Journal: Clinical and Translational Medicine

Article Title: Integrated spatial multi‐omics profiling of Fusobacterium nucleatum in breast cancer unveils its role in tumour microenvironment modulation and cancer progression

doi: 10.1002/ctm2.70273

Figure Lengend Snippet: Key proteins and their role in signalling pathways. (A) Venn diagram showing intersecting genes of the MAPK and focal adhesion signalling pathways at the transcriptomic and proteomic levels. (B) Pathway network analysis of the MAPK signalling, focal adhesion, and necroptosis pathways at the transcriptomic and proteomic levels. (C) Differential expression of VEGFD and PAK1 in F. nucleatum‐ positive and F. nucleatum ‐negative tumour regions (* p < .05; ** p < .01). (D) Protein–protein interaction (PPI) network displaying associations among the top 20 ranked proteins. (E) Network heatmap showing the association of VEGFD and PAK1 with the top 20 important proteins. (F) Changes in significantly different proteins in the MAPK signalling pathway. Red indicates upregulation; green indicates downregulation. The statistical significance was calculated using the Student's t ‐test.

Article Snippet: Sequential immunoblotting included incubation with primary antibodies targeting VEGFD (MCE, HY‐P82640, 1:500), PAK1 (MCE, HY‐P81618, 1:500), c‐Myc (CST, 5606S, 1:1000), phospho‐p38 MAPK (CST, 4511S, 1:1000), total p38 MAPK (CST, 9212S, 1:1000), phospho‐JNK1 (abcam, ab215208, 1:1000), with ACTIN (CST, #4970L, 1:1000) serving as loading control.

Techniques: Quantitative Proteomics

Journal: Clinical and Translational Medicine

Article Title: Integrated spatial multi‐omics profiling of Fusobacterium nucleatum in breast cancer unveils its role in tumour microenvironment modulation and cancer progression

doi: 10.1002/ctm2.70273

Figure Lengend Snippet: Mechanism of F. nucleatum promoting proliferation and migration of breast cancer cells. (A) Confocal microscopy showing spatial interaction between breast cancer cells (green: cytoskeleton; blue: nuclei) and F. nucleatum (red). Scale bar: 5 µm. (B) Line graph of CCK‐8 cell proliferation assay ( n = 5; Student's t ‐test). Statistical significance was determined by Student's t ‐test (* p < .05; ** p < .01; *** p < .001). (C) The wound‐healing assay was initiated with a uniform scratch width of .5 mm. (D) Migration distance quantification. Scratch width was measured at four predefined equidistant points per well at 0 and 24h using ImageJ, and distance was normalized to the initial width (0 h) ( n = 4; Student's t ‐test). (E, F) Line graph of CCK‐8 cell proliferation assay ( n = 5; Student's t ‐test). * p < .05; ** p < .01; *** p < .001. (G) Western blot analysis of VEGFD, PAK1, and MAPK pathway proteins in MDA‐MB‐231 and MCF‐7 cells co‐cultured with F. nucleatum . Blots are representative of three biological replicates. (H, I) EdU proliferation assay: EdU staining (red: proliferating cells; blue: DAPI) (H) and quantification of EdU‐positive cells (I). Three independent replicates were analyzed ( n = 3; Student's t ‐test). (J) Line graph of CCK‐8 proliferation assay after siRNA interference ( n = 5; Student's t ‐test). * p < .05; ** p < .01; *** p < .001. (K, M) Wound‐healing assay: Representative scratch images (K), schematic of the plate insert (L), and migration distance quantification (M). Scratch closure was measured at four equidistant positions per well ( n = 4; Student's t‐test). * p < .05; ** p < .01; *** p < .001. (N, O) Transwell migration assay: Crystal violet‐stained migrated cells (N) and quantification (O). Four independent experiments were performed ( n = 4; Student's t ‐test). * p < .05; ** p < .01; *** p < .001.

Article Snippet: Sequential immunoblotting included incubation with primary antibodies targeting VEGFD (MCE, HY‐P82640, 1:500), PAK1 (MCE, HY‐P81618, 1:500), c‐Myc (CST, 5606S, 1:1000), phospho‐p38 MAPK (CST, 4511S, 1:1000), total p38 MAPK (CST, 9212S, 1:1000), phospho‐JNK1 (abcam, ab215208, 1:1000), with ACTIN (CST, #4970L, 1:1000) serving as loading control.

Techniques: Migration, Confocal Microscopy, CCK-8 Assay, Proliferation Assay, Wound Healing Assay, Western Blot, Cell Culture, Staining, Transwell Migration Assay

Journal: Translational Stroke Research

Article Title: The Key Regulator of Necroptosis, RIP1 Kinase, Contributes to the Formation of Astrogliosis and Glial Scar in Ischemic Stroke

doi: 10.1007/s12975-021-00888-3

Figure Lengend Snippet: Knockdown of RIP1K reduces the gene and protein level of VEGF-D in astrocytes after I/R or OGD/Re. a Volcano plot of gene microarray analysis showed that 148 genes were down-regulated and 128 genes were up-regulated in RIPK1-knocked down astrocytes treated with OGD. Figf , the gene of VEGF-D, was found to be notably down-regulated ( n = 3). b Knockdown of RIP1K reduces the protein level of VEGF-D in astrocytes after OGD for 6 h and reoxygenation for 24 h with western blotting analysis. Data are mean ± SD, n = 3. ## P < 0.01 vs. non-OGD-Re24h + scr shRNA group; ** P < 0.01 vs. OGD-6h-Re24h + scr shRNA group. c Representative images of cerebral cortex double staining for GFAP (red) and VEGF-D (green) in rats 7 days after I/R or in sham-operated rats. Hoechst (blue) was used to stain the nucleus. The white dotted line represents the edge between the infarct area and the peri-infarct area, and the white boxes indicate the corresponding area of the enlarged images shown below. Mander’s overlap coefficient demonstrated the colocalization between GFAP and VEGF-D. Data are mean ± SD, n = 3. ## P < 0.01 vs. sham + scr shRNA group; ** P < 0.01 vs. I/R + scr shRNA group. Statistical analysis was carried out with one-way ANOVA followed by a post hoc Tukey’s test

Article Snippet: When astrocytes grew to 70% ~ 80% convergence, the astrocytes were treated with rat recombinant VEGF-D (400 ng/ml, Sino Biological, 80104-R01H) for 48 h, and then, the cells were harvested and the glial scar markers were detected with western blotting analysis.

Techniques: Microarray, Western Blot, shRNA, Double Staining, Staining

Journal: Translational Stroke Research

Article Title: The Key Regulator of Necroptosis, RIP1 Kinase, Contributes to the Formation of Astrogliosis and Glial Scar in Ischemic Stroke

doi: 10.1007/s12975-021-00888-3

Figure Lengend Snippet: The VEGF-D level is increased in astrocytes after I/R injury or in astrocytes and cell cultured medium after OGD/Re injury. a , b The time-course changes of VEGF-D expression after I/R injury. Transient ischemic stroke was induced by MCAO for 90 min (I/R) followed by reperfusion (I/R). a Representative images of double staining for GFAP (red) and VEGF-D (green) in the cerebral cortex of rats. Hoechst (blue) was used to stain the nucleus. The white dotted line represents the edge between the infarct area and the peri-infarct area, and the white boxes indicate the corresponding area of the enlarged images shown below. b Quantification of green fluorescence intensity of VEGF-D and red fluorescence intensity of GFAP immunostaining in a . Mander’s overlap coefficient demonstrated the colocalization between GFAP and VEGF-D. Data are mean ± SD, n = 3. # P < 0.05, ## P < 0.01 vs. sham group. c The time-course changes of VEGF-D level after OGD/Re injury with western blotting analysis. Astrocytes were exposed to OGD for 6 h followed by reoxygenation for 12 h or 24 h. Data are mean ± SD, n = 3. ## P < 0.01 vs. non-OGD group. d ELISA results showed that VEGF-D levels were increased both in astrocytes and in cell medium after OGD for 6 h and reoxygenation for 24 h. Data are mean ± SD, n = 3. ## P < 0.01 vs. non-OGD group. Statistical analysis was carried out with one-way ANOVA followed by a post hoc Tukey’s test ( b , c ) or with Student’s t test ( d )

Article Snippet: When astrocytes grew to 70% ~ 80% convergence, the astrocytes were treated with rat recombinant VEGF-D (400 ng/ml, Sino Biological, 80104-R01H) for 48 h, and then, the cells were harvested and the glial scar markers were detected with western blotting analysis.

Techniques: Cell Culture, Expressing, Double Staining, Staining, Fluorescence, Immunostaining, Western Blot, Enzyme-linked Immunosorbent Assay

Journal: Translational Stroke Research

Article Title: The Key Regulator of Necroptosis, RIP1 Kinase, Contributes to the Formation of Astrogliosis and Glial Scar in Ischemic Stroke

doi: 10.1007/s12975-021-00888-3

Figure Lengend Snippet: (a) Recombinant VEGF-D can induce the formation of glial scar. The astrocytes were treated with recombinant VEGF-D (400 ng/ml) for 48 h, and then, the levels of GFAP and phosphacan were detected with western blotting analysis. Data are mean ± SD, n = 3. ## P < 0.01 vs. non-OGD-Re24h group. Statistical analysis was carried out with Student’s t test. b SAR131675, a specific VEGFR-3 inhibitor reduces the levels of VEGFR-3, GFAP, neurocan, and phosphacan in astrocytes after OGD/Re with western blotting analysis. Astrocytes were exposed to OGD for 6 h followed by reoxygenation for 24 h. Astrocytes were treated with SAR131675 (20 nM) upon reoxygenation. Data are mean ± SD, n = 3. ## P < 0.01 vs. non-OGD group; ** P < 0.01 vs. OGD-6h-Re24h group. Statistical analysis was carried out with one-way ANOVA followed by a post hoc Tukey test

Article Snippet: When astrocytes grew to 70% ~ 80% convergence, the astrocytes were treated with rat recombinant VEGF-D (400 ng/ml, Sino Biological, 80104-R01H) for 48 h, and then, the cells were harvested and the glial scar markers were detected with western blotting analysis.

Techniques: Recombinant, Western Blot

Journal: The EMBO Journal

Article Title: VEGF receptor 2/-3 heterodimers detected in situ by proximity ligation on angiogenic sprouts

doi: 10.1038/emboj.2010.30

Figure Lengend Snippet: In situ PLA detection of VEGFR2/-3 heterodimers in intact HSaVECs. ( A ) Schematic outline of the in situ PLA strategy showing: (i) dimerized receptors (VEGFR2 in blue and VEGFR3 in grey) reacting with primary antibodies; (ii) close proximity of oligonucleotide-ligated secondary antibodies allows a rolling-circle amplification (RCA); (iii) detection of the RCA product by a fluorescently labelled probe. ( B ) Detection of heterodimers (in red) in HSaVECs treated with vehicle (–), VEGFA or VEGFC for 8 min on cells labelled with FITC-conjugated phalloidin (green). Inset in the VEGFC panel shows high magnification to clearly visualize the PLA spots representing heterodimers. Scale bar=10 μm. ( C ) Quantification of VEGFR2/-3 heterodimers in HSaVECs treated with vehicle (–), VEGFA (A) or VEGFC (C) in cells preincubated or not with neutralizing antibodies blocking ligand binding to VEGFR2 or VEGFR3. n =6. ( D ) Quantification of VEGFR2/-3 heterodimers in HSaVECs treated with different human VEGF isoforms (VEGFA121, 145, 165 or 189) or VEGFC for 8 min. n =6. ( E ) Quantification of VEGFR2/-3 heterodimers in response to VEGFA, VEGFC, VEGFD or PDGFB. Growth factors are indicated as A (VEGFA), C (VEGFC), D (VEGFD) and P (PDGFB). n =6. Note that a different batch of PLA probes was used in this analysis compared with other panels in the figure (see Materials and methods). ( F ) Turnover of VEGFR2/-3 heterodimers in HSaVECs. Cells were treated with VEGFC for different time periods from 10 min to 24 h and samples were processed for detection of in situ PLA signals. n =6. Asterisks in panels C – F indicate the degree of significance ( ** P <0.01, *** P <0.001).

Article Snippet: Effects were compared with those of VEGFC (in-house production), VEGFD (Vegenics Limited, Toorak, Australia), human platelet-derived growth factor (PDGFB; PeproTech), all given at 100 ng/ml.

Techniques: In Situ, Amplification, Blocking Assay, Ligand Binding Assay

Journal: PLoS ONE

Article Title: Blood Profile of Proteins and Steroid Hormones Predicts Weight Change after Weight Loss with Interactions of Dietary Protein Level and Glycemic Index

doi: 10.1371/journal.pone.0016773

Figure Lengend Snippet: List of analyzed blood proteins and steroid hormones.

Article Snippet: , VEGFD , Vascular endothelial growth factor-D , Aushon SearchLight.

Techniques: Coagulation, Binding Assay, Migration