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: 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: 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

Journal: ACS Omega

Article Title: G-Protein-Coupled Receptor 120 Agonist Mitigates Steatotic and Fibrotic Features Triggered in Obese Mice by the Administration of a High-Fat and High-Carbohydrate Diet

doi: 10.1021/acsomega.4c03507

Figure Lengend Snippet: Evaluation of steatotic profile in the liver. (A) Evaluation of gene expression in the liver. RT-PCR on mRNA extracted from livers of animals fed with WSD for 30 weeks (NASH) treated with vehicle (Veh) or GprA 90 mg/kg (GprA 90) for evaluating the expression of ffar4 , ffar1 . (B) Biochemical evaluation of triglycerides. Liver triglycerides were extracted from liver homogenates and titrated by spectrometric analysis. Optical density (O.D.) values were normalized for tissue fragment weight (around 30 mg for sample). Statistics, ANOVA ( p < 0.05) on AUC between Vehicle and GprA. (C) Histological analysis for fat infiltration. Oil red staining showed fat deposition in liver parenchyma (head arrows) at 26 and 30 weeks. (D) Quantification of oil red staining. The number and area of lipid droplets were assessed by oil red staining and normalized for section area. Statistics, ANOVA, p < 0.05). (E) RT-PCR on mRNA extracted from livers of animals fed with WSD for 26 weeks (NASH) treated with vehicle (Veh) or GprA 90 mg/kg (GprA 90) for evaluating the expression of per2 . Results are the mean + SD ( n = 7/group). Statistics, Student’s t test, p < 0.05.

Article Snippet: The receptor compound profiling experiments were done using Chinese hamster ovary (CHO) cells stably transfected by neomycin selection with GPR120 (full length, Origene, CAT#: RC214159, NCBI reference sequence: NM_181745.4) or GPR40 (CAT#: MR222997, NM_194057).

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

Journal: Endocrinology

Article Title: Selenoprotein P deletion ameliorates metabolic stress-associated anxiety-like behavior in male mice

doi: 10.1210/endocr/bqag010

Figure Lengend Snippet: Selenop expression in the amygdala. RNAscope ISH was performed in Selenop fl/fl mice and b Selenop -/- mice in the STD and the HFHSD groups. (A-B) RNAscope ISH of coronal sections of the amygdala for Selenop and immunostaining with anti-GS antibody (A) and the number of Selenop puncta per astrocyte (B), (C) RNAscope ISH of coronal sections of the amygdala for Selenop and immunostaining with anti-Iba1 antibody, (D) RNAscope ISH of coronal sections of the choroid plexus for Selenop without immunostaining, (E) Quantification of the fluorescence intensity of the Selenop signal in the amygdala regions of Selenop fl/fl mice. In (B, E), the data represent the mean ± SD; n = 3 mice for each condition; white boxes = control, black boxes = b Selenop -/- . STD, standard diet, HFHSD, high-fat high-sucrose diet. Statistical significance between different diets in the same genotype and different genotypes in the same diet was calculated using a 2-tailed unpaired Mann–Whitney test, significant P -values are indicated as exact numbers. Scale bars (A, C) 20 µm, (D) 150 µm. Scale bars (A, C) 20 µm, (D) 150 µm.

Article Snippet: Quantitative polymerase chain reaction (qPCR) was done using Applied Biosystems TaqMan probes (Sepp1, Mm00486048_m1, NCBI RefSeq NM_001042613.1, NM_001042614.1; Gpx1, Mm00656767_g1, NCBI RefSeq: NM_008160.6; Gapdh, 4351309, NCBI RefSeq: NM_008084; and the StepOnePlus real-time PCR system (Applied Biosystems by Life Technologies).

Techniques: Expressing, RNAscope, Immunostaining, Fluorescence, Control, MANN-WHITNEY

Journal: Endocrinology

Article Title: Selenoprotein P deletion ameliorates metabolic stress-associated anxiety-like behavior in male mice

doi: 10.1210/endocr/bqag010

Figure Lengend Snippet: Selenop expression in various brain regions. RT-PCR was performed in Selenop fl/fl mice and b Selenop -/- mice in the STD and the HFHSD groups. (A-E) Selenop mRNA expression in the (A) hippocampus, (B) cortex, (C) hypothalamus, (D) cerebellum, and (E) olfactory bulb of the brain. Gene expression was measured by real-time PCR. Expression values were normalized to that of Gapdh mRNA. n = 8 for Selenop fl/fl on STD; n = 13 for b Selenop -/- on STD, n = 5 for Selenop fl/fl on HFHSD; n = 10 for b Selenop -/- on HFHSD. Dots are individual values, and error bars are SD; white circles = Selenop fl/fl ; black circles = b Selenop -/- ; STD, standard diet, HFHSD, high-fat high-sucrose diet; Statistical significance between different diets in the same genotype and different genotypes in the same diet were calculated using 2-tailed unpaired Mann–Whitney test, significant P -values are indicated as exact numbers.

Article Snippet: Quantitative polymerase chain reaction (qPCR) was done using Applied Biosystems TaqMan probes (Sepp1, Mm00486048_m1, NCBI RefSeq NM_001042613.1, NM_001042614.1; Gpx1, Mm00656767_g1, NCBI RefSeq: NM_008160.6; Gapdh, 4351309, NCBI RefSeq: NM_008084; and the StepOnePlus real-time PCR system (Applied Biosystems by Life Technologies).

Techniques: Expressing, Reverse Transcription Polymerase Chain Reaction, Olfactory, Gene Expression, Real-time Polymerase Chain Reaction, MANN-WHITNEY

Journal: Endocrinology

Article Title: Selenoprotein P deletion ameliorates metabolic stress-associated anxiety-like behavior in male mice

doi: 10.1210/endocr/bqag010

Figure Lengend Snippet: Effect of brain-specific Selenop KO on body weight and glucose tolerance. (A) Body weight of Selenop fl/fl ( n = 3) and b Selenop -/- ( n = 7) on STD. (B) Body weight of Selenop fl/fl ( n = 6) and b Selenop -/- ( n = 9) on HFHSD. (C) Glucose tolerance of Selenop fl/fl ( n = 3) and b Selenop -/- ( n = 7) on STD. (D) Glucose tolerance of Selenop fl/fl ( n = 6) and b Selenop -/- ( n = 9) on HFHSD. (E) Baseline glucose level of Selenop fl/fl on STD ( n = 3) and HFHSD ( n = 6). (F) Baseline glucose level of b Selenop -/- on STD ( n = 7) and HFHSD ( n = 9). Mice fed STD and HFHSD received intraperitoneal glucose injections of 0.375 and 1.5 g/kg, respectively. In (A-B) and (E-F) dots are individual values and error bars are SD; in (C) and (D), dots represent mean values ± SD, white circles = Selenop fl/fl , black circles = b Selenop -/- . STD, standard diet, HFHSD, high-fat high-sucrose diet. Statistical significance was calculated by a 2-tailed unpaired Mann–Whitney test (A-B, E-F), and by area under the curve (AUC) analysis (C-D). There was no statistical difference in body weight (A, B) and glucose tolerance (C, D) between genotypes. Significant P -values are indicated as exact numbers (E-F).

Article Snippet: Quantitative polymerase chain reaction (qPCR) was done using Applied Biosystems TaqMan probes (Sepp1, Mm00486048_m1, NCBI RefSeq NM_001042613.1, NM_001042614.1; Gpx1, Mm00656767_g1, NCBI RefSeq: NM_008160.6; Gapdh, 4351309, NCBI RefSeq: NM_008084; and the StepOnePlus real-time PCR system (Applied Biosystems by Life Technologies).

Techniques: MANN-WHITNEY

Journal: Endocrinology

Article Title: Selenoprotein P deletion ameliorates metabolic stress-associated anxiety-like behavior in male mice

doi: 10.1210/endocr/bqag010

Figure Lengend Snippet: Anxiety-like behavior testing in the elevated plus maze (EPM) and open field (OF) tests. EPM and OF were performed in Selenop fl/fl mice and b Selenop -/- mice fed an STD and retested after 8 weeks of HFHSD feeding. (A) Timeline for anxiety-like behavior testing: Selenop fl/fl and b Selenop -/- mice were maintained on an STD for baseline assessment of anxiety-like behavior, followed by 8 weeks of an HFHSD feeding, after which behavioral testing was repeated. (B) Representative track plots of EPM, (C) time in the open arms, (D) distance traveled in the arena by Selenop fl/fl and b Selenop -/- mice in the EPM test before and after HFHSD feeding. (E) Representative track plots for OF, (F) time in the inner zone, (G) distance traveled by mice in the OF before and after HFHSD feeding. ( n = 7 for Selenop fl/fl mice, n = 11 for b Selenop -/- mice). Dots represent individual values, and error bars are SD; white circles = Selenop fl/fl ; black circles = b Selenop -/- ; STD, standard diet, HFHSD, high-fat high-sucrose diet. Statistical significance between different diets in the same genotype was calculated using a 2-tailed Wilcoxon matched-pairs signed rank test. Statistical significance between different genotypes in the same diet was calculated using a 2-tailed unpaired Mann–Whitney test, significant P -values are indicated as exact numbers.

Article Snippet: Quantitative polymerase chain reaction (qPCR) was done using Applied Biosystems TaqMan probes (Sepp1, Mm00486048_m1, NCBI RefSeq NM_001042613.1, NM_001042614.1; Gpx1, Mm00656767_g1, NCBI RefSeq: NM_008160.6; Gapdh, 4351309, NCBI RefSeq: NM_008084; and the StepOnePlus real-time PCR system (Applied Biosystems by Life Technologies).

Techniques: MANN-WHITNEY

Journal: Endocrinology

Article Title: Selenoprotein P deletion ameliorates metabolic stress-associated anxiety-like behavior in male mice

doi: 10.1210/endocr/bqag010

Figure Lengend Snippet: Spatial memory assessment using the Barnes Maze (BM). BM was performed in Selenop fl/fl mice and b Selenop -/- mice in the STD and retested after 8 weeks of HFHSD feeding. A) Number of errors during acquisition. (B) The area under the curve (AUC) analysis for the number of primary errors in each diet group in (A). The number of errors of Selenop fl/fl and b Selenop -/- mice in each diet group was combined to calculate the AUC. (C) Number of errors on the test day. (D) Latency to the first entry to the escape hole during acquisition in Selenop fl/fl mice and b Selenop -/- mice in the STD and the HFHSD groups. (E) The AUC analysis for the primary latency in each diet group in (D). The number of errors of Selenop fl/fl and b Selenop -/- mice in each diet group was combined to calculate the AUC. (F) Latency to reach the zone where the escape hole used to be on the test day. N = 7 for Selenop fl/fl mice, n = 11 for b Selenop -/- mice. In (A, B, D, E), dots and bars represent the mean ± SD; in (C) and (F), dots are individual values, and error bars are SD; white circles = Selenop fl/fl ; black circles = b Selenop -/- ; STD, standard diet, HFHSD, high-fat high-sucrose diet. Statistical significance between different diets in the same genotype was calculated using 2-tailed Wilcoxon matched-pairs signed rank test. Statistical significance between different genotypes in the same diet was calculated using a 2-tailed unpaired Mann–Whitney test. Significant P -values are indicated as exact numbers.

Article Snippet: Quantitative polymerase chain reaction (qPCR) was done using Applied Biosystems TaqMan probes (Sepp1, Mm00486048_m1, NCBI RefSeq NM_001042613.1, NM_001042614.1; Gpx1, Mm00656767_g1, NCBI RefSeq: NM_008160.6; Gapdh, 4351309, NCBI RefSeq: NM_008084; and the StepOnePlus real-time PCR system (Applied Biosystems by Life Technologies).

Techniques: MANN-WHITNEY

Journal: Endocrinology

Article Title: Selenoprotein P deletion ameliorates metabolic stress-associated anxiety-like behavior in male mice

doi: 10.1210/endocr/bqag010

Figure Lengend Snippet: Gpx1 expression in various brain regions. RT-PCR was performed in Selenop fl/fl mice and b Selenop -/- mice in the STD and the HFHSD groups. The mRNA expression of Gpx1 in the (A) hippocampus, (B) cortex, (C) hypothalamus, (D) cerebellum, and (E) olfactory bulb of the mouse brain. Each gene expression was assessed by real-time PCR. Expression values were normalized to Gapdh mRNA. n = 8 for Selenop fl/fl on STD; n = 10 for b Selenop -/- on STD, n = 6 for Selenop fl/fl on HFHSD; n = 9 for b Selenop -/- on HFHSD. Dots are individual values and error bars are SD; white circles = Selenop fl/fl ; black circles = b Selenop -/- ; STD, standard diet; HFHSD, high-fat high-sucrose diet; Gpx1 encodes glutathione peroxidase 1; Statistical significance between different diets in the same genotype and different genotypes in the same diet was calculated using 2-tailed unpaired Mann–Whitney test, significant P -values are indicated as exact numbers.

Article Snippet: Quantitative polymerase chain reaction (qPCR) was done using Applied Biosystems TaqMan probes (Sepp1, Mm00486048_m1, NCBI RefSeq NM_001042613.1, NM_001042614.1; Gpx1, Mm00656767_g1, NCBI RefSeq: NM_008160.6; Gapdh, 4351309, NCBI RefSeq: NM_008084; and the StepOnePlus real-time PCR system (Applied Biosystems by Life Technologies).

Techniques: Expressing, Reverse Transcription Polymerase Chain Reaction, Olfactory, Gene Expression, Real-time Polymerase Chain Reaction, MANN-WHITNEY