RNA helicase during the conversion of B act to B AQR , and of the helicase Aquarius during the transition of B act /B AQR complexes to the C-stage spliceosome.

Article Snippet: Human PRP2 (NCBI Reference Sequence identifier: NM_003587 , transcript variant 1) and human PPIL4 (NCBI Sequence identifiers BC020986 and NM_139126.4 ) open reading frame clones were obtained from OriGene (RC202912) and Applied Biological Materials (373710120000), whereas human GPKOW and SKIP were codon-optimized for expression in insect cells and synthesized by GeneArt (ThermoFisher Scientific).

Techniques: Cryo-EM Sample Prep, Labeling

Journal: Nature

Article Title: Structural basis of catalytic activation in human splicing

doi: 10.1038/s41586-023-06049-w

Figure Lengend Snippet: a , Domain composition and interactions of PRP2 in the B AQR complex. b , c , Structure and conformation of PRP2, the intron and SF3B1 in B AQR . Note that PRP2 NTD (the N-terminal domain) is not visible in c . d , In B AQR , PRP2 is moved in a cavity framed by PRP8 and SF3B1. The previous location and conformation of PRP2 and the intron in B act are shown in grey and black, respectively.

Article Snippet: Human PRP2 (NCBI Reference Sequence identifier: NM_003587 , transcript variant 1) and human PPIL4 (NCBI Sequence identifiers BC020986 and NM_139126.4 ) open reading frame clones were obtained from OriGene (RC202912) and Applied Biological Materials (373710120000), whereas human GPKOW and SKIP were codon-optimized for expression in insect cells and synthesized by GeneArt (ThermoFisher Scientific).

Techniques:

Journal: Nature

Article Title: Structural basis of catalytic activation in human splicing

doi: 10.1038/s41586-023-06049-w

Figure Lengend Snippet: a–f , Interactions between PRP2, PRP8, SF3B1, CWC22 and SKIP. Interacting distances corresponding to polar and hydrophobic contacts are indicated as dashed lines. g , Overview of PRP2 and the composite molecular brake. PRP2’s conserved domains are color-coded. HB – helix-bundle; OB – oligonucleotide/oligosaccharide- binding fold; WH – winged-helix. h , Interactions between PPIL4, SKIP, and PRP2. Residues involved in contacts are depicted as spheres and shown in the same color as their interacting partner. i , Interactions between the wedge element of SKIP and PRP2 core . j , The wedge element of SKIP appears to lock the RecA domains of PRP2. The ADP molecule is modeled by superposition with Ct PRP2 (PDB 6zm2) and is shown for the sake of orientation. k , Superposition between human PRP2 in the open conformation (observed in B AQR ) and Ct PRP2 in the closed conformation (colored in teal) (PDB 6zm2). Equivalent residues of PRP2 interacting with SKIP in B AQR are shown for Ct PRP2. Structures from panels i and k are depicted in the same orientation. The red arrow indicates the movement of the RecA2-like domain during the helicase transition from the open to the closed conformation. l , Genetic interactions from yeast mapped on the structure of human B AQR . The cold-sensitive allele of Prp2p Q548N genetically interacts with the D450G and V502F substitutions of Hsh155p. Residues involved in genetic interactions in yeast (blue) are depicted as spheres and mapped on the B AQR model. m-o , Structural superposition of the human ( Hs ) and budding yeast ( Sc ) MA3 domain of CWC22, composed from HEAT repeats. The 10 th HEAT repeat (residues 454–491) of Cwc22p is required for the productive function of yeast Prp2p and interacts with the “hook” motif of PRP2 NTD and BUD13 in B AQR . This suggests that Prp2p, Cwc22p, and Bud13p may interact in a similar fashion in budding yeast spliceosomes, thus explaining the functional connection between Prp2p and Cwc22p. Note that human CWC22 residues that interact with PRP2 and BUD13 are conserved in budding yeast Cwc22p (shown in n and o). The protein subunits, U2 snRNA and the pre-mRNA substrate are colored and labeled.

Article Snippet: Human PRP2 (NCBI Reference Sequence identifier: NM_003587 , transcript variant 1) and human PPIL4 (NCBI Sequence identifiers BC020986 and NM_139126.4 ) open reading frame clones were obtained from OriGene (RC202912) and Applied Biological Materials (373710120000), whereas human GPKOW and SKIP were codon-optimized for expression in insect cells and synthesized by GeneArt (ThermoFisher Scientific).

Techniques: Binding Assay, Functional Assay, Labeling

Journal: Nature

Article Title: Structural basis of catalytic activation in human splicing

doi: 10.1038/s41586-023-06049-w

Figure Lengend Snippet: a,b , Superposition between budding yeast (PDB 7DCO) and human (PDB 5Z56) B act complexes over equivalent residues from PHF5A. For clarity’s sake, only SF3B1, PRP2, the intron and U2 snRNA are depicted. All subunits are labeled accordingly. c , PRP2:RNA from human B AQR , as the only available structure of the human counterpart, was superimposed onto the Prp2p helicase from budding yeast B act and shown in the same orientation as in b. Note that the conformation of the RNA strand bound by human PRP2 or budding yeast Prp2p is virtually unchanged. d , Assignment of nucleotides bound by human PRP2 in B act based on the superposition. e , PRP2 translocates about 19 nucleotides towards the branch helix during the B act -to-B AQR transition. PRP2, RBMX2 and PPIL4 positions on the intron in different spliceosomal complexes are depicted. f , PRP2 translocation results in a substantial change in the intron’s conformation, dissociation of RBMX2 and recruitment of PPIL4 to the intron.

Article Snippet: Human PRP2 (NCBI Reference Sequence identifier: NM_003587 , transcript variant 1) and human PPIL4 (NCBI Sequence identifiers BC020986 and NM_139126.4 ) open reading frame clones were obtained from OriGene (RC202912) and Applied Biological Materials (373710120000), whereas human GPKOW and SKIP were codon-optimized for expression in insect cells and synthesized by GeneArt (ThermoFisher Scientific).

Techniques: Labeling, Translocation Assay

Journal: Nature

Article Title: Structural basis of catalytic activation in human splicing

doi: 10.1038/s41586-023-06049-w

Figure Lengend Snippet: a , Relocation of PRP2 on the intron, RES complex dissociation from the translocated RNA and subsequent binding of PPIL4 to the latter. Pink, green and broken lines represent the PPT, the PPT region bound by SF3B1 within B act , and the PPT-equivalent region visible only within the budding yeast B act structure, respectively. b , Close-up view of the interfaces between PRP2 and other subunits in the B act complex. c , The equivalent view and orientation of the B AQR complex. d , SF3B1 exhibits primary and secondary hinged pockets for binding of BS-A and PPT, respectively. SF3B1 adopts a loose conformation in B AQR , whereby the primary and secondary pockets are occupied and free, respectively.

Article Snippet: Human PRP2 (NCBI Reference Sequence identifier: NM_003587 , transcript variant 1) and human PPIL4 (NCBI Sequence identifiers BC020986 and NM_139126.4 ) open reading frame clones were obtained from OriGene (RC202912) and Applied Biological Materials (373710120000), whereas human GPKOW and SKIP were codon-optimized for expression in insect cells and synthesized by GeneArt (ThermoFisher Scientific).

Techniques: Binding Assay

Journal: Nature

Article Title: Structural basis of catalytic activation in human splicing

doi: 10.1038/s41586-023-06049-w

Figure Lengend Snippet: a , Destabilization of SF3B6, de-structuring of SF3B1 NTD , and the reorientation of PRP8’s EN (endonuclease-like, PRP8 EN ) and RH (RNase H-like, PRP8 RH ) domains upon PRP2 translocation. PRP2 is located on the plane above SF3B1 HEAT and is not shown for clarity’s sake. The reactive BS-A, the guanosine of the 5’SS, and the catalytic metal ions are shown as spheres and labeled. b , Close-up view of SF3B1, the intron, and the U2 snRNA from B AQR (color-coded) and B act (grey and black) after superposition of SF3B1’s equivalent residues. c , The binding pocket of the BS-A (primary pocket) is virtually unchanged in B act and B AQR . d , Cycle of SF3B1 transitions in splicing. The conformational transitions of SF3B1 are depicted based on cryo-EM structures and biochemical analyses. Here we refer to the intermediates II and III as pre-A1 and pre-A2 for clarity. The key spliceosome complexes representative for the SF3B1 intermediates shown here are: 17S U2 snRNP , , pre-A1 (A-like cross-exon complex bound by spliceostatin A ), pre-A2 (ref. ), A-to-B act (reviewed in refs. , , ), B AQR (this work), the SF3B complex , . Helicases that facilitate the transitions are shown in red. The helicase DHX15, colored magenta, mediates the disassembly of kinetically-slowed complexes (e.g. formed on suboptimal introns, weak splice sites and PPTs, multiple branch sites or cryptic sites – ).

Article Snippet: Human PRP2 (NCBI Reference Sequence identifier: NM_003587 , transcript variant 1) and human PPIL4 (NCBI Sequence identifiers BC020986 and NM_139126.4 ) open reading frame clones were obtained from OriGene (RC202912) and Applied Biological Materials (373710120000), whereas human GPKOW and SKIP were codon-optimized for expression in insect cells and synthesized by GeneArt (ThermoFisher Scientific).

Techniques: Translocation Assay, Labeling, Binding Assay, Cryo-EM Sample Prep

Journal: Nature

Article Title: Structural basis of catalytic activation in human splicing

doi: 10.1038/s41586-023-06049-w

Figure Lengend Snippet: a , Interactions between the subunits of the molecular brake. Interacting residues are shown as spheres depicted in the same colour as their binding partner. The domains of PRP2 RecA1, RecA2, HB and NTD are depicted. The anchoring surfaces of the brake elements to PRP8 (grey oval) are indicated. The inset shows a schematic representation of the molecular brake, in which the subunits are coloured as in the surface and cartoon representation. CWC15 is yellow. b , Interactions of the intron with PRP2 NTD , PPIL4 and SKIP. c , Interactions of PPIL4 with CWC15 and SKIP. d , e , The wedge element of SKIP intercalates between RecA1, RecA2 and the HB domains of PRP2, indicating inhibition of translocation. PPIL4, SKIP and CWC15 are red, cyan and yellow, respectively.

Article Snippet: Human PRP2 (NCBI Reference Sequence identifier: NM_003587 , transcript variant 1) and human PPIL4 (NCBI Sequence identifiers BC020986 and NM_139126.4 ) open reading frame clones were obtained from OriGene (RC202912) and Applied Biological Materials (373710120000), whereas human GPKOW and SKIP were codon-optimized for expression in insect cells and synthesized by GeneArt (ThermoFisher Scientific).

Techniques: Binding Assay, Inhibition, Translocation Assay

Journal: Nature

Article Title: Structural basis of catalytic activation in human splicing

doi: 10.1038/s41586-023-06049-w

Figure Lengend Snippet: a , Size-exclusion chromatography (SEC) profiles of PRP2 (137-1022), PPIL4 and the mixture of the two proteins shows that PRP2 (137-1022) forms a stable complex with PPIL4, in an RNA-independent manner. PRP2 (137-1022) comprises both the modeled PRP2 NTD ( i.e ., the pin, clip, and hook elements) and the helicase core. b , SDS-PAGE gels corresponding to the fractions from a . The in vitro reconstitution experiments were repeated two times, using two independent preparations of PRP2 (137–1022) and PPIL4. c , SDS-PAGE gel of SEC fractions showing the reconstitution of a stable PRP2-PPIL4 complex in vivo by co-expression in insect cells. PRP2 (137–1022) and PPIL4 were co-expressed in Sf9 insect cells from individual baculoviruses. The complex was captured by Strep-Tactin affinity followed by SEC. The preparation was performed twice with similar results d , The RNA-binding activity of human PRP2 in the presence of GPKOW and PPIL4. The ability of PRP2 (137–1022) and of the purified PRP2 (137–1022)-PPIL4 and PRP2 (137-1022)-GPKOW complexes to bind a Cyanine 5 (Cy5)-labeled RNA substrate was evidenced by EMSA. The RNA substrate comprised an RNA duplex, followed by 30 nucleotides 3′ single-stranded overhang, mimicking PRP2’s spliceosome substrate observed in B AQR . The free RNA substrate was separated from PRP2-bound (or cofactor-bound) species on a 5% polyacrylamide native gel. The EMSA gels were imaged at the Cy5 excitation peak. The assays were repeated two times. e , The RNA binding activity of human PPIL4 in the absence and presence of PRP2 (137–1022). Compared to the EMSA shown in d , PPIL4 was added in a 5-fold excess over PRP2 (137–1022) and PRP2’s final assay concentrations are indicated above the last three gels lanes. Note the apparent increase in PRP2’s RNA affinity in the presence of PPIL4. The EMSAs were repeated three times. f , SEC profiles showing the formation of a stable complex between PRP2 (137–1022) and GPKOW, in an RNA-independent manner. The SDS-PAGE corresponding to the complex purified by SEC is shown. g , The RNA-binding activity of human GPKOW assessed by an EMSA. The same RNA substrate was used as in d and the assay was repeated four times. h , The helicase activity of human PRP2 was investigated using a fluorescence-based assay. Compared to the gel-based helicase assays shown in i-l , the fluorescence-based assay employs a dual-labeled helicase substrate. Displacement of the labeled strand by the helicase leads to the formation of an intramolecular hairpin, which brings in proximity the Cy5 fluorophore and its spectrally overlapping quencher (BHQ-2). The decrease in substrate’s fluorescence upon unwinding is monitored as a function of time. Several representative fluorescence traces of PRP2, recorded under different experimental conditions and in the presence/absence of helicase cofactors, are shown together with the unwinding curves of Prp22p, used as a positive control. i , The helicase/unwinding activity of human PRP2 and of the PRP2-GPKOW complex. To assess the ability of PRP2 (137–1022) and of the purified, in vitro reconstituted PRP2 (137–1022)-GPKOW complex to unwind RNA-RNA duplexes, the purified protein samples were mixed with the Cy5-labeled helicase substrate (depicted on the right with the labeled strand colored in red) in the presence (or absence) of ATP and of a competitor DNA (green). Following a 1-hour incubation, the samples were analyzed on a native 14% polyacrylamide gel and imaged by in-gel fluorescence. Budding yeast Prp22p was used as a positive control. All gel-based helicase assays were repeated at least two times. j , The helicase activity of the in vivo reconstituted PRP2 (137–1022)-PPIL4 complex, in the absence or presence of GPKOW. k , The helicase activity of human PRP2 (137–1022) in the presence or absence of its cofactor GPKOW. Compared to the assay shown in i , the GPKOW cofactor was added to the purified helicase in a 5-fold excess. The concentrations indicated above the native gel represent the final assay concentrations of PRP2 (137–1022) or Prp22p. The RNA bands labeled with an asterisk represent, most likely, degradation products. l , Comparative helicase activities of the PRP2 (137–1022)-PPIL4 and PRP2 (137–1022)-GPKOW complexes. For gel source data, see Supplementary Figs. and .

Article Snippet: Human PRP2 (NCBI Reference Sequence identifier: NM_003587 , transcript variant 1) and human PPIL4 (NCBI Sequence identifiers BC020986 and NM_139126.4 ) open reading frame clones were obtained from OriGene (RC202912) and Applied Biological Materials (373710120000), whereas human GPKOW and SKIP were codon-optimized for expression in insect cells and synthesized by GeneArt (ThermoFisher Scientific).

Techniques: Size-exclusion Chromatography, SDS Page, In Vitro, In Vivo, Expressing, RNA Binding Assay, Activity Assay, Purification, Labeling, Fluorescence, Positive Control, Incubation

Journal: Nature

Article Title: Structural basis of catalytic activation in human splicing

doi: 10.1038/s41586-023-06049-w

Figure Lengend Snippet: a , During the B act (PDB 5Z57) to the B AQR transition, translocation of PRP2 in a 3′-to-5′ direction results in the destabilization of the RES complex (RBMX2, BUD13, SNIP1). In addition, SRRM1 and SF3B6/p14 are no longer observed in B AQR due to PRP2-induced SF3B1 opening. During the remodeling of B AQR and transition to the C complex (PDB 5yzg, PDB 6zym), the remaining SF3B/SF3A subunits and CWC24 are released from the branch helix and the 5’SS. The branch helix then moves to the catalytic center, bringing the BS-A and the 5’SS GU nucleotides in proximity for the branching reaction. The different human spliceosome states were structurally aligned by using the PRP8 subunit as a reference and are depicted in two different orientations. The spliceosome subunits are color-coded and shown in cartoon representation. Spliceosome subunits not undergoing significant rearrangement were omitted for the sake of simplicity. b , Repositioning of the branch helix (U2/BS) during the transition from B act to B AQR and then to C complexes. The different spliceosome states, B act (PDB 5Z57), B AQR (this work), and C complex (PDB 5yzg, PDB 6zym), were aligned using the PRP8 subunit as a reference. All protein subunits, except PRP8, were omitted and the RNA moieties were color-coded. The reactive BS-A and the 5’SS GU nucleotides are shown as spheres and colored red and light green, respectively.

Article Snippet: Human PRP2 (NCBI Reference Sequence identifier: NM_003587 , transcript variant 1) and human PPIL4 (NCBI Sequence identifiers BC020986 and NM_139126.4 ) open reading frame clones were obtained from OriGene (RC202912) and Applied Biological Materials (373710120000), whereas human GPKOW and SKIP were codon-optimized for expression in insect cells and synthesized by GeneArt (ThermoFisher Scientific).

Techniques: Translocation Assay

Journal: Nature

Article Title: Structural basis of catalytic activation in human splicing

doi: 10.1038/s41586-023-06049-w

Figure Lengend Snippet: a , Catalytic activation depicted as structural transitions between B act , B AQR and B*. B* and the post-branching C complex have similar structures. Most subunits are shown as surface representations. SYF1 and SYF3 are shown as ribbons to enable better visualization of Aquarius. b , Structure-based model of human spliceosome remodelling by PRP2 and Aquarius. The hypothetical intermediate between B act and B AQR considers the following: (1) the molecular brake can form only after the binding of PPIL4 to the RNA, the binding site of which becomes available after dissociation of the RES complex by the translocation of PRP2; and (2) the advance of PRP2 should start stripping the intron from SF3B1 and promote the loose conformation of SF3B1. The hypothetical intermediate between B AQR and B* was generated by considering that Aquarius should induce complete displacement of the branch duplex from SF3B1, liberating BS-A from its binding pocket. Consequently, SF3B1 will transit from the loose to the open conformation. Because the HEAT repeats that bind PRP8 (in B AQR ) rearrange after loose-to-open conformation, SF3B1 dissociates from PRP8. This likely causes the complete destabilization of the SF3A–SF3B complexes and PRP2 from the spliceosome. c , Model of the spliceosome remodelling by Prp2p in budding yeast (S. cerevisiae) , based on the similarities and differences with the human counterpart.

Article Snippet: Human PRP2 (NCBI Reference Sequence identifier: NM_003587 , transcript variant 1) and human PPIL4 (NCBI Sequence identifiers BC020986 and NM_139126.4 ) open reading frame clones were obtained from OriGene (RC202912) and Applied Biological Materials (373710120000), whereas human GPKOW and SKIP were codon-optimized for expression in insect cells and synthesized by GeneArt (ThermoFisher Scientific).

Techniques: Activation Assay, Binding Assay, Translocation Assay, Stripping Membranes, Generated

Journal: Nature

Article Title: Structural basis of catalytic activation in human splicing

doi: 10.1038/s41586-023-06049-w

Figure Lengend Snippet: a , Lines of structural communication between Aquarius and the branch duplex in B AQR . Aquarius and PRP2 are in diametrically-opposed locations of the B AQR spliceosome. A continuous bridge of proteins is present between Aquarius and the SF3B1:branch duplex. Most of these proteins are SF3A/B subunits and PPIE. Another bridge that primarily involves the RBM22 protein is between Aquarius and U6 catalytic core. The intron region not visible in the density is dashed. Aquarius is depicted in red (RecA-like domains) and light blue (accessory domains). The first nucleotide of the intron (G +1 ) is positioned in the catalytic center. Other subunits of the spliceosome, including SYF1 and ISY1, are not shown for the sake of clarity b , Subunits of the C complex (pdb 5yzg) are shown in the same orientation as in a . The U6 snRNA was used as a reference for the superposition between the C and B AQR complexes. Note that PRP2 and the SF3A/B complex have dissociated and the BS-A has been relocated to the catalytic centre (see also c , below). RBM22’s orientation has remained virtually the same in B AQR and C complexes. The subunits are colored as in a and labeled. The B AQR and C complexes were superimposed over PRP8 (not shown) and U6 snRNA c , The BS-A juxtaposed to the first nucleotide of the intron in the catalytic center of the C complex. The catalytic metal ions are shown as magenta spheres. d , Superposition between the “open” (observed in the apo SF3B complex, PDB 5IFE) and the “loose” states (observed in B AQR ) of SF3B1. Except for SF3B1 OPEN , all shown subunits belong to the B AQR complex. e , B AQR and the SF3B complex in its apo form (PDB 5IFE) were superimposed over equivalent residues from PHF5A (not shown in the figures d, f and g for clarity’s sake). Note that the HEAT repeats H16-H20 of SF3B1 adopt virtually identical conformations in the “loose” and “open” states, while the other helical repeats are substantially reorganized. f–g , The BS-A’s release from the primary pocket of SF3B1 likely induces rearrangement of the HEAT repeats upon the “loose” to “open” conformational transition. Consequently, the contacts between HEAT repeats and PRP8, and those between SF3B2 (attached to the HEAT repeats) and the U6 snRNA might get disrupted, causing the complete dissociation of the SF3A/B complexes from the spliceosome.

Article Snippet: Human PRP2 (NCBI Reference Sequence identifier: NM_003587 , transcript variant 1) and human PPIL4 (NCBI Sequence identifiers BC020986 and NM_139126.4 ) open reading frame clones were obtained from OriGene (RC202912) and Applied Biological Materials (373710120000), whereas human GPKOW and SKIP were codon-optimized for expression in insect cells and synthesized by GeneArt (ThermoFisher Scientific).

Techniques: Labeling

Journal: eLife

Article Title: CD56 regulates human NK cell cytotoxicity through Pyk2

doi: 10.7554/eLife.57346

Figure Lengend Snippet: ( A ) Western blot analysis of CD56 from wild-type (WT) and CD56-knockout (KO) YTS (left) and NK92 (right) cell lines or primary human NK cells with actin as a loading control. ( B ) Flow cytometry analysis of CD56 expression in NK92 or YTS WT (filled histogram, dark grey) or CD56-KO (filled histogram, light grey) cells compared to unstained cells (dashed line). ( C ) NK92 or YTS cells were treated with PNGase F to remove polysialic acid. Following treatment, lysates were separated by SDS-PAGE and CD56 or actin as a loading control were detected by Western blotting. ( D ) NK cell lines (left) or Jurkat or Raji cells as a positive control (right) were treated with PI-PLC to cleave GPI anchored proteins from the cell surface. PI-PLC activity was confirmed by cleavage of GPI-anchored CD55 (right). All data shown are representative of 3 technical replicates performed on different days.

Article Snippet: NCAM reporter plasmids were generated by Epoch Life Sciences Inc and were made by subcloning NCAM1 (NCBI reference sequence: NM_000615.6, transcript variant 1; Origene) into BamHI and SalI digested pBABE-puro-mApple retroviral plasmid.

Techniques: Western Blot, Knock-Out, Control, Flow Cytometry, Expressing, SDS Page, Positive Control, Activity Assay

Journal: eLife

Article Title: CD56 regulates human NK cell cytotoxicity through Pyk2

doi: 10.7554/eLife.57346

Figure Lengend Snippet: 51 Cr-release assays were performed using NK92 ( A, C, D ) or YTS ( B, C ) WT and CD56-KO cell lines as effectors against susceptible targets. ( A ) 4 hr assays were performed with NK92 cell lines using K562 (left) or 721.221 (right) target cells. ( B ) 4 hr assays were performed with YTS cell lines using 721.221 (left) or KT86 (right) target cells. ( C ) 1 hr 51 Cr-release assays were performed using NK92 (left) or YTS (right) cells as effectors. ( D ) CD56 (NCAM-140) was re-expressed in NK92 CD56-KO cells and these cells, NK92 or NK92 CD56-KO cells were used for 4 hr cytotoxicity assays against K562 (left) or 721.221 (right) target cells. ( E ) CD56-KO NK92 cells were transfected with chimeric CD56 constructs fused to an mApple fluorescent reporter as described in Materials and methods. Flow cytometry was used to confirm the expression of CD56 and/or mApple. ( F ) Cytotoxicity assays were performed with chimeric cell lines using K562 cells as targets. Mean ± S.D. of three independent experiments pooled. **p<0.01, ***p<0.001, ****p<0.0001 by Ordinary one-way ANOVA with multiple corrections test or unpaired student t-test with Welch’s correction. ΔECD: chimeric construct lacking extracellular domain, ΔICD: chimeric construct lacking intracellular domain.

Article Snippet: NCAM reporter plasmids were generated by Epoch Life Sciences Inc and were made by subcloning NCAM1 (NCBI reference sequence: NM_000615.6, transcript variant 1; Origene) into BamHI and SalI digested pBABE-puro-mApple retroviral plasmid.

Techniques: Transfection, Construct, Flow Cytometry, Expressing

Journal: eLife

Article Title: CD56 regulates human NK cell cytotoxicity through Pyk2

doi: 10.7554/eLife.57346

Figure Lengend Snippet: NK92 and YTS WT and CD56-KO cells were analyzed for expression of cell surface receptors and intracellular effector molecules using five panels as described in Materials and methods. Effector functions were evaluated in the presence (activated) or absence (rest) of activation by PMA and ionomycin. Mean fluorescence intensity was measured and % positive cells based on fluorescence minus one gating was also calculated. Shown is one representative of 3 independent experiments.

Article Snippet: NCAM reporter plasmids were generated by Epoch Life Sciences Inc and were made by subcloning NCAM1 (NCBI reference sequence: NM_000615.6, transcript variant 1; Origene) into BamHI and SalI digested pBABE-puro-mApple retroviral plasmid.

Techniques: Expressing, Activation Assay, Fluorescence

Journal: eLife

Article Title: CD56 regulates human NK cell cytotoxicity through Pyk2

doi: 10.7554/eLife.57346

Figure Lengend Snippet: CD56 (left) or PSA-NCAM (right) were detected by flow cytometry on WT (solid line), CD56-KO (dashed line) or CD56-KO cells reconstituted with NCAM140 (dot-dashed line). Isotype antibody was used as a negative control (solid filled histogram). Shown is one representative of two independent repeats.

Article Snippet: NCAM reporter plasmids were generated by Epoch Life Sciences Inc and were made by subcloning NCAM1 (NCBI reference sequence: NM_000615.6, transcript variant 1; Origene) into BamHI and SalI digested pBABE-puro-mApple retroviral plasmid.

Techniques: Flow Cytometry, Negative Control

Journal: eLife

Article Title: CD56 regulates human NK cell cytotoxicity through Pyk2

doi: 10.7554/eLife.57346

Figure Lengend Snippet: ( A ) WT NK92, CD56-KO or CD56-KO reconstituted cells were incubated for 60–90 min on plates pre-coated with 10 μg/ml of anti-CD18 and anti-NKp30 antibodies. Supernatant was collected and granzyme A secretion was measured by a BLT esterase assay. Secretory potential was measured as a readout of the % maximum of granzyme A activity in the supernatant. ( B ) WT NK92, CD56-KO or CD56-KO reconstituted cells were incubated for 1–2 hr on plates pre-coated with 10 µg/ml of anti-CD18 and anti-NKp30 antibodies. Cells were harvested and degranulation was measured by CD107a expression using flow cytometry. ( C ) WT NK92, CD56-KO or CD56-KO reconstituted cells were co-cultured with 721.221 target cells. Cells were harvested and CD107a expression was measured by flow cytometry. For co-culture experiments the average background of media only was subtracted from samples. Mean ± SD. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001 by one-way ANOVA with Tukey’s multiple comparisons post-hoc test. ( D ) WT YTS and CD56-KO cells were incubated with 721.221 target cells at a 2:1 ratio for 22 hr. Supernatant was collected and used in a human IFN gamma ELISA. ****p<0.0001 by unpaired student t-test with Welch’s correction. All data are representative of 3 independent experiments performed in duplicate or triplicate.

Article Snippet: NCAM reporter plasmids were generated by Epoch Life Sciences Inc and were made by subcloning NCAM1 (NCBI reference sequence: NM_000615.6, transcript variant 1; Origene) into BamHI and SalI digested pBABE-puro-mApple retroviral plasmid.

Techniques: Incubation, Esterase Assay, Activity Assay, Expressing, Flow Cytometry, Cell Culture, Co-Culture Assay, Enzyme-linked Immunosorbent Assay

Journal: eLife

Article Title: CD56 regulates human NK cell cytotoxicity through Pyk2

doi: 10.7554/eLife.57346

Figure Lengend Snippet: WT or CD56-KO effector cells were cultured at a 2:1 ratio with K562 target cells for 45 min then fixed, immunostained and visualized by confocal microscopy. ( A ) Representative images from >30 cells from three independent experiments immunostained as indicated. ( B ) Integrated intensity of actin at the immune synapse for WT or CD56-KO cells. Data are representative from one experiment performed three times. n = 30, 39. NS = not significant by unpaired t-test. ( C ) MTOC to synapse distance (µm) calculated from WT or CD56-KO conjugates. n = 70, 83. Data pooled from three independently replicated experiments. ( D ) Mean granule to centroid distance for WT or CD56-KO conjugates. Each data point represents the mean distance granule to centroid distance from one conjugate. n = 33, 45 from one representative experiment of >3 experiments. NS = not significant by unpaired T test with Welch’s correction. ( E ) WT or CD56-KO NK92 effector cells were differentially labeled then conjugated at a 2:1 ratio with K562 target cells for the times indicated then fixed and analyzed by flow cytometry. The frequency of NK92-K562 conjugates was calculated for each timepoint. Each point represents a single experiment performed on different days in triplicate (n = 4 replicates). Error bars indicate mean ± SD; *p<0.05 by Mann-Whitney test.

Article Snippet: NCAM reporter plasmids were generated by Epoch Life Sciences Inc and were made by subcloning NCAM1 (NCBI reference sequence: NM_000615.6, transcript variant 1; Origene) into BamHI and SalI digested pBABE-puro-mApple retroviral plasmid.

Techniques: Cell Culture, Confocal Microscopy, Labeling, Flow Cytometry, MANN-WHITNEY

Journal: eLife

Article Title: CD56 regulates human NK cell cytotoxicity through Pyk2

doi: 10.7554/eLife.57346

Figure Lengend Snippet: ( A ) NK92 WT, CD56-KO or CD56 reconstituted (KO+CD56) cells were incubated for 25–30 min on plates pre-coated with 10 µg/ml of anti-CD18 and anti-NKp30 antibodies. Cells were permeabilized and immunostained for pPyk2 Y402 then data were acquired by flow cytometry. Relative fluorescent intensity (RFI) of pPyk2 was calculated based upon the intensity of the WT NK92 condition. Shown are the pooled data from three independent experiments. ( B ) WT or CD56-KO NK92 or YTS cells were permeabilized and immunostained for pPyk2 Y402 then data were acquired by flow cytometry. Shown is pooled data from 2 (YTS) or 3 (NK92) independent experiments. **p<0.01 by one-way ANOVA with multiple comparisons. ( C ) 4 hr 51 Cr assays were performed with WT (black) or CD56-KO (red) NK92 cells as effectors. Assays were performed in the presence of Pyk2 inhibitor PF431396 or vehicle control (DMSO) following brief pre-incubation of effectors with inhibitor. Shown are representative data from three independent repeats. ( D ) Pooled data from the 10:1 effector to target cell ratio of the experiments described in ( C ) normalized to the WT YTS condition without inhibitor. ***p<0.001 by one-way ANOVA with multiple comparisons. ( E ) Representative confocal microscopy images of WT or CD56-KO NK92 effectors conjugated to K562 target cells in the presence of non-blocking CD56 antibody then fixed and immunostained for perforin, pPyk2 Y402 and actin. ( F ) Fluorescent intensity of pPYK2 Y402 at the immune synapse of WT or CD56-KO effector cells. n = 24, 28 from one representative experiment of 3 independent repeats.

Article Snippet: NCAM reporter plasmids were generated by Epoch Life Sciences Inc and were made by subcloning NCAM1 (NCBI reference sequence: NM_000615.6, transcript variant 1; Origene) into BamHI and SalI digested pBABE-puro-mApple retroviral plasmid.

Techniques: Incubation, Flow Cytometry, Control, Confocal Microscopy, Blocking Assay

Journal: eLife

Article Title: CD56 regulates human NK cell cytotoxicity through Pyk2

doi: 10.7554/eLife.57346

Figure Lengend Snippet: WT (top) or CD56-KO (bottom) NK92 cells were conjugated to K562 targets then fixed, permeabliized and immunostained for tubulin, Pyk2, actin (phalloidin) and CD56. Images were acquired on a spinning disk confocal microscope. Shown is one representative of 60 cells from two independent technical replicates.

Article Snippet: NCAM reporter plasmids were generated by Epoch Life Sciences Inc and were made by subcloning NCAM1 (NCBI reference sequence: NM_000615.6, transcript variant 1; Origene) into BamHI and SalI digested pBABE-puro-mApple retroviral plasmid.

Techniques: Microscopy

Journal: eLife

Article Title: CD56 regulates human NK cell cytotoxicity through Pyk2

doi: 10.7554/eLife.57346

Figure Lengend Snippet: Primary NK cells were isolated and allowed to rest overnight in the presence of low-dose IL-15 prior to delivery of CD56 CRISPR-Cas9. Cells were further expanded in the presence of 25 ng/ml IL-15 for 15 days and cytotoxicity against K562 targets was measured. ( A ) Representative FACS plot of CD56-deficient (blue) or control primary cells (red) after 15 days of IL-15 expansion. Shown also is the fluorescence minus one control (yellow). ( B ) K562 target cell lysis by primary NK cells shown in ( A ). ( C ) Control or CD56-deficient NK cells from three healthy donors were incubated for 1 week after CD56 CRISPR-Cas9 delivery in 25 ng/mL IL-15 then cells were isolated by FACS and cultured for an additional 8 days and the MFI of CD56 was measured by flow cytometry. ( D ) Specific lysis of K562 target cells by isolated and expanded CD56 bright NK cells from the three healthy donors shown in ( C ). ( E ) Primary NK cells were incubated and expanded for 14 days in the presence of 50 ng/ml IL-15 then cytotoxicity against K562 target cells was tested in the presence or absence of Pyk2 inhibitor PF431396. Freshly isolated, non-expanded NK cells were used as a control and similarly treated with PF431396. ( F ) WT or CD56-KO NK92 cells were incubated for 7 days in the presence of 50 ng/ml IL-15 then cytotoxicity was tested in the presence or absence of PF431396. Shown is one representative experiment from three independent biological repeats. Error bars represent 3 technical repeats, SEM.

Article Snippet: NCAM reporter plasmids were generated by Epoch Life Sciences Inc and were made by subcloning NCAM1 (NCBI reference sequence: NM_000615.6, transcript variant 1; Origene) into BamHI and SalI digested pBABE-puro-mApple retroviral plasmid.

Techniques: Isolation, CRISPR, Control, Fluorescence, Lysis, Incubation, Cell Culture, Flow Cytometry

Journal: eLife

Article Title: CD56 regulates human NK cell cytotoxicity through Pyk2

doi: 10.7554/eLife.57346

Figure Lengend Snippet: Primary NK cells were enriched from peripheral blood then incubated with K562 target cells for 45 min on poly-L-lysine coated coverslips in the presence of non-blocking anti-CD56 antibody. Following incubation cells were fixed, permeabilized, and immunostained for pPyk2 Y402 (magenta), perforin (cyan) and actin (phalloidin, greyscale). 3D volumetric images were acquired by spinning disk confocal microscopy. ( A ) Representative images from one of three healthy donors. Shown is a maximum projection of 13 planes taken with 0.5 µm steps. ( B ) Fluorescence intensity of CD56 (left) or pPyk2 Y402 (right) measured at the synaptic (IS) or non-synaptic (NK) cell cortex of primary NK cells conjugated to target cells. n = 22 from one independent experiment of 3 using three different healthy donors. **p<0.005, ****p<0.0001 by paired t-test.

Article Snippet: NCAM reporter plasmids were generated by Epoch Life Sciences Inc and were made by subcloning NCAM1 (NCBI reference sequence: NM_000615.6, transcript variant 1; Origene) into BamHI and SalI digested pBABE-puro-mApple retroviral plasmid.

Techniques: Incubation, Blocking Assay, Confocal Microscopy, Fluorescence