Journal:

Article Title: Genetic Diversity of nifH Gene Sequences in Paenibacillus azotofixans Strains and Soil Samples Analyzed by Denaturing Gradient Gel Electrophoresis of PCR-Amplified Gene Fragments

doi:

Figure Lengend Snippet: Bacterial strains and specificity of the PCR based on the nifH gene of P. azotofixans

Article Snippet: For database accession numbers see Fig. . table ft1 table-wrap mode="anchored" t5 TABLE 3 caption a7 Strain or sequence % Nucleotide or amino acid identity a P. azotofixans BE1 P. azotofixans RCPG7 P. azotofixans 2RC1 P. azotofixans ATCC 35681 P. azotofixans F102 P. azotofixans SD20 P. azotofixans C3L4 Azotobacter vinelandii Klebsiella pneumoniae Rhodobacter capsulata Lyngbya lagerheimii Rhizobium meliloti P. polymyxa P. macerans P. durum P. azotofixans P3E20 P. azotofixans RBN4 Clostridium pasteurianum nifH3 alternative ( anf ) Methanococcus thermolithotrophicus alternative ( anf ) P. azotofixans BE1 99 97 96 96 95 93 76 76 76 72 73 72 71 67 65 66 59 57 P. azotofixans RCPG7 100 98 97 96 96 94 76 76 76 72 73 72 71 67 66 67 59 58 P. azotofixans 2RC1 99 99 98 97 95 94 76 77 76 73 73 72 72 66 65 66 59 57 P. azotofixans ATCC 35681 99 99 100 96 95 93 76 77 75 72 73 72 71 65 65 65 59 57 P. azotofixans F102 99 99 98 98 94 94 77 76 76 72 73 73 72 67 66 66 59 56 P. azotofixans SD20 99 99 98 98 98 95 76 76 76 72 73 73 72 67 66 66 60 59 P. azotofixans C3L4 99 99 98 98 98 100 76 75 75 72 72 73 71 67 66 66 59 56 Azotobacter vinelandii 85 85 86 86 86 85 85 83 79 79 78 71 73 66 64 64 59 56 Klebsiella pneumoniae 86 86 87 87 85 86 86 93 80 78 76 71 72 67 66 66 58 57 Rhodobacter capsulata 88 88 87 87 89 88 88 86 86 76 76 70 72 64 63 64 57 56 Lyngbya lagerheimii 84 84 85 85 84 84 84 87 84 83 74 70 67 63 62 62 56 55 Rhizobium meliloti 85 85 86 86 86 85 85 84 83 91 82 70 71 63 63 63 58 57 P. polymyxa 75 75 74 74 74 75 75 68 68 68 68 65 74 59 58 59 57 56 P. macerans 48 48 48 48 49 49 49 44 42 46 46 44 49 60 59 60 53 54 P. durum 74 74 73 73 75 74 74 78 76 76 74 72 59 41 98 97 72 66 P. azotofixans P3E20 73 73 72 72 74 73 73 77 75 75 73 72 58 41 99 97 71 65 P. azotofixans RBN4 73 73 72 72 74 73 73 77 75 75 73 72 58 41 97 98 73 66 Clostridium pasteurianum nifH3 alternative ( anf ) 72 72 72 72 72 72 72 74 76 73 72 71 57 38 86 86 86 68 Methanococcus thermolithotrophicus alternative ( anf ) 68 68 68 68 67 68 68 72 72 69 69 67 54 35 80 79 79 79 Open in a separate window a The values on the upper right are levels of DNA identity, and the values on the lower left are levels of amino acid identity.

Techniques:

Journal:

Article Title: Genetic Diversity of nifH Gene Sequences in Paenibacillus azotofixans Strains and Soil Samples Analyzed by Denaturing Gradient Gel Electrophoresis of PCR-Amplified Gene Fragments

doi:

Figure Lengend Snippet: Southern hybridization of EcoRI-restricted genomic DNA of P. azotofixans and Paenibacillus sp. strains with a nifH probe (PCR product from strain ATCC 35681 generated with primers NHA1 and NHA2). The figure is a composite of two blots. Lane 1, P. durum DSMZ 1735; lane 2, P. azotofixans SD20; lane 3, BE1; lane 4, P3E20; lane 5, RCPG7; lane 6, SD17; lane 7, 2RC1; lane 8, P. macerans LMD24.3; lane 9, P. polymyxa DSMZ 356; lane 10, ATCC 35681; lane 11, F102; lane 12, C3L4; lane 13, RBN4.

Article Snippet: For database accession numbers see Fig. . table ft1 table-wrap mode="anchored" t5 TABLE 3 caption a7 Strain or sequence % Nucleotide or amino acid identity a P. azotofixans BE1 P. azotofixans RCPG7 P. azotofixans 2RC1 P. azotofixans ATCC 35681 P. azotofixans F102 P. azotofixans SD20 P. azotofixans C3L4 Azotobacter vinelandii Klebsiella pneumoniae Rhodobacter capsulata Lyngbya lagerheimii Rhizobium meliloti P. polymyxa P. macerans P. durum P. azotofixans P3E20 P. azotofixans RBN4 Clostridium pasteurianum nifH3 alternative ( anf ) Methanococcus thermolithotrophicus alternative ( anf ) P. azotofixans BE1 99 97 96 96 95 93 76 76 76 72 73 72 71 67 65 66 59 57 P. azotofixans RCPG7 100 98 97 96 96 94 76 76 76 72 73 72 71 67 66 67 59 58 P. azotofixans 2RC1 99 99 98 97 95 94 76 77 76 73 73 72 72 66 65 66 59 57 P. azotofixans ATCC 35681 99 99 100 96 95 93 76 77 75 72 73 72 71 65 65 65 59 57 P. azotofixans F102 99 99 98 98 94 94 77 76 76 72 73 73 72 67 66 66 59 56 P. azotofixans SD20 99 99 98 98 98 95 76 76 76 72 73 73 72 67 66 66 60 59 P. azotofixans C3L4 99 99 98 98 98 100 76 75 75 72 72 73 71 67 66 66 59 56 Azotobacter vinelandii 85 85 86 86 86 85 85 83 79 79 78 71 73 66 64 64 59 56 Klebsiella pneumoniae 86 86 87 87 85 86 86 93 80 78 76 71 72 67 66 66 58 57 Rhodobacter capsulata 88 88 87 87 89 88 88 86 86 76 76 70 72 64 63 64 57 56 Lyngbya lagerheimii 84 84 85 85 84 84 84 87 84 83 74 70 67 63 62 62 56 55 Rhizobium meliloti 85 85 86 86 86 85 85 84 83 91 82 70 71 63 63 63 58 57 P. polymyxa 75 75 74 74 74 75 75 68 68 68 68 65 74 59 58 59 57 56 P. macerans 48 48 48 48 49 49 49 44 42 46 46 44 49 60 59 60 53 54 P. durum 74 74 73 73 75 74 74 78 76 76 74 72 59 41 98 97 72 66 P. azotofixans P3E20 73 73 72 72 74 73 73 77 75 75 73 72 58 41 99 97 71 65 P. azotofixans RBN4 73 73 72 72 74 73 73 77 75 75 73 72 58 41 97 98 73 66 Clostridium pasteurianum nifH3 alternative ( anf ) 72 72 72 72 72 72 72 74 76 73 72 71 57 38 86 86 86 68 Methanococcus thermolithotrophicus alternative ( anf ) 68 68 68 68 67 68 68 72 72 69 69 67 54 35 80 79 79 79 Open in a separate window a The values on the upper right are levels of DNA identity, and the values on the lower left are levels of amino acid identity.

Techniques: Hybridization, Generated

Journal:

Article Title: Genetic Diversity of nifH Gene Sequences in Paenibacillus azotofixans Strains and Soil Samples Analyzed by Denaturing Gradient Gel Electrophoresis of PCR-Amplified Gene Fragments

doi:

Figure Lengend Snippet: Alignment of amino acid sequences deduced from the nifH sequence of Paenibacillus sp. and from the sequences of nifH genes of other organisms in the database. The sequences compared correspond to residues 48 to 155 of the Anabaena (Nostoc) sp. strain 7120 sequence (GenBank accession no. A00534). Abbreviations: av, Azotobacter vinelandii; kp, Klebsiella pneumoniae; C3l4, sd20, 2rc1, atcc, be1, rcpg7, and f102, P. azotofixans C3L4, SD20, 2RC1, ATCC 35681, BE1, RCPG7, and F102, respectively; rc, Rhodobacter capsulata; rm, Rhizobium meliloti; LL, Lyngbya lagerheimii; durum, P. durum; p3e20 and frbn4, P. azotofixans P3E20 and RBN4, respectively; cp3, C. pasteurianum nifH3 alternative (anf); cp1, C. pasteurianum nifH1; mtal, Methanococcus thermolithotrophicus alternative (anf); pol, P. polymyxa; mac, P. macerans. For database accession numbers see Fig. ​Fig.33.

Article Snippet: For database accession numbers see Fig. . table ft1 table-wrap mode="anchored" t5 TABLE 3 caption a7 Strain or sequence % Nucleotide or amino acid identity a P. azotofixans BE1 P. azotofixans RCPG7 P. azotofixans 2RC1 P. azotofixans ATCC 35681 P. azotofixans F102 P. azotofixans SD20 P. azotofixans C3L4 Azotobacter vinelandii Klebsiella pneumoniae Rhodobacter capsulata Lyngbya lagerheimii Rhizobium meliloti P. polymyxa P. macerans P. durum P. azotofixans P3E20 P. azotofixans RBN4 Clostridium pasteurianum nifH3 alternative ( anf ) Methanococcus thermolithotrophicus alternative ( anf ) P. azotofixans BE1 99 97 96 96 95 93 76 76 76 72 73 72 71 67 65 66 59 57 P. azotofixans RCPG7 100 98 97 96 96 94 76 76 76 72 73 72 71 67 66 67 59 58 P. azotofixans 2RC1 99 99 98 97 95 94 76 77 76 73 73 72 72 66 65 66 59 57 P. azotofixans ATCC 35681 99 99 100 96 95 93 76 77 75 72 73 72 71 65 65 65 59 57 P. azotofixans F102 99 99 98 98 94 94 77 76 76 72 73 73 72 67 66 66 59 56 P. azotofixans SD20 99 99 98 98 98 95 76 76 76 72 73 73 72 67 66 66 60 59 P. azotofixans C3L4 99 99 98 98 98 100 76 75 75 72 72 73 71 67 66 66 59 56 Azotobacter vinelandii 85 85 86 86 86 85 85 83 79 79 78 71 73 66 64 64 59 56 Klebsiella pneumoniae 86 86 87 87 85 86 86 93 80 78 76 71 72 67 66 66 58 57 Rhodobacter capsulata 88 88 87 87 89 88 88 86 86 76 76 70 72 64 63 64 57 56 Lyngbya lagerheimii 84 84 85 85 84 84 84 87 84 83 74 70 67 63 62 62 56 55 Rhizobium meliloti 85 85 86 86 86 85 85 84 83 91 82 70 71 63 63 63 58 57 P. polymyxa 75 75 74 74 74 75 75 68 68 68 68 65 74 59 58 59 57 56 P. macerans 48 48 48 48 49 49 49 44 42 46 46 44 49 60 59 60 53 54 P. durum 74 74 73 73 75 74 74 78 76 76 74 72 59 41 98 97 72 66 P. azotofixans P3E20 73 73 72 72 74 73 73 77 75 75 73 72 58 41 99 97 71 65 P. azotofixans RBN4 73 73 72 72 74 73 73 77 75 75 73 72 58 41 97 98 73 66 Clostridium pasteurianum nifH3 alternative ( anf ) 72 72 72 72 72 72 72 74 76 73 72 71 57 38 86 86 86 68 Methanococcus thermolithotrophicus alternative ( anf ) 68 68 68 68 67 68 68 72 72 69 69 67 54 35 80 79 79 79 Open in a separate window a The values on the upper right are levels of DNA identity, and the values on the lower left are levels of amino acid identity.

Techniques: Sequencing

Journal:

Article Title: Genetic Diversity of nifH Gene Sequences in Paenibacillus azotofixans Strains and Soil Samples Analyzed by Denaturing Gradient Gel Electrophoresis of PCR-Amplified Gene Fragments

doi:

Figure Lengend Snippet: Levels of nucleotide and amino acid sequence identity for nifH genes of P. azotofixans and nifH sequences of other bacterial species

Article Snippet: For database accession numbers see Fig. . table ft1 table-wrap mode="anchored" t5 TABLE 3 caption a7 Strain or sequence % Nucleotide or amino acid identity a P. azotofixans BE1 P. azotofixans RCPG7 P. azotofixans 2RC1 P. azotofixans ATCC 35681 P. azotofixans F102 P. azotofixans SD20 P. azotofixans C3L4 Azotobacter vinelandii Klebsiella pneumoniae Rhodobacter capsulata Lyngbya lagerheimii Rhizobium meliloti P. polymyxa P. macerans P. durum P. azotofixans P3E20 P. azotofixans RBN4 Clostridium pasteurianum nifH3 alternative ( anf ) Methanococcus thermolithotrophicus alternative ( anf ) P. azotofixans BE1 99 97 96 96 95 93 76 76 76 72 73 72 71 67 65 66 59 57 P. azotofixans RCPG7 100 98 97 96 96 94 76 76 76 72 73 72 71 67 66 67 59 58 P. azotofixans 2RC1 99 99 98 97 95 94 76 77 76 73 73 72 72 66 65 66 59 57 P. azotofixans ATCC 35681 99 99 100 96 95 93 76 77 75 72 73 72 71 65 65 65 59 57 P. azotofixans F102 99 99 98 98 94 94 77 76 76 72 73 73 72 67 66 66 59 56 P. azotofixans SD20 99 99 98 98 98 95 76 76 76 72 73 73 72 67 66 66 60 59 P. azotofixans C3L4 99 99 98 98 98 100 76 75 75 72 72 73 71 67 66 66 59 56 Azotobacter vinelandii 85 85 86 86 86 85 85 83 79 79 78 71 73 66 64 64 59 56 Klebsiella pneumoniae 86 86 87 87 85 86 86 93 80 78 76 71 72 67 66 66 58 57 Rhodobacter capsulata 88 88 87 87 89 88 88 86 86 76 76 70 72 64 63 64 57 56 Lyngbya lagerheimii 84 84 85 85 84 84 84 87 84 83 74 70 67 63 62 62 56 55 Rhizobium meliloti 85 85 86 86 86 85 85 84 83 91 82 70 71 63 63 63 58 57 P. polymyxa 75 75 74 74 74 75 75 68 68 68 68 65 74 59 58 59 57 56 P. macerans 48 48 48 48 49 49 49 44 42 46 46 44 49 60 59 60 53 54 P. durum 74 74 73 73 75 74 74 78 76 76 74 72 59 41 98 97 72 66 P. azotofixans P3E20 73 73 72 72 74 73 73 77 75 75 73 72 58 41 99 97 71 65 P. azotofixans RBN4 73 73 72 72 74 73 73 77 75 75 73 72 58 41 97 98 73 66 Clostridium pasteurianum nifH3 alternative ( anf ) 72 72 72 72 72 72 72 74 76 73 72 71 57 38 86 86 86 68 Methanococcus thermolithotrophicus alternative ( anf ) 68 68 68 68 67 68 68 72 72 69 69 67 54 35 80 79 79 79 Open in a separate window a The values on the upper right are levels of DNA identity, and the values on the lower left are levels of amino acid identity.

Techniques: Sequencing

Journal:

Article Title: Genetic Diversity of nifH Gene Sequences in Paenibacillus azotofixans Strains and Soil Samples Analyzed by Denaturing Gradient Gel Electrophoresis of PCR-Amplified Gene Fragments

doi:

Figure Lengend Snippet: DGGE analysis of PCR-amplified nifH gene fragments from P. azotofixans and P. durum strains: parallel DGGE separation patterns of gene fragments obtained with genomic DNA from strains DSMZ 1735 (lane 1), BE1 (lane 2), SD20 (lane 3), SD17 (lane 4), RCPG7 (lane 5), F102 (lane 6), 2RC1 (lane 7), C3L4 (lane 8), RBN4 (lane 9), P3E20 (lane 10), and ATCC 35681 (lane 11).

Article Snippet: For database accession numbers see Fig. . table ft1 table-wrap mode="anchored" t5 TABLE 3 caption a7 Strain or sequence % Nucleotide or amino acid identity a P. azotofixans BE1 P. azotofixans RCPG7 P. azotofixans 2RC1 P. azotofixans ATCC 35681 P. azotofixans F102 P. azotofixans SD20 P. azotofixans C3L4 Azotobacter vinelandii Klebsiella pneumoniae Rhodobacter capsulata Lyngbya lagerheimii Rhizobium meliloti P. polymyxa P. macerans P. durum P. azotofixans P3E20 P. azotofixans RBN4 Clostridium pasteurianum nifH3 alternative ( anf ) Methanococcus thermolithotrophicus alternative ( anf ) P. azotofixans BE1 99 97 96 96 95 93 76 76 76 72 73 72 71 67 65 66 59 57 P. azotofixans RCPG7 100 98 97 96 96 94 76 76 76 72 73 72 71 67 66 67 59 58 P. azotofixans 2RC1 99 99 98 97 95 94 76 77 76 73 73 72 72 66 65 66 59 57 P. azotofixans ATCC 35681 99 99 100 96 95 93 76 77 75 72 73 72 71 65 65 65 59 57 P. azotofixans F102 99 99 98 98 94 94 77 76 76 72 73 73 72 67 66 66 59 56 P. azotofixans SD20 99 99 98 98 98 95 76 76 76 72 73 73 72 67 66 66 60 59 P. azotofixans C3L4 99 99 98 98 98 100 76 75 75 72 72 73 71 67 66 66 59 56 Azotobacter vinelandii 85 85 86 86 86 85 85 83 79 79 78 71 73 66 64 64 59 56 Klebsiella pneumoniae 86 86 87 87 85 86 86 93 80 78 76 71 72 67 66 66 58 57 Rhodobacter capsulata 88 88 87 87 89 88 88 86 86 76 76 70 72 64 63 64 57 56 Lyngbya lagerheimii 84 84 85 85 84 84 84 87 84 83 74 70 67 63 62 62 56 55 Rhizobium meliloti 85 85 86 86 86 85 85 84 83 91 82 70 71 63 63 63 58 57 P. polymyxa 75 75 74 74 74 75 75 68 68 68 68 65 74 59 58 59 57 56 P. macerans 48 48 48 48 49 49 49 44 42 46 46 44 49 60 59 60 53 54 P. durum 74 74 73 73 75 74 74 78 76 76 74 72 59 41 98 97 72 66 P. azotofixans P3E20 73 73 72 72 74 73 73 77 75 75 73 72 58 41 99 97 71 65 P. azotofixans RBN4 73 73 72 72 74 73 73 77 75 75 73 72 58 41 97 98 73 66 Clostridium pasteurianum nifH3 alternative ( anf ) 72 72 72 72 72 72 72 74 76 73 72 71 57 38 86 86 86 68 Methanococcus thermolithotrophicus alternative ( anf ) 68 68 68 68 67 68 68 72 72 69 69 67 54 35 80 79 79 79 Open in a separate window a The values on the upper right are levels of DNA identity, and the values on the lower left are levels of amino acid identity.

Techniques: Amplification

Journal: Science Advances

Article Title: HSP90 inhibition suppresses tumor glycolytic flux to potentiate the therapeutic efficacy of radiotherapy for head and neck cancer

doi: 10.1126/sciadv.adk3663

Figure Lengend Snippet: ( A ) Effect of ganetespib on alterations in cytokine profile in HN12 cells determined by Human Cytokine Array. A total of 500 μg of proteins were collected from cells treated with or without 0.1 μM ganetespib for 24 hours. Quantitative dot intensity ( n = 2) is shown in the right panel. VEGF, vascular endothelial growth factor; TSP, thrombospondin 1; TSG14, tumor necrosis factor-inducible gene 14 protein, DKK1; dickkopf-Related Protein 1; uPAR, urokinase-type plasminogen activator receptor; MMP9, matrix metalloproteinase 9; GDF15, growth and differentiation factor 15. ( B ) Effect of ganetespib on IL-1α, IL-1β, and IL-8 protein levels in HN6 and HN12 cells validated by Western blot. ( C ) Effect of ganetespib on IL-8 secretion in HN6 and HN12 cell supernatants determined by ELISA. In (B) and (C), HN6 and HN12 cells were treated with or without 0.1 μM ganetespib for 24 hours. ( D ) Effect of HSP90AA1 knockdown on IL-8 protein levels in HN6 and HN12 cells determined by Western blot. ( E and F ) Effect of different glucose concentrations on IL-8 protein levels in HN6 and HN12 cells and cell supernatant determined by Western blot and ELISA, respectively. ( G and H ) Effect of ganetespib on IL-8 protein levels in HN6 and HN12 cells and cell supernatant under low (500 mg/liter) or high (2000 mg/liter) glucose concentrations determined by Western blot and ELISA, respectively. ( I ) Effect of PKM or PFKP knockdown on IL-8 protein levels in HN6 and HN12 cells. ( J ) Effect of PKM or PFKP knockdown on IL-8 levels in HN12 cells under low (500 mg/liter) and high (2000 mg/liter) glucose concentrations. In (E), (F), (G), (H), and (J), intracellular and secreted IL-8 levels were determined 24 hours after glucose addition. shCTRL, a scrambled shRNA; shPKM-1 and shPKM-2, two shRNAs against the PKM gene; shPFKP-1 and shPFKP-2, two shRNAs against the PFKP gene. Data are presented as mean values ± SD. For statistical comparisons, P values were assessed by unpaired, two-tailed Student’s t test in (C), (F), and (H). ** P < 0.01.

Article Snippet: The full-length Flag-tagged human IL8 was cloned into mammalian expression vector pCMV3 and obtained from SinoBiological Inc. (catalog no. HG10098-CF).

Techniques: Western Blot, Enzyme-linked Immunosorbent Assay, shRNA, Two Tailed Test

Journal: Science Advances

Article Title: HSP90 inhibition suppresses tumor glycolytic flux to potentiate the therapeutic efficacy of radiotherapy for head and neck cancer

doi: 10.1126/sciadv.adk3663

Figure Lengend Snippet: ( A ) Correlation between IL-8 expression and survival in patients in TCGA HNSCC cohorts ( n = 479). HR, hazard ratio. ( B and C ) Growth curve and weight of HN12 tumors treated with vehicle or ganetespib ( n = 5 mice per group). ( D ) Mouse survival curves for groups receiving vehicle and ganetespib ( n = 5 mice per group, log-rank test). ( E ) Immunostaining of PKM2, PFKP, and IL-8 in HN12 tumors treated with vehicle or ganetespib. Quantitative data ( n = 5 mice per group) are shown in the bottom panel, respectively. ( F ) Correlation between IL-8 expression and infiltration level of CD8 + T cells in TCGA HNSCC cohort analyzed via TIMER. Correlation was analyzed using Pearson correlation coefficient. TPM, transcripts per million. ( G ) Immunostaining of CD8α and IL-8 in paired primary HNSCC patient tumor tissues ( n = 2) obtained before and after ganetespib treatment in the clinical trial (NCT02334319). Quantitative data are shown in the right panel. ( H ) Percent of CD8 + T cell subsets in monocytes isolated from human peripheral blood mononuclear cells (PBMCs) determined by flow cytometry. ( I ) Schematic diagram of in vitro CD8 + T cell migration assays in the presence or absence rhIL-8. ( J ) Effect of rhIL-8 on suppressing CD8 + T cell migration to HN12 cells. ( K ) Effect of ganetespib on promoting CD8 + T cell migration to IL8 overexpressing HN12 cells (HN12–IL-8) and HN12 cells. ( L ) Overexpression of huIL8 gene in MOC2 cells (MOC2–huIL-8). ( M ) Cell proliferation of MOC2–huIL-8 cells compared with MOC2 cells. ( N ) Growth curve and weight of MOC2–huIL-8 tumors and MOC2 tumors. ( O ) Percent of CD8 + CD4 − T cell subsets in MOC2–huIL-8 tumors and MOC2 tumors. CD4 + and CD8 + T subsets were gated from CD3 + population. Data are presented as mean values ± SD. For statistical comparisons, P values were assessed by unpaired, two-tailed Student’s t test. * P < 0.05; ** P < 0.01.

Article Snippet: The full-length Flag-tagged human IL8 was cloned into mammalian expression vector pCMV3 and obtained from SinoBiological Inc. (catalog no. HG10098-CF).

Techniques: Expressing, Immunostaining, Isolation, Flow Cytometry, In Vitro, Migration, Over Expression, Two Tailed Test

Journal: Science Advances

Article Title: HSP90 inhibition suppresses tumor glycolytic flux to potentiate the therapeutic efficacy of radiotherapy for head and neck cancer

doi: 10.1126/sciadv.adk3663

Figure Lengend Snippet: ( A ) Effect of RT on PKM2, PFKP, and IL-8 protein levels in HN6 and HN12 cells. ( B ) Effect of ganetespib on RT-induced PKM2/PFKP–IL-8 axis in HN6 and HN12 cells. ( C ) Effect of ganetespib and/or RT on clonogenic ability of HN6 and HN12 cells in culture medium with low (500 mg/liter) and high dose (2000 mg/liter) of glucose. Quantitative data from three independent experiments are shown in the bottom panels. ( D ) Effect of PKM or PFKP knockdown on clonogenic ability of HN6 and HN12 cells with or without RT [4 gray (Gy)]. Quantitative data from three independent experiments are shown in the right panel. ( E ) Simultaneous overexpression of PKM2 and PFKP in HN6 and HN12 cells. ( F ) Effect of individual or concurrent overexpression of PKM2 and PFKP on IL-8 levels in HN6 and HN12 cells in the presence of 0.1 μM ganetespib. ( G ) Effect of individual or concurrent overexpression of PKM2 and PFKP on clonogenic ability of HN6 and HN12 cells exposed to 4-Gy RT in the presence or absence of ganetespib. Quantitative data from three independent experiments are shown in the right panel. shPKM, a specific shRNA against the PKM gene; shPFKP, a specific shRNA against the PFKP gene; EV, empty vector; PKM2 O/E, PKM2 overexpression vector; PFKP O/E, PKFP overexpression vector. Data are presented as mean values ± SD. For statistical comparisons, P values were assessed by unpaired, two-tailed Student’s t test in (C) and (G) and by one-way ANOVA analysis with Turkey’s multiple comparisons in (D). * P < 0.05; ** P < 0.01.

Article Snippet: The full-length Flag-tagged human IL8 was cloned into mammalian expression vector pCMV3 and obtained from SinoBiological Inc. (catalog no. HG10098-CF).

Techniques: Over Expression, shRNA, Plasmid Preparation, Two Tailed Test

Journal: Nature Communications

Article Title: Expansion of human megakaryocyte-biased hematopoietic stem cells by biomimetic Microniche

doi: 10.1038/s41467-023-37954-3

Figure Lengend Snippet: a REVIGO interactive graph of major Gene Ontology molecular function (GO MF) terms in RNA-seq of sorted CD34 + cells after culture. Bubble color indicates −Log 10 p value; bubble size indicates GO term frequency. b GSEA of the indicated gene sets enriched in Microniche vs. control. Bubble size indicates normalized enrichment score (NES); bubble color indicates p value. c Heatmap of average relative expression for cytokine-cytokine receptor genes enriched in Microniche-cultured CD34 + cells. Color indicates fold change in expression levels quantified by qPCR. d Heatmap showing the average relative secretion of chemokines enriched in Microniche culture supernatant. Color indicates fold change in chemokine levels determined by Luminex liquid suspension chip. e Identification of nine cell clusters visualized by UMAP in 10X Genomics single-cell RNA-seq profile of CD34 + cells sorted after culture. Each dot represents one cell, and colors represent distinct cell clusters (C0–C8). Pie chart showing the percentages of each cell cluster in control and Microniche cultures. f Dot plot showing the expression of feature genes related to Mk and Er lineage, HSC stemness, chemokines, cytokines, and receptors in each cell cluster. Bubble color indicates average relative expression; bubble size indicates percentage of relative expression. g CellPhoneDB analysis of crosstalk between cell clusters. h Representative FACS profiles of phenotype-defined cell subsets in UCB CD34 + cells before (fresh) and after cultured with or without cytokines (CXCL8, CCL24, IL-10, IL-1β, IL-1α) for 7 days. P1: CD34 + , P2: CD34 + CD38 − , P3: CD34 + CD38 − CD45RA − CD90 + , P4: CD34 + CD38 − CD45RA − CD90 + CD49f low , P5: CD34 + CD38 − CD45RA − CD90 + CD49f low CD62L − CD133 + . i Dot plot showing the absolute numbers and percentages of phenotype-defined cell subpopulations. For each subpopulation, their changes after culture are summarized as Log number and Log percentage relative to fresh, with color (red) depth indicating the size of absolute numbers and bubble size indicating the size of percentages in living cells. Fresh, n = 3 technical replicates; control and cytokines, n = 4 biological replicates. Data were normalized with data of fresh group.

Article Snippet: Cultures were performed in a 2 ml medium/well supplemented with or without a cytokine mixture [6.2416 ng/ml CXCL8 (SinoBiological, 10098-HNCH2), 0.2379 ng/ml CCL24 (SinoBiological, 10901-H08B), 0.0107 ng/ml IL-10 (SinoBiological, 10947-H07H), 0.0055 ng/ml IL-1β (SinoBiological, 10139-HNAE), and 0.0055 ng/ml IL-1α (SinoBiological, 10128-HNCH)] at 37 °C with 5% CO 2 for 7 days.

Techniques: RNA Sequencing Assay, Control, Expressing, Cell Culture, Luminex, Suspension

Journal: BMB Reports

Article Title: Expression profiling identified IL-8 as a regulator of homotypic cell-in-cell formation

doi: 10.5483/BMBRep.2018.51.8.089

Figure Lengend Snippet: Expression profiling of genes associated with CIC formation. (A) A sequential screening for genes associated with CIC formation. The differentially expressed genes shared by three analysis sets were selected as the candidate genes. The number in brackets are probes differentially expressed in each comparison set. (B) A heat map of the 73 probes potentially involved in CIC formation. The up-regulated and down-regulated probes are marked as up (26) and down (47). The number in brackets are the differentially expressed probes. (C) A gene network centered on IL-8 constructed by the “Pathway” algorithm of GeneSpring software under the condition of “simple algorithm” and “direct interaction”. (D) Validation of candidate gene expression by quantitative RT-PCR. Five down-regulated genes NUPR1, STAT6, SERPINA1, DLC1 and PRSS23 and one up-regulated gene IL-8 were selected for qRT-PCR analysis. Data are mean ± SD of three independent experiments.

Article Snippet: Human IL-8 / CXCL8 protein (aa 23–99) was purchased from Sino Biological (Beijing, China).

Techniques: Expressing, Construct, Software, Quantitative RT-PCR

Journal: BMB Reports

Article Title: Expression profiling identified IL-8 as a regulator of homotypic cell-in-cell formation

doi: 10.5483/BMBRep.2018.51.8.089

Figure Lengend Snippet: Regulation of CIC formation by NUPR1 and IL-8 . (A) CIC formation in MDA-MB-436 cells upon NUPR1 knockdown. Representative images show the cytospins of MDA-MB-436 cells transfected with control and NUPR1 siRNAs. Nuclei were stained with DAPI. Scale bar: 100 μm. (B) CIC formation in FENT and FK12 cells with IL-8 knockdown. (C) CIC formation in MDA-MB-436 and ZR75-1 cells treated with recombinant IL-8. IL-8 activity was determined by Akt phosphorylation. The black bar graphs show relative mRNA level examined by qRT-PCR. Data are mean ± SD of three independent experiments. The white bar graphs show the quantification of CIC formation. Data are mean ± SD of cells analyzed in triplicate and are representative of three independent experiments. “*” for P < 0.05. “**” for P < 0.01. “MM436” for MDA-MB-436. “si” for siRNA. “NC” for negative control.

Article Snippet: Human IL-8 / CXCL8 protein (aa 23–99) was purchased from Sino Biological (Beijing, China).

Techniques: Transfection, Staining, Recombinant, Activity Assay, Quantitative RT-PCR, Negative Control

Journal: BMB Reports

Article Title: Expression profiling identified IL-8 as a regulator of homotypic cell-in-cell formation

doi: 10.5483/BMBRep.2018.51.8.089

Figure Lengend Snippet: Regulation of cell-cell adhesion by IL-8. (A) RNAi-mediated IL-8 knockdown and IL-8 treatment regulates cluster formation. Scale bar: 100 μm. The graph shows the percentage of cells forming clusters. Cells in cluster = cells forming cluster / total cells counted. Cell cluster was defined as a cell colony that contains more than 6 cells. Data are mean ± SD of cells in three fields of view, n > 200 for each field. (B) Relative mRNA level of CDH1, CDH3, CTNNA1, CTNNB1, CTNNG and CTNND1 by quantitative real-time PCR. Data are mean ± SD of three independent experiments. (C and E) Expression of adhesion molecules as detected by western blot. (D and F) Quantification of the blots of C and E. Data are mean ± SD of triplicate quantification. “*” for P < 0.05. “**” for P < 0.01. “MM436” for MDA-MB-436. “si” for siRNA. “NC” for negative control.

Article Snippet: Human IL-8 / CXCL8 protein (aa 23–99) was purchased from Sino Biological (Beijing, China).

Techniques: Real-time Polymerase Chain Reaction, Expressing, Western Blot, Negative Control