Journal: Journal of Clinical Microbiology

Article Title: Identification and Characterization of “ Haemophilus quentini ” Strains Causing Invasive Disease in Ontario, Canada (2016 to 2018)

doi: 10.1128/JCM.01254-19

Figure Lengend Snippet: Initial description of suspected Haemophilus quentini isolates and controls

Article Snippet: The seventh isolate (HQ7) was biotype III and fermented glucose and mannose but not sucrose, lactose, or xylose ( ). table ft1 table-wrap mode="anchored" t5 TABLE 3 caption a7 Strain PCR result for the following gene target: H. influenzae MLST allele no. b hpd ompP6 sodC H. quentini -specific 16S rRNA gene adk atpG frdB fucK mdh pgi recA HQ1 + + + + 8 27 27 No product 37 15 24 HQ2 + + + + 8 27 27 No product 37 15 24 HQ3 + + + + 8 (99.8% match) 27 27 (99.8% match) No product 37 (99.3% match) 15 24 HQ4 + + + + 8 (99.8% match) 27 27 (99.8% match) No product 37 (99.3% match) 15 24 HQ5 + + + + 8 27 27 No product 37 15 24 HQ6 + + + + 8 (99.8% match) 27 27 (99.8% match) No product 37 (99.3% match) 15 24 HQ7 + + + + 81 (99.8% match) 63 (99.3% match) 86 (98.2% match) No product 63 (98.0% match) 84 (97.4% match) 60 (96.5% match) NTHi1 + + – – 11 43 35 11 51 31 18 NTHi2 + + – – 30 2 15 7 22 38 3 H. quentini HK-1 + + + + 8 27 27 No product 37 15 24 H. haemolyticus ATCC 33390 T + + + – 80 66 83 No product 126 101 74 Open in a separate window a PCR and MLST descriptions of seven invasive clinical isolates of suspected Haemophilus quentini from Ontario, Canada (HQ1 to HQ7), two isolates of invasive nontypeable H. influenzae biotype IV from Ontario, Canada (NTHi1 and NTHi2), a clinical blood isolate of H. quentini from Hong Kong, China (HK-1), and the type strain of H. haemolyticus (ATCC 33390 T ). b The percentage in parentheses refers to the percent match of the query to the nucleotide sequences of the indicated allele number.

Techniques: Sequencing

Journal: Journal of Clinical Microbiology

Article Title: Identification and Characterization of “ Haemophilus quentini ” Strains Causing Invasive Disease in Ontario, Canada (2016 to 2018)

doi: 10.1128/JCM.01254-19

Figure Lengend Snippet: PCR and MLST results a

Article Snippet: The seventh isolate (HQ7) was biotype III and fermented glucose and mannose but not sucrose, lactose, or xylose ( ). table ft1 table-wrap mode="anchored" t5 TABLE 3 caption a7 Strain PCR result for the following gene target: H. influenzae MLST allele no. b hpd ompP6 sodC H. quentini -specific 16S rRNA gene adk atpG frdB fucK mdh pgi recA HQ1 + + + + 8 27 27 No product 37 15 24 HQ2 + + + + 8 27 27 No product 37 15 24 HQ3 + + + + 8 (99.8% match) 27 27 (99.8% match) No product 37 (99.3% match) 15 24 HQ4 + + + + 8 (99.8% match) 27 27 (99.8% match) No product 37 (99.3% match) 15 24 HQ5 + + + + 8 27 27 No product 37 15 24 HQ6 + + + + 8 (99.8% match) 27 27 (99.8% match) No product 37 (99.3% match) 15 24 HQ7 + + + + 81 (99.8% match) 63 (99.3% match) 86 (98.2% match) No product 63 (98.0% match) 84 (97.4% match) 60 (96.5% match) NTHi1 + + – – 11 43 35 11 51 31 18 NTHi2 + + – – 30 2 15 7 22 38 3 H. quentini HK-1 + + + + 8 27 27 No product 37 15 24 H. haemolyticus ATCC 33390 T + + + – 80 66 83 No product 126 101 74 Open in a separate window a PCR and MLST descriptions of seven invasive clinical isolates of suspected Haemophilus quentini from Ontario, Canada (HQ1 to HQ7), two isolates of invasive nontypeable H. influenzae biotype IV from Ontario, Canada (NTHi1 and NTHi2), a clinical blood isolate of H. quentini from Hong Kong, China (HK-1), and the type strain of H. haemolyticus (ATCC 33390 T ). b The percentage in parentheses refers to the percent match of the query to the nucleotide sequences of the indicated allele number.

Techniques:

Journal: Journal of Clinical Microbiology

Article Title: Identification and Characterization of “ Haemophilus quentini ” Strains Causing Invasive Disease in Ontario, Canada (2016 to 2018)

doi: 10.1128/JCM.01254-19

Figure Lengend Snippet: Phylogenetic trees constructed on the phylogeny.fr platform showing the relatedness of seven invasive Haemophilus quentini clinical isolates from Ontario, Canada (HQ1 to HQ7), clinical H. quentini isolates from Hong Kong (HK-1) and the United Kingdom (MP1), H. haemolyticus reference (M19346) and type (ATCC 33390T) strains, two nontypeable H. influenzae biotype IV clinical isolates from Ontario, Canada (NTHi1, NTHi2), and an H. influenzae reference strain (Rd KW20) based on hpd (A), ompP6 (B), sodC (C), the 16S rRNA gene (D), and concatenated sequences of six MLST genes (adk, atpG, frdB, mdh, pgi, and recA) (E). NTHi1, NTHi2, and Rd KW20 were negative for sodC.

Article Snippet: The seventh isolate (HQ7) was biotype III and fermented glucose and mannose but not sucrose, lactose, or xylose ( ). table ft1 table-wrap mode="anchored" t5 TABLE 3 caption a7 Strain PCR result for the following gene target: H. influenzae MLST allele no. b hpd ompP6 sodC H. quentini -specific 16S rRNA gene adk atpG frdB fucK mdh pgi recA HQ1 + + + + 8 27 27 No product 37 15 24 HQ2 + + + + 8 27 27 No product 37 15 24 HQ3 + + + + 8 (99.8% match) 27 27 (99.8% match) No product 37 (99.3% match) 15 24 HQ4 + + + + 8 (99.8% match) 27 27 (99.8% match) No product 37 (99.3% match) 15 24 HQ5 + + + + 8 27 27 No product 37 15 24 HQ6 + + + + 8 (99.8% match) 27 27 (99.8% match) No product 37 (99.3% match) 15 24 HQ7 + + + + 81 (99.8% match) 63 (99.3% match) 86 (98.2% match) No product 63 (98.0% match) 84 (97.4% match) 60 (96.5% match) NTHi1 + + – – 11 43 35 11 51 31 18 NTHi2 + + – – 30 2 15 7 22 38 3 H. quentini HK-1 + + + + 8 27 27 No product 37 15 24 H. haemolyticus ATCC 33390 T + + + – 80 66 83 No product 126 101 74 Open in a separate window a PCR and MLST descriptions of seven invasive clinical isolates of suspected Haemophilus quentini from Ontario, Canada (HQ1 to HQ7), two isolates of invasive nontypeable H. influenzae biotype IV from Ontario, Canada (NTHi1 and NTHi2), a clinical blood isolate of H. quentini from Hong Kong, China (HK-1), and the type strain of H. haemolyticus (ATCC 33390 T ). b The percentage in parentheses refers to the percent match of the query to the nucleotide sequences of the indicated allele number.

Techniques: Construct

Journal: The Journal of Biological Chemistry

Article Title: Structure and Evolution of the Archaeal Lipid Synthesis Enzyme sn -Glycerol-1-phosphate Dehydrogenase *

doi: 10.1074/jbc.M115.647461

Figure Lengend Snippet: Expanded gap version of Dali-lite pairwise structural alignment of MJ G1PDH (36). Selected members of the larger enzyme superfamily including GDHs (Protein Data Bank codes 3CE9 and 1JQ5), ADHs (Protein Data Bank codes 1RRM, 3JZD, 3BFJ, and 4FR2), and DHQSs (Protein Data Bank codes 3QBE, 1XAG, and 1UJN) were used in the alignment. Secondary structural elements and residue numbering correspond to G1PDH. Residues that coordinate metals are highlighted in blue. The coenzyme binding motif is in italics, whereas residues that interact with coenzyme (NADP(H)) and substrate (DHAP) with respect to MJ G1PDH are highlighted in yellow and green, respectively. Reported intersubunit contacts between monomers are in red. Uppercase lettering indicates structurally equivalent positions with G1PDH, whereas lowercase indicates insertions relative to G1PDH.

Article Snippet: Previous biochemical characterizations of archaeal G1PDHs have shown the enzyme to be multimeric ( 17 , 42 ). table ft1 table-wrap mode="anchored" t5 TABLE 3 caption a7 Organism Class Protein Data Bank code-monomer Z-score a r.m.s.d. b lali c %id d C. acetobutylicum ATCC 824 GDH 3CE9-A 39.1 2.0 310 34 Sinorhizobium meliloti GDH 3UHJ-A 31.7 2.6 305 21 Geobacillus stearothermophilus GDH 1JQ5-A 30.3 2.5 300 21 Serratia plymuthica A30 GDH 4MCA-A 30.1 2.7 302 21 Schizosaccharomyces pombe GDH 1TA9-B 29.7 2.7 306 20 T. maritima GDH 1KQ3-A 29.5 2.6 301 26 E. coli Lactaldehyde reductase 1RRM-A 27.2 3.1 301 16 Ralstonia eutropha Fe-ADH 3JZD-A 26.9 2.9 302 15 Rhizobium sp. MTP-10005 MR 3W5S-A 26.8 3.2 300 15 Klebsiella pneumoniae POR 3BFJ-A 26.7 3.1 302 18 Agrobacterium tumefaciens MR 3HL0-A 26.8 3.0 302 15 Zymomonas mobilis ADH 2 3OWO-A 26.4 3.2 302 16 O. oeni POR 4FR2-A 26.4 3.2 302 20 Corynebacterium glutamicum ADH IV 3IV7-A 26.0 3.1 303 16 T. maritima Butanol dehydrogenase 1VLJ-B 25.2 3.5 299 18 T. maritima Fe-ADH 1VHD-A 25.1 3.2 294 18 Geobacillus thermoglucosidasius ADH 3ZDR-A 24.2 3.2 299 16 Shewanella denitrificans Fe-ADH 3RF7-A 24.2 3.4 290 17 E. coli Hypothetical oxidoreductase YqhD 1OJ7-A 23.6 3.9 300 17 Actinidia chinensis DHQS 3ZOK-D 23.0 3.5 294 16 Mycobacterium tuberculosis DHQS 3QBE-A 22.9 2.9 287 14 Aspergillus nidulans DHQS 1NVB-B 22.3 3.3 290 17 Streptomyces hygroscopicus Cyclase 4P53-A 21.0 2.9 277 16 Staphylococcus aureus DHQS 1XAG-A 20.9 3.3 282 17 Bacillus circulans 2-Deoxy- scyllo -inosose synthase 2GRU-A 20.3 3.2 283 18 Staphylococcus aureus DHQS 1XAH-A 19.5 3.2 264 16 Vibrio cholerae DHQS 3OKF-A 19.4 3.4 280 18 T. thermophilus DHQS 1UJN-A 18.8 3.4 271 14 Helicobacter pylori DHQS 3CLH-A 17.6 3.4 255 21 Open in a separate window a A measure of the statistical significance of the result relative to an alignment of random structures. b Root mean square deviation of α-carbon atoms. c Number of aligned residues. d Sequence identity between the two chains.

Techniques: Binding Assay

Journal: The Journal of Biological Chemistry

Article Title: Structure and Evolution of the Archaeal Lipid Synthesis Enzyme sn -Glycerol-1-phosphate Dehydrogenase *

doi: 10.1074/jbc.M115.647461

Figure Lengend Snippet: Top structural alignment hits from the Dali-based structural alignment of MJ G1PDH ( 36 ) MR, maleylacetate reductase; POR, 1,3-propanediol oxidoreductase.

Article Snippet: Previous biochemical characterizations of archaeal G1PDHs have shown the enzyme to be multimeric ( 17 , 42 ). table ft1 table-wrap mode="anchored" t5 TABLE 3 caption a7 Organism Class Protein Data Bank code-monomer Z-score a r.m.s.d. b lali c %id d C. acetobutylicum ATCC 824 GDH 3CE9-A 39.1 2.0 310 34 Sinorhizobium meliloti GDH 3UHJ-A 31.7 2.6 305 21 Geobacillus stearothermophilus GDH 1JQ5-A 30.3 2.5 300 21 Serratia plymuthica A30 GDH 4MCA-A 30.1 2.7 302 21 Schizosaccharomyces pombe GDH 1TA9-B 29.7 2.7 306 20 T. maritima GDH 1KQ3-A 29.5 2.6 301 26 E. coli Lactaldehyde reductase 1RRM-A 27.2 3.1 301 16 Ralstonia eutropha Fe-ADH 3JZD-A 26.9 2.9 302 15 Rhizobium sp. MTP-10005 MR 3W5S-A 26.8 3.2 300 15 Klebsiella pneumoniae POR 3BFJ-A 26.7 3.1 302 18 Agrobacterium tumefaciens MR 3HL0-A 26.8 3.0 302 15 Zymomonas mobilis ADH 2 3OWO-A 26.4 3.2 302 16 O. oeni POR 4FR2-A 26.4 3.2 302 20 Corynebacterium glutamicum ADH IV 3IV7-A 26.0 3.1 303 16 T. maritima Butanol dehydrogenase 1VLJ-B 25.2 3.5 299 18 T. maritima Fe-ADH 1VHD-A 25.1 3.2 294 18 Geobacillus thermoglucosidasius ADH 3ZDR-A 24.2 3.2 299 16 Shewanella denitrificans Fe-ADH 3RF7-A 24.2 3.4 290 17 E. coli Hypothetical oxidoreductase YqhD 1OJ7-A 23.6 3.9 300 17 Actinidia chinensis DHQS 3ZOK-D 23.0 3.5 294 16 Mycobacterium tuberculosis DHQS 3QBE-A 22.9 2.9 287 14 Aspergillus nidulans DHQS 1NVB-B 22.3 3.3 290 17 Streptomyces hygroscopicus Cyclase 4P53-A 21.0 2.9 277 16 Staphylococcus aureus DHQS 1XAG-A 20.9 3.3 282 17 Bacillus circulans 2-Deoxy- scyllo -inosose synthase 2GRU-A 20.3 3.2 283 18 Staphylococcus aureus DHQS 1XAH-A 19.5 3.2 264 16 Vibrio cholerae DHQS 3OKF-A 19.4 3.4 280 18 T. thermophilus DHQS 1UJN-A 18.8 3.4 271 14 Helicobacter pylori DHQS 3CLH-A 17.6 3.4 255 21 Open in a separate window a A measure of the statistical significance of the result relative to an alignment of random structures. b Root mean square deviation of α-carbon atoms. c Number of aligned residues. d Sequence identity between the two chains.

Techniques:

Journal: BMC Microbiology

Article Title: Antibiotics resistance and toxin profiles of Bacillus cereus -group isolates from fresh vegetables from German retail markets

doi: 10.1186/s12866-019-1632-2

Figure Lengend Snippet: Characteristics of whole genome datasets of selective B. cereus s.l. strains

Article Snippet: A homologous group filtering and a group alignment were performed by PATRIC pipeline [ ] and an estimated phylogenetic tree from concatenated alignment sequences was calculated within the type strains B. cereus ATCC 14579 T (DSM 31 T ), B. thuringiensis ATCC 10792 T (DSM 2046 T ), B. toyonensis BCT-7112 T (CECT 876 T ), B. weihenstephanensis WSBC 10204 T (DSM 11821 T ), B. mycoides ATCC 6462 T (DSM 2048 T ) and Bacillus pseudomycoides AFS069374 T using a FastTree method [ ].

Techniques:

Journal: BMC Microbiology

Article Title: Antibiotics resistance and toxin profiles of Bacillus cereus -group isolates from fresh vegetables from German retail markets

doi: 10.1186/s12866-019-1632-2

Figure Lengend Snippet: The amino acid sequences of the hblC gene of Bacillus cereus s.l. strains were clustered in this study. Most of strains reacted Hbl-positive with the Duopath test but two strains (B26 and MS17) were Hbl-negative. The phylogenetic analysis was carried out using the Jukes-Cantor model for genetic distance and the unweighted pair-group method using arithmetic averages (UPGMA) algorithm of the Geneious® program. The branch length and branch support values indicate the units of substitutions per site of the sequence alignment

Article Snippet: A homologous group filtering and a group alignment were performed by PATRIC pipeline [ ] and an estimated phylogenetic tree from concatenated alignment sequences was calculated within the type strains B. cereus ATCC 14579 T (DSM 31 T ), B. thuringiensis ATCC 10792 T (DSM 2046 T ), B. toyonensis BCT-7112 T (CECT 876 T ), B. weihenstephanensis WSBC 10204 T (DSM 11821 T ), B. mycoides ATCC 6462 T (DSM 2048 T ) and Bacillus pseudomycoides AFS069374 T using a FastTree method [ ].

Techniques: Sequencing

Journal: BMC Microbiology

Article Title: Antibiotics resistance and toxin profiles of Bacillus cereus -group isolates from fresh vegetables from German retail markets

doi: 10.1186/s12866-019-1632-2

Figure Lengend Snippet: Origin of Bacillus cereus -groupstrains investigated in this study

Article Snippet: A homologous group filtering and a group alignment were performed by PATRIC pipeline [ ] and an estimated phylogenetic tree from concatenated alignment sequences was calculated within the type strains B. cereus ATCC 14579 T (DSM 31 T ), B. thuringiensis ATCC 10792 T (DSM 2046 T ), B. toyonensis BCT-7112 T (CECT 876 T ), B. weihenstephanensis WSBC 10204 T (DSM 11821 T ), B. mycoides ATCC 6462 T (DSM 2048 T ) and Bacillus pseudomycoides AFS069374 T using a FastTree method [ ].

Techniques: Isolation

Journal: Journal for Immunotherapy of Cancer

Article Title: SPI1+CD68+ macrophages as a biomarker for gastric cancer metastasis: a rationale for combined antiangiogenic and immunotherapy strategies

doi: 10.1136/jitc-2024-009983

Figure Lengend Snippet: SPI1 was highly expressed in macrophages within the metastatic lesions of gastric cancer. ( A ) UMAP representation colored according to different cell types. ( B ) Density plot of SPI1 expression distribution in scRNA sequencing data. ( C ) UMAP indicated SPI1 staining of macrophages in primary and metastasis gastric cancer (GC). ( D ) Differential analysis of SPI1 expression in the primary and metastasis tumor of GC in scRNA sequencing data. ( E ) Infiltration of SPI1 + CD68 + TAMs in normal tissue, primary tumor, and metastasis sites of GC. ( F ) Differential analysis of SPI1 + CD68 + TAMs infiltration between primary and metastasis tumor according to double immunohistochemical staining. ( G ) SPI1 + CD68 + TAMs were screened by flow cytometry in primary and metastasis tumor of patients with GC. ( H ) Quantitative analysis of SPI1 + CD68 + TAMs infiltration based on flow cytometry. scRNA, single-cell RNA; SPI1, Spi-1 proto-oncogene; TAMs, tumor-associated macrophages; UMAP, Uniform Manifold Approximation and Projection.

Article Snippet: Subsequently, a mixture of anti-SPI1 antibody (mouse, Proteintech, 66618–2-Ig, diluted 1:250) and anti-CD68 antibody (rabbit, Proteintech, 25747–1-AP, diluted 1:500) was incubated on the slides overnight at 4°C.

Techniques: Expressing, Sequencing, Staining, Immunohistochemical staining, Flow Cytometry

Journal: Journal for Immunotherapy of Cancer

Article Title: SPI1+CD68+ macrophages as a biomarker for gastric cancer metastasis: a rationale for combined antiangiogenic and immunotherapy strategies

doi: 10.1136/jitc-2024-009983

Figure Lengend Snippet: SPI1 + CD68 + TAMs was an independent prognostic factor in patients with metastatic gastric cancer. ( A ) Overall survival (OS) and disease-free survival (DFS) of patients with gastric cancer (GC) in different SPI1 + CD68 + TAMs groups in the training cohort. ( B ) OS and DFS of patients with GC in different SPI1 + CD68 + TAMs groups in the external validation cohort. ( C ) OS and DFS of patients with GC in different SPI1 − CD68 + TAMs groups in the training cohort. ( D ) OS and DFS of patients with GC in different SPI1 − CD68 + TAMs groups in the external validation cohort. ( E ) Univariate cox regression analysis of patients with GC in the training cohort. ( F ) SPI1 + CD68 + TAMs was an independent prognostic factor of patients with GC in the training cohort. ( G ) Univariate cox regression analysis of patients with GC in the external validation cohort. ( H ) SPI1 + CD68 + TAMs was an independent prognostic factor of patients with GC in the external validation cohort. SPI1, Spi-1 proto-oncogene; TAMs, tumor-associated macrophages.

Article Snippet: Subsequently, a mixture of anti-SPI1 antibody (mouse, Proteintech, 66618–2-Ig, diluted 1:250) and anti-CD68 antibody (rabbit, Proteintech, 25747–1-AP, diluted 1:500) was incubated on the slides overnight at 4°C.

Techniques: Biomarker Discovery

Journal: Journal for Immunotherapy of Cancer

Article Title: SPI1+CD68+ macrophages as a biomarker for gastric cancer metastasis: a rationale for combined antiangiogenic and immunotherapy strategies

doi: 10.1136/jitc-2024-009983

Figure Lengend Snippet: SPI1 was associated with M2 polarization of macrophages. ( A ) Screening M1/M2 macrophages used flow cytometry with CD45, CD68, CD80, CD206 and SPI1 in patients with gastric cancer (GC). ( B ) Quantitative analysis of SPI1 + CD68 + TAMs infiltration in M1 and M2 type based on flow cytometry of patients with GC. ( C ) The markers of induced M1 and M2-type macrophages were detected by qRT-PCR. ( D ) Detecting the macrophage-related markers with qRT-PCR after SPI1 knockdown. ( E ) qRT-PCR was used to detect the macrophage-associated markers after SPI1 overexpression. ( F ) Western blot was used to detect the macrophage-related markers after SPI1 knockdown. ( G ) Macrophage-related markers were detected with western blot after SPI1 overexpression. ( H ) Flow cytometry was used to detect the macrophage-related markers after SPI1 knockdown. ( I ) Quantitative analysis of flow cytometry in SPI1 knockdown macrophages. ( J ) Flow cytometry was used to detect the macrophage-related markers after SPI1 overexpression. ( K ) Quantitative analysis of flow cytometry in SPI1 overexpression macrophages. IL-10, interleukin-10; mRNA, messenger RNA; qRT-PCR, quantitative Reverse Transcription Polymerase Chain Reaction; SPI1, Spi-1 proto-oncogene; TAMs, tumor-associated macrophages.

Article Snippet: Subsequently, a mixture of anti-SPI1 antibody (mouse, Proteintech, 66618–2-Ig, diluted 1:250) and anti-CD68 antibody (rabbit, Proteintech, 25747–1-AP, diluted 1:500) was incubated on the slides overnight at 4°C.

Techniques: Flow Cytometry, Quantitative RT-PCR, Knockdown, Over Expression, Western Blot, Reverse Transcription, Polymerase Chain Reaction

Journal: Journal for Immunotherapy of Cancer

Article Title: SPI1+CD68+ macrophages as a biomarker for gastric cancer metastasis: a rationale for combined antiangiogenic and immunotherapy strategies

doi: 10.1136/jitc-2024-009983

Figure Lengend Snippet: SPI1 + CD68 + TAMs promoted tumor angiogenesis through VEGF pathway. ( A ) GSVA analysis between SPI1-high and SPI1-low TAMs groups in scRNA sequencing data. ( B ) Incoming and outgoing interaction strength in different types of cells. ( C ) Cell–cell communication between different cell types in the VEGF signaling pathway. ( D ) Response to bevacizumab in patients with different TAMs infiltrates in the external validation cohort. ( E ) Immunofluorescence revealed the infiltration of SPI1 + CD68 + TAMs around the tumor blood vessels (CD31 marked). ( F ) Difference in the number of SPI1 + CD68 + TAMs and SPI1 − CD68 + TAMs around tumor vessels. ( G ) Difference in distance between blood vessels with SPI1 + CD68 + TAMs and SPI1 − CD68 + TAMs. DAPI, 4',6-Diamidino-2-Phenylindole; GSVA, gene set variation analysis; NK, Natural Killer cells; scRNA, single-cell RNA; SPI1, Spi-1 proto-oncogene; TAMs, tumor-associated macrophages; VEGFA, vascular endothelial growth factor A.

Article Snippet: Subsequently, a mixture of anti-SPI1 antibody (mouse, Proteintech, 66618–2-Ig, diluted 1:250) and anti-CD68 antibody (rabbit, Proteintech, 25747–1-AP, diluted 1:500) was incubated on the slides overnight at 4°C.

Techniques: Sequencing, Biomarker Discovery, Immunofluorescence

Journal: Journal for Immunotherapy of Cancer

Article Title: SPI1+CD68+ macrophages as a biomarker for gastric cancer metastasis: a rationale for combined antiangiogenic and immunotherapy strategies

doi: 10.1136/jitc-2024-009983

Figure Lengend Snippet: The role of SPI1 in GC cell growth and metastasis in vivo. ( A ) The representative images of the xenograft tumor. ( B ) Quantitative analysis of tumor growth curve. ( C ) Quantitative analysis of tumor weight in different SPI1 expression group. ( D ) Bioluminescence images of tumor-bearing mice individually treated with shNC, shSPI1, Vector, SPI1 transfected macrophages at day 5, 10, 15, 20, 25, 30, 40, and 50. ( E ) Representative photographs of peritoneum and mesentery metastasis lesions in different SPI1 expression groups. ( F ) The fluorescence intensity of tumors in various groups of mice. ( G ) Quantitative analysis of peritoneum nodules. ( H ) Survival curves of mice in different groups. GC, gastric cancer; SPI1, Spi-1 proto-oncogene.

Article Snippet: Subsequently, a mixture of anti-SPI1 antibody (mouse, Proteintech, 66618–2-Ig, diluted 1:250) and anti-CD68 antibody (rabbit, Proteintech, 25747–1-AP, diluted 1:500) was incubated on the slides overnight at 4°C.

Techniques: In Vivo, Expressing, Plasmid Preparation, Transfection, Fluorescence

Journal: Journal for Immunotherapy of Cancer

Article Title: SPI1+CD68+ macrophages as a biomarker for gastric cancer metastasis: a rationale for combined antiangiogenic and immunotherapy strategies

doi: 10.1136/jitc-2024-009983

Figure Lengend Snippet: SPI1 closely interacted with endothelial cells and regulated VEGFA transcription. ( A ) Wound healing assay was used to detect the migration ability of HUVEC cells cultured with conditioned medium of SPI1 knockdown macrophages. ( B ) Compared with the control group, the overexpression of SPI1 enhanced the migration ability of HUVEC cells. ( C ) Tube formation assay was used to detect the angiogenic ability of HUVEC cells after cultured with conditioned medium of SPI1 knockdown macrophages. ( D ) On overexpressing SPI1, the angiogenesis ability of HUVEC cells was augmented. ( E ) The mRNA level of VEGFA changed after knockdown or overexpression of SPI1. ( F ) The protein level of VEGFA changed after knockdown or overexpression of SPI1. ( G ) Motif sequence logo plot of SPI1 according to JASPAR database. ( H ) Putative SPI1 and VEGFA promoter binding sites. ( I ) ChIP analysis of the direct interaction between SPI1 and the promoter of VEGFA. ( J ) The binding site of SPI1 to the VEGFA promoter was assessed using ChIP-qPCR. ChIP, chromatin immunoprecipitation; HUVEC, Human Umbilical Vein Endothelial Cells; mRNA, messenger RNA; qPCR, quantitative PCR; SPI1, Spi-1 proto-oncogene; VEGFA, vascular endothelial growth factor A.

Article Snippet: Subsequently, a mixture of anti-SPI1 antibody (mouse, Proteintech, 66618–2-Ig, diluted 1:250) and anti-CD68 antibody (rabbit, Proteintech, 25747–1-AP, diluted 1:500) was incubated on the slides overnight at 4°C.

Techniques: Wound Healing Assay, Migration, Cell Culture, Knockdown, Control, Over Expression, Tube Formation Assay, Sequencing, Binding Assay, ChIP-qPCR, Chromatin Immunoprecipitation, Real-time Polymerase Chain Reaction

Journal: Journal for Immunotherapy of Cancer

Article Title: SPI1+CD68+ macrophages as a biomarker for gastric cancer metastasis: a rationale for combined antiangiogenic and immunotherapy strategies

doi: 10.1136/jitc-2024-009983

Figure Lengend Snippet: In vivo response to anti-PD-1 immunotherapy and antiangiogenesis treatment. ( A ) Response to immunotherapy in patients with different CPS scores and SPI1 + CD68 + TAMs infiltration. ( B ) Difference analysis of immunotherapy response in patients with different SPI1 + CD68 + TAMs infiltration. ( C ) Correlation analysis of SPI1 and PD-L1. ( D–G ) ROC curve of CPS score, CD68 + cells, SPI1 + CD68 + TAMs, and CPS score plus SPI1 + CD68 + TAMs. ( H ) Multiple immunofluorescences staining of PD-1 + CD8 + T cells and SPI1 + CD68 + TAMs. ( I ) Spatial distribution of SPI1 + CD68 + TAMs, SPI1 − CD68 + TAMs and PD-1 + CD8 + T cells analyzed by HALO. ( J ) Average distance from PD-1 + CD8 + T cells to SPI1 + CD68 + TAMs and SPI1 − CD68 + TAMs (p<0.05). ( K ) The discrepancy in the quantity of SPI1 + CD68 + TAMs and SPI1 − CD68 + TAMs surrounding PD-1 + CD8 + T cells (p<0.05). ( L ) Bioluminescence images of NOD/SCID mice with different treatment in week 1, 3, and 6. ( M ) The fluorescence intensity of intraperitoneal tumors in various groups of mice. CPS, Combined Positive Score; DAPI, 4',6-Diamidino-2-Phenylindole; PD-1, programmed cell death protein-1; PD-L1, programmed death-ligand 1; ROC, receiver operating characteristic; SPI1, Spi-1 proto-oncogene; TAMs, tumor-associated macrophages.

Article Snippet: Subsequently, a mixture of anti-SPI1 antibody (mouse, Proteintech, 66618–2-Ig, diluted 1:250) and anti-CD68 antibody (rabbit, Proteintech, 25747–1-AP, diluted 1:500) was incubated on the slides overnight at 4°C.

Techniques: In Vivo, Staining, Fluorescence

Journal: Cell Communication and Signaling : CCS

Article Title: Src family kinases interfere with dimerization of STAT5A through a phosphotyrosine-SH2 domain interaction

doi: 10.1186/s12964-014-0081-7

Figure Lengend Snippet: The SH2 domain of STAT5A is required for efficient binding to Src kinases. (A) Domain structure of fluorescently labeled STAT5A-eYFP. (B) Subcellular localization of STAT5A-eYFP in the absence or presence of Epo. HeLa T-REx HA-EpoR cells stably transfected with STAT5A-eYFP were stimulated with 1 U/ml Epo for 30 min and the localization of STAT5A-eYFP was analyzed by confocal microscopy. Scale bars: 20 μm. (C) Subcellular localization of STAT5A-eYFP (upper panel), STAT5A R618Q -eYFP (middle panel) and STAT3-eYFP (lower panel) was investigated in the presence of vSrc-dsRed. HeLa T-REx vSrc-dsRed cells were treated with 5 ng/ml doxycycline and transfected with the indicated constructs and the distribution of fluorescently labeled fusion proteins was analyzed after 24 h by confocal microscopy. Scale bars: 20 μm. (D) Quantification of the relative subcellular distribution of eYFP-labeled STAT3 and STAT5A constructs in HeLa T-REx HA-EpoR cells stably expressing STAT5A-eYFP (B) and HeLa T-REx vSrc-dsRed cells transfected with STAT5A-eYFP, STAT5A R618Q -eYFP or STAT3-eYFP (C) . The expression of the HA-EpoR and vSrc-dsRed was induced with 5 ng/ml doxycycline for 24 hours. Mean fluorescence intensities (MFI) of the cytoplasm and nucleus were determined using the Zen 2012 software and changes in the ratio between the compartments were plotted. The data shown are means ± SD of n = 30 cells and were statistically evaluated by Student’s t -test. ***p < 0.0005. n.s. = not significant. (E + F) HeLa T-REx FRT cells were co-transfected with plasmids coding for STAT5A-eYFP or STAT5A R618Q -eYFP and vSrc-dsRed or Hck-dsRed. Fluorescently labeled STAT5 was immunoprecipitated from cell lysates using a GFP antibody and analyzed by immunoblotting for the presence of vSrc-dsRed or Hck-dsRed 24 h after transfection. The expression and phosphorylation of STAT5A and vSrc/Hck proteins was analyzed in the whole cellular lysates (WCL) using antibodies against pY 416 -Src, Src, Hck, pY 694/699 -STAT5A/B and GFP.

Article Snippet: Anti-pY 694/699 -STAT5A/B (#9351), anti-pY 416 -Src (#2101), anti-pY 412 -Abl (#2865), anti-Hsp70 (#4872, Cell Signaling, Beverly, USA), anti-STAT5A (clone #5073, rabbit polyclonal antiserum was kindly provided by Richard Moriggl, Ludwig Boltzmann Institute for Cancer Research (LBI-CR), Vienna, Austria), anti-GFP (600-103-215, Rockland, Gilbertsville, USA), anti-FLAG (F3165), anti-α-tubulin (T5168, Sigma, St. Louis, USA), anti-GAPDH (sc-32233), anti-Abl (sc-131), anti-Hck (sc-72), anti-cSrc (sc-19, Santa Cruz Biotechnology, Santa Cruz, USA), anti-vSrc (MABS193, Millipore, Billerica, MA, USA) and anti-HA (MMS-101R, Covance, Princeton, New Jersey, USA) antibodies were used for immunoblotting.

Techniques: Binding Assay, Labeling, Stable Transfection, Transfection, Confocal Microscopy, Construct, Expressing, Fluorescence, Software, Immunoprecipitation, Western Blot

Journal: Cell Communication and Signaling : CCS

Article Title: Src family kinases interfere with dimerization of STAT5A through a phosphotyrosine-SH2 domain interaction

doi: 10.1186/s12964-014-0081-7

Figure Lengend Snippet: STAT5A binds to the phosphorylated activation loop of SFK. (A) Domain structure of vSrc-dsRed. Selected amino acids are highlighted. A multiple sequence alignment of the activation loop of SFK is shown. Autophosphorylation site is highlighted (red). (*) conserved amino acids, (:) similar properties . Bold characters highlight peptide sequence used for precipitation. (B + C) HeLa T-REx FRT cells stably expressing STAT5A-eYFP were transfected with the indicated vSrc-dsRed variants. Phosphorylation was analyzed 24 h after transfection using antibodies against pY 416 -Src, Src, pY 694/699 -STAT5A/B and STAT5A. CTRL = untransfected cells. (D) Quantification of relative subcellular distribution of STAT5A in HeLa T-REx FRT stably expressing STAT5A-eYFP and the indicated vSrc-dsRed mutants. Mean fluorescence intensity (MFI) of eYFP-fluorescence in the cytoplasm and nucleus were determined using the Zen 2012 software and changes in the ratio between the compartments were plotted. Data show means ± SD of n = 10 cells and were statistically evaluated by Student’s t -test. ***p < 0.0005, **p < 0.005, *p < 0.05, n.s. = not significant. (E) HeLa T-REx FRT cells stably expressing STAT5A-eYFP were transfected with vSrc K295N -dsRed or vSrc Y416F -dsRed. Subcellular distribution of STAT5A-eYFP was analyzed 24 h after transfection by confocal microscopy. Scale bars: 20 μm. (F + G) HeLa T-REx FRT cells were co-transfected with plasmids coding for vSrc-dsRed (Hck-dsRed), vSrc K295N -dsRed (Hck K269N -dsRed) or vSrc Y416F -dsRed (Hck Y390F -dsRed) and STAT5A-eYFP. STAT5-eYFP was immunoprecipitated from cell lysates using a GFP antibody and analyzed by immunoblotting for the presence of vSrc-dsRed (Hck-dsRed) 24 h after transfection. Expression and phosphorylation of STAT5A and vSrc proteins was analyzed in the WCL using antibodies against pY 416 -Src, Src, Hck, pY 694/699 -STAT5A/B and GFP. (s) short exposure, (l) long exposure. (H) HeLa T-REx FRT cells expressing STAT5A-eYFP or STAT5A R618Q -eYFP were lysed and incubated with a Src-peptide containing tyrosine- or phosphotyrosine 416. Precipitates and WCL were analyzed by immunoblotting using a GFP-specific antibody.

Article Snippet: Anti-pY 694/699 -STAT5A/B (#9351), anti-pY 416 -Src (#2101), anti-pY 412 -Abl (#2865), anti-Hsp70 (#4872, Cell Signaling, Beverly, USA), anti-STAT5A (clone #5073, rabbit polyclonal antiserum was kindly provided by Richard Moriggl, Ludwig Boltzmann Institute for Cancer Research (LBI-CR), Vienna, Austria), anti-GFP (600-103-215, Rockland, Gilbertsville, USA), anti-FLAG (F3165), anti-α-tubulin (T5168, Sigma, St. Louis, USA), anti-GAPDH (sc-32233), anti-Abl (sc-131), anti-Hck (sc-72), anti-cSrc (sc-19, Santa Cruz Biotechnology, Santa Cruz, USA), anti-vSrc (MABS193, Millipore, Billerica, MA, USA) and anti-HA (MMS-101R, Covance, Princeton, New Jersey, USA) antibodies were used for immunoblotting.

Techniques: Activation Assay, Sequencing, Stable Transfection, Expressing, Transfection, Fluorescence, Software, Confocal Microscopy, Immunoprecipitation, Western Blot, Incubation

Journal: Cell Communication and Signaling : CCS

Article Title: Src family kinases interfere with dimerization of STAT5A through a phosphotyrosine-SH2 domain interaction

doi: 10.1186/s12964-014-0081-7

Figure Lengend Snippet: SFK-mediated cytoplasmic localization of STAT5A is dominant over BCR-ABL induced nuclear accumulation. (A) HeLa T-REx BCR-ABL cells were transiently transfected with STAT5A-eYFP and either treated with 5 ng/ml doxycycline for 24 h to induce BCR-ABL expression (lower panel) or left untreated (upper panel). Fixation was performed with methanol. Fixed cells were stained for BCR-ABL using a cABL-specific primary antibody and a secondary antibody conjugated to Alexa Fluor-405. The subcellular distribution of STAT5A-eYFP was analyzed by confocal microscopy. Scale bars: 20 μm. (B) The subcellular distribution of STAT5A-eYFP was investigated in the presence of vSrc-dsRed (upper panel), vSrc K295N -dsRed (middle panel) or vSrc Y416F -dsRed (lower panel) in HeLa T-REx BCR-ABL cells that were treated with 5 ng/ml doxycycline for 24 h. Fixation was performed with methanol. Fixed cells were stained for BCR-ABL using an Abl-specific primary antibody and a secondary antibody conjugated to Alexa Fluor-405. Scale bars: 20 μm. (C) HeLa T-REx BCR-ABL cells were co-transfected with vSrc-dsRed, or the respective kinase activity affecting mutants vSrc K295N -dsRed or vSrc Y416F -dsRed and STAT5A-eYFP. The cells were either treated with 5 ng/ml doxycycline for 24 h (lanes 1–3) to induce the expression of BCR-ABL or left untreated (lane 4). Protein expression and phosphorylation in the cellular extracts was investigated by immunoblotting with antibodies against pY 412 -cABL, cABL, pY 694/699 -STAT5A/B, STAT5A, pY 416 -Src and Src. α-Tubulin served as a loading control.

Article Snippet: Anti-pY 694/699 -STAT5A/B (#9351), anti-pY 416 -Src (#2101), anti-pY 412 -Abl (#2865), anti-Hsp70 (#4872, Cell Signaling, Beverly, USA), anti-STAT5A (clone #5073, rabbit polyclonal antiserum was kindly provided by Richard Moriggl, Ludwig Boltzmann Institute for Cancer Research (LBI-CR), Vienna, Austria), anti-GFP (600-103-215, Rockland, Gilbertsville, USA), anti-FLAG (F3165), anti-α-tubulin (T5168, Sigma, St. Louis, USA), anti-GAPDH (sc-32233), anti-Abl (sc-131), anti-Hck (sc-72), anti-cSrc (sc-19, Santa Cruz Biotechnology, Santa Cruz, USA), anti-vSrc (MABS193, Millipore, Billerica, MA, USA) and anti-HA (MMS-101R, Covance, Princeton, New Jersey, USA) antibodies were used for immunoblotting.

Techniques: Transfection, Expressing, Staining, Confocal Microscopy, Activity Assay, Western Blot

Journal: Cell Communication and Signaling : CCS

Article Title: Src family kinases interfere with dimerization of STAT5A through a phosphotyrosine-SH2 domain interaction

doi: 10.1186/s12964-014-0081-7

Figure Lengend Snippet: Binding of STAT5A to Src kinases interferes with dimerization. (A) HeLa T-REx HA-EpoR cells stably expressing STAT5A-eYFP were transfected with STAT5A-FLAG. The cells were treated with 5 ng/ml doxyxcyline for 24 h to induce the expression of the HA-tagged EpoR and stimulated with 5 U/ml Epo for 30 minutes or left untreated. HeLa T-REx vSrc-dsRed cells stably expressing STAT5A-eYFP were transfected with STAT5A-FLAG. The expression of vSrc-dsRed was induced for 8 h with 5 ng/ml doxycycline or the cells were left untreated. STAT5A-eYFP was immunoprecipitated from cell lysates using a GFP antibody and analyzed by immunoblotting for the presence of STAT5A-FLAG. The expression and phosphorylation of STAT5A-eYFP and STAT5A-FLAG was analyzed in the WCL using antibodies against pY 694/699 -STAT5A/B, GFP and the FLAG-tag. (B) HeLa T-REx HA-EpoR cells stably expressing STAT5A-eYFP were treated with 5 ng/ml doxyxcyline for 24 h to induce the expression of the human EpoR and stimulated with 5 U/ml Epo for 30 minutes or left untreated. HeLa T-REx vSrc-dsRed cells stably expressing STAT5A-eYFP or a STAT5A S710F -eYFP were treated with 5 ng/ml doxyxcline for 8 h or the cells were left untreated. Cellular extracts were prepared under native conditions and STAT5A-eYFP dimers were separated from monomers by blue native PAGE electrophoresis (NP). STAT5A-eYFP dimer complexes were measured by the detection of the eYFP fluorescence. The cellular extracts were subjected to immunoblotting using antibodies against pY 694/699 -STAT5A/B, STAT5A, Src and the HA-tag of the EpoR. (C) Confocal microscopy analysis of HeLa T-REx FRT cells co-expressing vSrc-dsRed together with STAT5A S710F -eYFP (upper panel), a serine phosphorylation mimicking mutant STAT5A S710D -eYFP (middle panel) or a serine phosphorylation deficient mutant STAT5A S710A -eYFP (lower panel). Methanol fixation was performed 24 h after transfection. Scale bars: 20 μm.

Article Snippet: Anti-pY 694/699 -STAT5A/B (#9351), anti-pY 416 -Src (#2101), anti-pY 412 -Abl (#2865), anti-Hsp70 (#4872, Cell Signaling, Beverly, USA), anti-STAT5A (clone #5073, rabbit polyclonal antiserum was kindly provided by Richard Moriggl, Ludwig Boltzmann Institute for Cancer Research (LBI-CR), Vienna, Austria), anti-GFP (600-103-215, Rockland, Gilbertsville, USA), anti-FLAG (F3165), anti-α-tubulin (T5168, Sigma, St. Louis, USA), anti-GAPDH (sc-32233), anti-Abl (sc-131), anti-Hck (sc-72), anti-cSrc (sc-19, Santa Cruz Biotechnology, Santa Cruz, USA), anti-vSrc (MABS193, Millipore, Billerica, MA, USA) and anti-HA (MMS-101R, Covance, Princeton, New Jersey, USA) antibodies were used for immunoblotting.

Techniques: Binding Assay, Stable Transfection, Expressing, Transfection, Immunoprecipitation, Western Blot, FLAG-tag, Blue Native PAGE, Electrophoresis, Fluorescence, Confocal Microscopy, Mutagenesis

Journal: Cell Communication and Signaling : CCS

Article Title: Src family kinases interfere with dimerization of STAT5A through a phosphotyrosine-SH2 domain interaction

doi: 10.1186/s12964-014-0081-7

Figure Lengend Snippet: Activated SFK interfere with dimerization and nuclear translocation of pSTAT5A in BCR-ABL expressing cells. Left scheme: Classical activation of the JAK2-STAT5A signaling pathway downstream of the EpoR. Right scheme: BCR-ABL directly phosphorylates STAT5A Y694 resulting in STAT5A dimerization, nuclear accumulation and finally target gene expression . In the presence of BCR-ABL, a predominantly cytoplasmic localization of pSTAT5A is achieved (i) upon binding to the scaffolding adaptor Gab2 resulting in pro-survival signaling through PI3K/Akt activation and (ii) through binding of the STAT5A SH2 domain to the phosphorylated activation loop of SFK, a mechanism that interferes with STAT5A dimerization and subsequent nuclear accumulation. Constitutively active STAT5A S710F escapes the SFK-mediated cytoplasmic retention. Flashes indicate phosphorylation events.

Article Snippet: Anti-pY 694/699 -STAT5A/B (#9351), anti-pY 416 -Src (#2101), anti-pY 412 -Abl (#2865), anti-Hsp70 (#4872, Cell Signaling, Beverly, USA), anti-STAT5A (clone #5073, rabbit polyclonal antiserum was kindly provided by Richard Moriggl, Ludwig Boltzmann Institute for Cancer Research (LBI-CR), Vienna, Austria), anti-GFP (600-103-215, Rockland, Gilbertsville, USA), anti-FLAG (F3165), anti-α-tubulin (T5168, Sigma, St. Louis, USA), anti-GAPDH (sc-32233), anti-Abl (sc-131), anti-Hck (sc-72), anti-cSrc (sc-19, Santa Cruz Biotechnology, Santa Cruz, USA), anti-vSrc (MABS193, Millipore, Billerica, MA, USA) and anti-HA (MMS-101R, Covance, Princeton, New Jersey, USA) antibodies were used for immunoblotting.

Techniques: Translocation Assay, Expressing, Activation Assay, Binding Assay, Scaffolding