Journal:

Article Title: Induction of human keratinocytes into enamel-secreting ameloblasts

doi: 10.1016/j.ydbio.2010.05.511

Figure Lengend Snippet: Differentiation of human keratinocytes into enamel-secreting ameloblsts in the presence of FGF8. (A) A recombinant tooth crown showed elongated ameloblasts (arrow in insert) and deposition of enamel and dentin. (B) Specific staining of human ameloblastin in the ameloblasts (arrows). (C) Immunostaining of human MMP-20 in the ameloblasts (arrow) and enamel (asterisks). (D) Immunostaining of FGF8 in the ameloblasts. Note the odontoblasts and dental pulp cells of mouse origin stained negatively. A, ameloblast; D, dentin; E, enamel; O, odontoblasts; P, pulp. Scale bar: 100 μm.

Article Snippet: The following antibodies were used: anti-human MHC I and anti-mouse MHC I (Biolegend); anti-human ameloblastin, anti-human FGF8, and anti-human MMP-20 (Santa Cruz); anti-human CD29, anti-human K15, and anti-human K19 (Neomarkers).

Techniques: Recombinant, Staining, Immunostaining

Journal:

Article Title: Induction of human keratinocytes into enamel-secreting ameloblasts

doi: 10.1016/j.ydbio.2010.05.511

Figure Lengend Snippet: Activation of PITX2 in the human keratinocytes in the recombinants in the presence of FGF8. (A) RT-PCR assays show the expression of PITX2 in 11-week old human embryonic premolar germ (11W), absence of PITX2 in human keratinocytes before tissue recombination (KSC) and in the tissue recombinant in the presence of FGF8 2-day (2d) after recombination, the expression of PITX2 in the tissue recombinants in the presence of FGF8 4-day (4d) and 6-day (6d) after tissue recombination. (B) In situ hybridization shows the expression of PITX2 in the human keratinocyte aggregate in a recombinant with the mouse dental mesenchyme in the presence of FGF8. (C) Negative control of in situ hybridization for PITX2 using sense probes.

Article Snippet: The following antibodies were used: anti-human MHC I and anti-mouse MHC I (Biolegend); anti-human ameloblastin, anti-human FGF8, and anti-human MMP-20 (Santa Cruz); anti-human CD29, anti-human K15, and anti-human K19 (Neomarkers).

Techniques: Activation Assay, Reverse Transcription Polymerase Chain Reaction, Expressing, Recombinant, In Situ Hybridization, Negative Control

Journal: Journal of Cachexia, Sarcopenia and Muscle

Article Title: Association of circulating PLA2G7 levels with cancer cachexia and assessment of darapladib as a therapy

doi: 10.1002/jcsm.12758

Figure Lengend Snippet: Darapladib treatment was not sufficient to counteract CCx in C26 tumour‐bearing mice despite a strong inhibition of PLA2G7 activity. (A–L) Mice were injected subcutaneously either with PBS (control mice) or C26 cancer cells and treated once daily either with vehicle (PBS mice, white bars, n = 6 animals; C26‐vehicle tumour‐bearing mice, dark grey bars/lines, n = 11 animals) or 50 mg/kg darapladib (C26‐darapladib tumour‐bearing mice, light grey bars/lines, n = 9 animals). (A) Tumour weights. (B) PAF‐AH activity in plasma, (C) tumours and GC muscles. (D) Kaplan–Meier curve depicting the percentage of mice developing cachexia over time. (E) Kinetic of body weight loss during days prior sacrifice. (F) Loss of body weight, and lean and fat mass (expressed as percentage of initial mass). (G) GC muscles, TA muscles, and heart weights. (H) mRNA levels of atrophy and autophagy markers in GC muscle and (I) heart. (J) Epididymal (eWAT), inguinal (iWAT), and brown (BAT) adipose tissues weights. (K) Spleen and lymph nodes weights. (L) Plasma interleukin‐6 (IL‐6) and platelet‐activating factor (PAF) levels ( n = 5 PBS animals, n = 11 C26‐shCTR animals, and n = 9 C26‐sh Pla2g7 animals). Data are mean ± standard error of the mean. Statistical analyses were performed using unpaired t ‐test ( A, C ), unpaired one‐way ANOVA or Kruskal Wallis with Bonferroni or Dunn's post hoc tests, respectively ( B, C, F–L ), paired two‐way ANOVA ( E ), and log‐rank (Mantel–Cox) test ( D ). Tests were two sided. * P < 0.05, ** P < 0.01, *** P < 0.001, and **** P < 0.0001.

Article Snippet: For the experiment including the PLA2G7 inhibitor treatment, mice received 50 mg/kg of darapladib (Biorbyt #ORB181231) or a DMSO/carboxymethylcellulose 0.5% solution (10:90, v/v, 50 μL per 10 g body weight) as vehicle control daily by oral gavage once tumours were palpable.

Techniques: Inhibition, Activity Assay, Injection, Control, Clinical Proteomics, Muscles

Journal: Journal of Cachexia, Sarcopenia and Muscle

Article Title: Association of circulating PLA2G7 levels with cancer cachexia and assessment of darapladib as a therapy

doi: 10.1002/jcsm.12758

Figure Lengend Snippet: Graphical summary of the study. Circulating PLA2G7 levels are increased in CCx in both mice and humans. Despite a significant contribution, tumour is probably not the only responsible for the increase in circulating PLA2G7 levels in CCx. Circulating leucocytes and tissues such as liver, spleen, lymph nodes, or adipose tissue also show increased Pla2g7 expression upon cachexia and may significantly contribute to its circulating levels. In tissues, liver Kupffer cells, lymphocytes, and infiltrating myeloid cells are considered to be the main cell types expressing Pla2g7 (according to publicly available single cell RNA sequencing data on the Tabula Muris website, https://tabula‐muris.ds.czbiohub.org/ ). Tumour‐secreted factors as well as increased circulating levels of pro‐inflammatory cytokines and/or platelet‐activating factor (PAF) may promote PLA2G7 expression and secretion by cells from the host. Chronic treatment with the specific PLA2G7 inhibitor darapladib was not sufficient to improve inflammation and to counteract tissue wasting. Future studies should focus on the potential of PLA2G7 as early biomarker for the diagnosis of CCx.

Article Snippet: For the experiment including the PLA2G7 inhibitor treatment, mice received 50 mg/kg of darapladib (Biorbyt #ORB181231) or a DMSO/carboxymethylcellulose 0.5% solution (10:90, v/v, 50 μL per 10 g body weight) as vehicle control daily by oral gavage once tumours were palpable.

Techniques: Expressing, RNA Sequencing, Biomarker Discovery

Journal: OncoImmunology

Article Title: Immunoreactivity against fibroblast growth factor 8 in alveolar rhabdomyosarcoma patients and its involvement in tumor aggressiveness

doi: 10.1080/2162402x.2022.2096349

Figure Lengend Snippet: Figure 1. FGF8 autoantibodies detection in very high-risk ARMS patients. (a) Left, experimental workflow followed for the identification of autoantibodies in ARMS patients using plasma samples and ProtoArrayTM technology.31 The immune response profile was obtained from 10 metastatic ARMS patients and 15 healthy subjects (HS), probing protein microarray chips with plasma. Reactivity of 9374 spotted antigens was evaluated after signal detection, filtration and normalization using robust linear model (RLM). Antigens median values were calculated for each group, compared and ranked according to significant p-value scale. Right, volcano plot of protein microarray data showing differentially immunoreactive antigens between patients and healthy subjects, plotted along dimension of fold change (abscissae) and statistical difference (ordinates). Antigens with significant p-values (≥0,05) are indicated by colors and names, while antigens with no significant difference in immunoreactivity between patients and controls are plotted uncolored on the bottom of the graph (gray dots). Antigens more reactive in patients or controls are distinguished by red or green dots, respectively. (b) Venn diagram showing the overlap between differential immunoreactive antigens (n = 55) and PAX3-FOXO1 target genes (n = 1010).32 (c) Box plot of FGF8 signal intensity revealed in patients and controls by protein microarray (RFU = relative fluorescence unit) and (d) validation by indirect ELISA assay. (e) Correlation of FGF8 autoantibody signal intensity and FGF8 IgG concentration obtained in the same samples cohort by protein microarrays and ELISA assay, respectively. p < 0,05 (*); p < 0,01 (**).

Article Snippet: Primary antibody against FGF8 (Proteintech) and in 1%BSA/PBS1X were probed at 37°C for 60 minutes, followed by secondary antibody Alexa Fluor® 546 conjugate (Thermo Fisher scientific) in PBS1X at 37°C for 60 minutes.

Techniques: Clinical Proteomics, Microarray, Filtration, Fluorescence, Biomarker Discovery, Indirect ELISA, Concentration Assay, Enzyme-linked Immunosorbent Assay

Journal: OncoImmunology

Article Title: Immunoreactivity against fibroblast growth factor 8 in alveolar rhabdomyosarcoma patients and its involvement in tumor aggressiveness

doi: 10.1080/2162402x.2022.2096349

Figure Lengend Snippet: Figure 2. Correlation between humoral immune response against FGF8 and patients’ outcome. Kaplan-Meier survival analysis representing (a) event-free survival (EFS) and (b) overall survival (OS) of ARMS patients distinguished accord ing to FGF8 autoantibodies median value.

Article Snippet: Primary antibody against FGF8 (Proteintech) and in 1%BSA/PBS1X were probed at 37°C for 60 minutes, followed by secondary antibody Alexa Fluor® 546 conjugate (Thermo Fisher scientific) in PBS1X at 37°C for 60 minutes.

Techniques:

Journal: OncoImmunology

Article Title: Immunoreactivity against fibroblast growth factor 8 in alveolar rhabdomyosarcoma patients and its involvement in tumor aggressiveness

doi: 10.1080/2162402x.2022.2096349

Figure Lengend Snippet: Figure 3. Expression of FGF8 in RMS cell lines. (a) Relative quantification of FGF8 mRNA by qRT-PCR in normal control cells (CTR, n = 4), alveolar RMS (ARMS, n = 5), embryonal RMS (ERMS, n = 5), Ewing sarcoma (EWS, n = 7), Non-Hodgkin lymphoma cell lines (NHL, n = 5), leukemia cell lines (Leukemia, n = 3) and cell lines of various origins (Others, n = 10). Statistical significance was calculated by Mann-Whitney U test, comparing each group of cell lines with ARMS group. Glyceraldehyde- 3-phosphate dehydrogenase (GAPDH) housekeeping gene was used for normalization, while normal skeletal muscle tissue extracts were used as external calibrator. (b) FGF8 protein expression and localization by immunocytochemistry and (c) immunofluorescence analysis in RH30 (PF+ ARMS) and RD (PF− ERMS) cell lines (magnifications of selected areas are shown apart). (d) Western blotting and (e) direct ELISA assay performed using serum-starved RH30 and RD total lysate and growth medium, respectively, to assess FGF8 protein at intracellular and at secreted level. A_SKM, adult skeletal muscle; p < 0,05 (*); p < 0,01 (**).

Article Snippet: Primary antibody against FGF8 (Proteintech) and in 1%BSA/PBS1X were probed at 37°C for 60 minutes, followed by secondary antibody Alexa Fluor® 546 conjugate (Thermo Fisher scientific) in PBS1X at 37°C for 60 minutes.

Techniques: Expressing, Quantitative Proteomics, Quantitative RT-PCR, Control, MANN-WHITNEY, Immunocytochemistry, Immunofluorescence, Western Blot, Direct ELISA

Journal: OncoImmunology

Article Title: Immunoreactivity against fibroblast growth factor 8 in alveolar rhabdomyosarcoma patients and its involvement in tumor aggressiveness

doi: 10.1080/2162402x.2022.2096349

Figure Lengend Snippet: Figure 4. FGF8 signaling in RMS cells. (a) Relative quantification of FGFR1-4 receptors mRNA in ARMS and ERMS cell lines. FGF8 mRNA levels are also displayed in graph (red open dots). Glyceraldehyde- 3-phosphate dehydrogenase (GAPDH) was used for signal intensity normalization, while skeletal muscle extracts were used as external calibrator. (b) FGFR2 and FGFR4 expression and phosphorylation in PF+ (RH30, RH4) and PF− (RD) cell lines, performed through immunoprecipitation of total FGFR2 and FGFR4 receptor proteins. GAPDH protein was used as gel-loading control. (c) Western blotting showing time-dependent phosphorylation of FRS2 and ERK1/2 proteins induced in RH4 and RD cells upon exposure to 50 ng/mL human recombinant FGF8 protein for the indicated time periods. Between blots a graph displaying phosphorylated FRS2 and ERK1/2 band densities, quantified using ImageJ software, was included. γ-Tubulin was used as gel loading control. (d) RH4 and RD cell lines wound healing assay performed in presence and absence of 100 ng/ml human FGF8. Images were taken up to 48 hours after the treatment. (e) Immunoblot analysis of phosphorylated ERK1/2 kinase in RMS cells exposed to increasing concentration of human FGF8 (25, 100 ng/mL), pretreated or not for 2 hours with 5 μM of NVP-BGJ398. γ-Tubulin was used as gel loading control. (f) MTT assay showing PF+ ARMS (RH30, RH4) and PF− ERMS (RH36, RD) cell viability in the presence of 5 μM of NVP-BGJ398 up to 72 hours.

Article Snippet: Primary antibody against FGF8 (Proteintech) and in 1%BSA/PBS1X were probed at 37°C for 60 minutes, followed by secondary antibody Alexa Fluor® 546 conjugate (Thermo Fisher scientific) in PBS1X at 37°C for 60 minutes.

Techniques: Quantitative Proteomics, Expressing, Phospho-proteomics, Immunoprecipitation, Control, Western Blot, Recombinant, Software, Wound Healing Assay, Concentration Assay, MTT Assay

Journal: OncoImmunology

Article Title: Immunoreactivity against fibroblast growth factor 8 in alveolar rhabdomyosarcoma patients and its involvement in tumor aggressiveness

doi: 10.1080/2162402x.2022.2096349

Figure Lengend Snippet: Figure 5. FGF8-induced gene expression in RMS cells. (a) Time-dependent expression of DUSP6, SPRY4, GDF15 and ETV5 FGF-target genes, upon treatment of RH4 and RD cells with 100 ng/ml of human recombinant FGF8. Glyceraldehyde- 3-phosphate dehydrogenase (GAPDH) housekeeping gene was used for normalization. (b) STRING analysis. (c) Time-dependent expression of PLAU and MMP-9 genes after treatment of RH4 and RD cell lines with 100 ng/ml of human recombinant FGF8.

Article Snippet: Primary antibody against FGF8 (Proteintech) and in 1%BSA/PBS1X were probed at 37°C for 60 minutes, followed by secondary antibody Alexa Fluor® 546 conjugate (Thermo Fisher scientific) in PBS1X at 37°C for 60 minutes.

Techniques: Gene Expression, Expressing, Recombinant

Journal: OncoImmunology

Article Title: Immunoreactivity against fibroblast growth factor 8 in alveolar rhabdomyosarcoma patients and its involvement in tumor aggressiveness

doi: 10.1080/2162402x.2022.2096349

Figure Lengend Snippet: Figure 6. FGF8 expression in RMS tumor biopsies. (a) Relative quantification of FGF8 mRNA in RMS primary tumors (n = 50) and normal controls (n = 4), and (b) in RMS primary tumors divided according to fusion status. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) gene was used for normalization. (c) Hematoxylin/Eosin (HE) and FGF8 staining of representative ARMS and ERMS cases. (d) Scatter plot showing the correlation between FGF8 mRNA levels and autoantibodies titer in 33 PF+ ARMS primary tumors and plasma samples, respectively. Vertical and horizontal dashed lines represent FGF8 mRNA and autoantibody median values, respectively, used to divide the plot in four regions (I–IV). Dots represent patients, labeled with different colors based on event occurrence (gray) or not (red) after frontline chemotherapy. p < 0,001 (***); p < 0,0001 (****).

Article Snippet: Primary antibody against FGF8 (Proteintech) and in 1%BSA/PBS1X were probed at 37°C for 60 minutes, followed by secondary antibody Alexa Fluor® 546 conjugate (Thermo Fisher scientific) in PBS1X at 37°C for 60 minutes.

Techniques: Expressing, Quantitative Proteomics, Staining, Clinical Proteomics, Labeling

Journal: OncoImmunology

Article Title: Immunoreactivity against fibroblast growth factor 8 in alveolar rhabdomyosarcoma patients and its involvement in tumor aggressiveness

doi: 10.1080/2162402x.2022.2096349

Figure Lengend Snippet: Figure 7. FGF8 expression in RMS recurrent tumors. (a) FGF8 and PAX3-FOXO1 or PAX7-FOXO1, mRNA at diagnosis and at relapse in 7 cases of ARMS, and (b) 5 ERMS cases. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) gene was used for normalization. n.s., not significant; p < 0,05 (*).

Article Snippet: Primary antibody against FGF8 (Proteintech) and in 1%BSA/PBS1X were probed at 37°C for 60 minutes, followed by secondary antibody Alexa Fluor® 546 conjugate (Thermo Fisher scientific) in PBS1X at 37°C for 60 minutes.

Techniques: Expressing, Biomarker Discovery

Journal:

Article Title: FGF-8 in the ventral pharynx alters development of myocardial calcium transients after neural crest ablation

doi:

Figure Lengend Snippet: In situ hybridization showing pharyngeal FGF-8 message expression in stage 14 (a–c) and stage 18 (d–f) chick embryos. (a and d) Sham-operated embryos with prominent FGF-8 message expression in the frontonasal process, midbrain-hindbrain boundary, and all of the pharyngeal arches and pouches. The expression in arches 1 and 2 appears to be less than that in arches 3, 4, and 6. (b and e) Embryos that have undergone cardiac neural crest ablations. The expression of FGF-8 message appears to be identical to that in the sham-operated embryos. (c and f) Embryos with double ablations of the cardiac neural crest and nodose placodes. These embryos show overall growth retardation although the expression of FGF-8 message in the pharyngeal region appears to be approximately the same as neural crest–ablated and sham-operated embryos at both stages.

Article Snippet: The heart tube was removed from this piece, which was cut into two pieces of equal size and cultured, with and without anti–FGF-2– or anti–FGF-8–neutralizing Ab (Atlanta Biologicals Inc., Norcross, Georgia, USA), under the conditions described above.

Techniques: In Situ Hybridization, Expressing

Journal:

Article Title: FGF-8 in the ventral pharynx alters development of myocardial calcium transients after neural crest ablation

doi:

Figure Lengend Snippet: Paraffin sections of whole-mount embryos in Figure ​Figure66 showing the location of FGF-8 message in pharyngeal arch 3. (a and c) Sham-operated embryos at stages 14 (a) and 18 (c). (b and d) Neural crest–ablated embryos at stages 14 (b) and 18 (d). (a) FGF-8 message is expressed strongly in the ectoderm covering the arch and weakly in the pharyngeal endoderm. (b) A similar pattern and level of expression of FGF-8 message after neural crest ablation to that of the sham-operated control. (c) Dramatic decrease in FGF-8 message expression at stage 18 in both the endoderm and ectoderm of a sham-operated embryo. (d) A similar decrease in expression in a neural crest–ablated embryo.

Article Snippet: The heart tube was removed from this piece, which was cut into two pieces of equal size and cultured, with and without anti–FGF-2– or anti–FGF-8–neutralizing Ab (Atlanta Biologicals Inc., Norcross, Georgia, USA), under the conditions described above.

Techniques: Expressing

Journal:

Article Title: FGF-8 in the ventral pharynx alters development of myocardial calcium transients after neural crest ablation

doi:

Figure Lengend Snippet: Ab to FGF-8 rescues the myocardial calcium transient in neural crest–ablated chick embryos

Article Snippet: The heart tube was removed from this piece, which was cut into two pieces of equal size and cultured, with and without anti–FGF-2– or anti–FGF-8–neutralizing Ab (Atlanta Biologicals Inc., Norcross, Georgia, USA), under the conditions described above.

Techniques: