Journal: Frontiers in Cell and Developmental Biology

Article Title: A differentiation protocol for generating pancreatic delta cells from human pluripotent stem cells

doi: 10.3389/fcell.2024.1490040

Figure Lengend Snippet: Final concentration of differentiation factors.

Article Snippet: • Activin A (StemCell Technologies, cat. no. 78001) • CHIR99021 (Stemgent, cat. no. 04-0004-10) • FGF7 (StemCell, cat. no. 78046) • FGF2 (Med Chem Express, cat. no. HY-P7004) • Retinoic acid (Sigma, cat. no. R2625) • SANT-1 (Sigma, cat. no. S4572) • LDN193189 (Reprocell, cat. no. 40074) • PdBU (Millipore, cat. no. 524390) • ALK5i II (Cell Guidance Systems, cat. no. SM09-50) • XXI (Millipore, cat. no. 595790) • Betacellulin (Med Chem Express, cat. no. HY-P7005) • Heparin (Sigma, cat. no. H3149-500KU) • N-acetyl cysteine (Sigma, cat. no. A9165-100G) • R428 (Selleck, cat. no. S2841) • α-Tocopherol (Sigma, cat. no. T3251) • Zinc sulfate (Sigma, cat. no. Z0251) • PBS for dissolving factors (HyClone, cat. no. SH30256.01) • Dimethyl sulfoxide for dissolving factors (DMSO) (MilliporeSigma, cat. no. D4540-100ML)

Techniques: Concentration Assay

Journal: Cells

Article Title: The Aberrant Expression of the Mesenchymal Variant of FGFR2 in the Epithelial Context Inhibits Autophagy

doi: 10.3390/cells8070653

Figure Lengend Snippet: The ectopic expression of FGFR2c and its signaling inhibit autophagy in human keratinocytes. HaCaT cells, stably transduced with pBp-FGFR2b or pBp-FGFR2c constructs or with the empty pBp retroviral vector as control, were left untreated or stimulated with FGF7 or FGF2 in presence or absence of the FGFR2 tyrosine kinase inhibitor SU5402 as described in Material and Methods. ( A ) Western blot analysis showed that, while FGF7 stimulation increases the levels of LC3-II in all clones, FGF2 stimulation significantly decreased them only in HaCaT pBp-FGFR2c cells. All the observed effects were abolished by the presence of SU5402. Equal loading was assessed with the anti-ACTB antibody. For the densitometric analysis the values from three independent experiments were normalized and expressed as fold increases and are reported as mean values ± standard deviations (SD). Student’s t test was performed, and significance levels are defined as P < 0.05. * p < 0.05 and *** p < 0.001 vs the corresponding FGF-unstimulated cells; ** p < 0.05 vs the corresponding SU5402-untreated cells; not significant (NS) vs the corresponding FGF-unstimulated, SU5402-untreated cells. ( B ) Real-time Reverse Transcriptase-Polymerase Chain Reaction (RT-PCR) analysis shows that while FGF7 stimulation induces the increases of LC3 mRNA transcripts in all clones, FGF2 treatment does not affect them. The results observed in HaCaT pBp and pBp-FGFR2b upon FGF7 stimulation were abolished by SU5402. Results are expressed as mean values ± SE. Student’s t test was performed, and significance levels were defined as P < 0.05. * p < 0.01, *** p < 0.05 and NS vs the corresponding FGF-unstimulated cells; ** p < 0.05 and NS vs the corresponding SU5402-untreated-cells. ( C ) Quantitative immunofluorescence analysis shows that LC3 signal intensity was increased by FGF7 stimulation in all clones, but it appears strongly reduced upon FGF2 treatment only in HaCaT pBp-FGFR2c cells. The observed effects were abolished by SU5402 treatment. Quantitative analysis of the fluorescence intensity and LC3 positive dots per cell were performed as described in Materials and Methods, and the results are expressed as mean values ± standard errors (SE). The student’s t test was performed, and significance levels were defined as P < 0.05. * p < 0.01, *** p < 0.001 and ^ p < 0.0001, vs the corresponding FGF-unstimulated cells; ** p < 0.001 and ^^ p < 0.0001 vs the corresponding SU5402-untreated cells.

Article Snippet: For growth factors stimulation, cells were left untreated or incubated with FGF7 (Upstate Biotechnology, Lake Placid, NY, 01-118) or with FGF2 (PeproTech, London, BFGF 100-188) 100 ng/mL for 24 h at 37 °C.

Techniques: Expressing, Stable Transfection, Transduction, Construct, Retroviral, Plasmid Preparation, Control, Western Blot, Clone Assay, Reverse Transcription, Polymerase Chain Reaction, Reverse Transcription Polymerase Chain Reaction, Immunofluorescence, Fluorescence

Journal: Cells

Article Title: The Aberrant Expression of the Mesenchymal Variant of FGFR2 in the Epithelial Context Inhibits Autophagy

doi: 10.3390/cells8070653

Figure Lengend Snippet: FGFR2c expression and signaling inhibits the autophagosome formation. ( A ) HaCaT pBp, pBp-FGFR2b or pBp-FGFR2c cells were left untreated or stimulated with FGF7 or FGF2 in presence or absence of the FGFR2 tyrosine kinase inhibitor SU5402 as above. Western blot analysis shows that, the 62 kDa band corresponding to SQSTM1 was significantly increased only in HaCaT pBp-FGFR2c clones after FGF2 stimulation, while it decreases in all clones upon FGF7 stimulation. All the observed effects were abolished by SU5402. Equal loading was assessed with anti-ACTB antibody. Densitometric analysis and the student’s t test were performed as reported above. * p < 0.05 vs the corresponding FGF-unstimulated cells; ** p < 0.05 and *** p < 0. 001 vs the corresponding SU5402-untreated cells. ( B ) HaCaT clones were stimulated with FGFR2 ligands as above. Real-time RT-PCR analysis shows that FGF7 stimulation decreased SQSTM1 mRNA transcripts in all clones, while FGF2 treatment did not affect them. Results are expressed as mean values ± SE. The student’s t test was performed, and significance levels were defined as P < 0.05. * p < 0.01 vs the corresponding FGF7-unstimulated cells; NS vs the corresponding FGF7-unstimulated cells. ( C ) HaCaT clones were transiently transfected with mCherry-EGFP-LC3 construct. Cells were then left untreated or stimulated with FGF7 or FGF2 as above. Quantitative fluorescence analysis showed that, while FGF7 stimulation increased both yellow and red dots in HaCaT pBp and HaCaT pBp-FGFR2b cells, FGF2 stimulation decreased them in HaCaT pBp-FGFR2c cells. Quantitative analysis was performed as described in Materials and Methods, and results were expressed as mean values ± SE. The student’s t test was performed as reported in the legend to B. * p < 0.05 and ** p < 0.001 vs the corresponding FGF7-unstimulated cells; *** p < 0.01 vs the corresponding FGF2-unstimulated cells. ( D ) HaCaT pBp-FGFR2b and pBp-FGFR2c clones were left untreated or stimulated with FGF7 or FGF2 in the presence or absence of bafilomycin A1 for the last 3 h. Western blot analysis showed that the increase in the levels of LC3-II upon FGF7 stimulation observed in HaCaT pBp-FGFR2b was further enhanced by bafilomycin while the decrease of LC3-II observed in HaCaT pBp-FGFR2c cells upon FGF2 stimulation was not recovered in the presence of the drug. Equal loading was assessed with anti-ACTB antibody. Densitometric analysis and the student’s t test were performed as reported above. * p < 0.05 vs the corresponding FGF-unstimulated cells; ** p < 0.05, *** p < 0.01 and NS vs the corresponding bafilomycin-untreated cells.

Article Snippet: For growth factors stimulation, cells were left untreated or incubated with FGF7 (Upstate Biotechnology, Lake Placid, NY, 01-118) or with FGF2 (PeproTech, London, BFGF 100-188) 100 ng/mL for 24 h at 37 °C.

Techniques: Expressing, Western Blot, Clone Assay, Quantitative RT-PCR, Transfection, Construct, Fluorescence

Journal: Cells

Article Title: The Aberrant Expression of the Mesenchymal Variant of FGFR2 in the Epithelial Context Inhibits Autophagy

doi: 10.3390/cells8070653

Figure Lengend Snippet: ESRP1 depletion by siRNA in haCaT cells results in aberrant FGFR2 splicing and FGF2-mediated inhibition of autophagy. ( A ) HaCaT cells were transiently transfected with ESRP1 siRNA or an unrelated siRNA as a control. Cells were then left in complete medium (left panel). Alternatively, cells were left untreated or stimulated with FGF7 or FGF2 in the presence or absence of the FGFR2 tyrosine kinase inhibitor SU5402 as above (right panel). Real-time RT-PCR analysis (left panel) and Western blot analysis (right panel) show the efficiency of ESRP1 depletion. Results are expressed as mean values ± SE. The student’s t test was performed, and significance levels are defined as P values of ± 0.05. * p < 0.01 vs the corresponding control siRNA cells. For the Western blot analysis, equal loading was assessed with anti-ACTB antibody. ( B ) HaCaT cells were transiently transfected with ESRP1 siRNA or an unrelated siRNA as a control and then left in complete medium. Real-time RT-PCR analysis shows that ESRP1 depletion leads to a significant decrease of FGFR2b expression (left panel) and to the appearance of FGFR2c (right panel). Results are expressed as mean values ± SE. The student’s t test was performed, and significance levels are defined as P values of 0.05. * p < 0.05 vs the corresponding control siRNA cells. ( C ) HaCaT cells were transiently transfected with ESRP1 siRNA or an unrelated siRNA as a control. Cells were left untreated or stimulated with FGF7 or FGF2 in the presence or absence of the FGFR2 tyrosine kinase inhibitor SU5402 as above. Western blot analysis shows that ESRP1 depletion abolishes the increase of LC3 induced by FGF7 while it induces a decrease of LC3 upon FGF2 stimulation. Equal loading was assessed with anti-ACTB antibody. Densitometric analysis and the student’s t test were performed as reported in the legend to A. * p < 0.05 and ** p < 0.01 vs the corresponding FGF-unstimulated cells; *** p < 0.01 and **** p < 0.05 vs the corresponding SU5402-untreated cells. ( D ) HaCaT cells were transiently transfected with ESRP1 siRNA or an unrelated siRNA as a control and then left untreated or stimulated with FGF2 in presence or absence of SU5402 as above. Western blot analysis performed using antibody directed against the phosphorylated form of MTOR shows the phosphorylation of this substrates upon FGF2 stimulation in HaCaT ESRP1 siRNA cells. Equal loading was assessed with anti-ACTB antibody. Densitometric analysis and the student’s t test were performed as reported in the legend to A. * p < 0.05 vs the corresponding FGF-unstimulated cells; ** p < 0. 001 vs the corresponding SU5402-untreated cells. ( E ) HKs cells were transiently transfected with ESRP1 siRNA or an unrelated siRNA as a control and left in complete medium (left panel) or stimulated with FGF7 or FGF2 as above (right panel). Real-time RT-PCR analysis (left panel) and Western blot analysis (right panel) showed the efficiency of ESRP1 depletion. Results are expressed as mean values ± SE. The student’s t test was performed, and significance levels are defined as P values of ± 0.05. * p < 0.05 vs the corresponding control siRNA cells. For the Western blot analysis, equal loading was assessed with anti-ACTB antibody. ( F ) HKs cells were transiently transfected with ESRP1 siRNA or an unrelated siRNA as a control and then left in complete medium. Real-time RT-PCR analysis showed that ESRP1 depletion lead to a significant decrease of FGFR2b expression (left panel) and to the appearance of FGFR2c (right panel). Results are expressed as mean values ± SE. The student’s t test was performed, and significance levels are defined as P values of ± 0.05. * p < 0.01 vs the corresponding control siRNA cells. ( G ) HKs cells were transiently transfected with ESRP1 siRNA or an unrelated siRNA as a control. Cells were left untreated or stimulated with FGF7 or FGF2 as above. Western blot analysis shows that ESRP1 depletion abolished the increase of LC3 induced by FGF7 while it induced a decrease of LC3 upon FGF2 stimulation. Equal loading was assessed with anti-ACTB antibody. Densitometric analysis and the student’s t test were performed as reported in the legend to A. * p < 0.05 vs the corresponding FGF-unstimulated cells. ( H ) HKs cells were transiently transfected with ESRP1 siRNA or an unrelated siRNA as a control and then left untreated or stimulated with FGF2. Western blot analysis performed using antibody directed against the phosphorylated form of MTOR shows that ESRP1 depletion induces the phosphorylation of this substrates upon FGF2 stimulation. Equal loading was assessed with anti-ACTB antibody. Densitometric analysis and the student’s t test were performed as reported in the legend to A. * p < 0.05 vs the corresponding control siRNA cells.

Article Snippet: For growth factors stimulation, cells were left untreated or incubated with FGF7 (Upstate Biotechnology, Lake Placid, NY, 01-118) or with FGF2 (PeproTech, London, BFGF 100-188) 100 ng/mL for 24 h at 37 °C.

Techniques: Inhibition, Transfection, Control, Quantitative RT-PCR, Western Blot, Expressing, Phospho-proteomics

Journal: Advanced Science

Article Title: Targeting Tumor Heterogeneity by Breaking a Stem Cell and Epithelial Niche Interaction Loop

doi: 10.1002/advs.202307452

Figure Lengend Snippet: BMP7 is a basal‐to‐luminal paracrine candidate regulated by stromal FGFs. A) Diagram depicting the experimental procedures of single‐cell analyses of transplants from mixtures of wild‐type luminal cells with either wild‐type or Fgfr2 ∆/∆ null basal cells five days after surgery. B) Identification of basal and luminal cells were based on expression of published marker gene sets for these two cell types. [ ⁴⁸ ] C) GSEA analysis of pathways related to epithelial cell proliferation in the luminal cells of the control group and the experimental group, in which basal cells were wild type or Fgfr2 ∆/∆ null cells, respectively. D) Comparison of the number of ligand‐receptor pairs between basal and luminal cells based on CellphoneDB analysis in control and experimental samples. E) Select ligand‐receptor pairs between basal and luminal cells based on CellphoneDB analysis. F) Bmp7 mRNA expression in control ( Fgfr2 +/+ ) and experimental ( Fgfr2 ∆/∆ ) basal cells, confirming its reduced experiment in mutant basal cells. G) Western blotting and H) quantification analysis of BMP7 and the internal control GAPDH protein in the absence or presence of FGF2 stimulation using wild‐type and Fgfr2 null primary mammary epithelial basal cells. Ø denotes control group without FGF2 addition. Graph shows mean ± SD. Unpaired Student's t‐test was performed for statistical analysis, n.s., not significant, P ≥ 0.05; * P < 0.05.

Article Snippet: After the cells were cultured as spheres in the stem cell medium, they were placed in a sphere containing 2% Growth factor, starved for 24 h in the basic medium of reduced Matrigel, treated with FGF2, FGF7 (GenScript, Z03154), or FGF10 (GenScript, Z03155) (50 ng mL −1 ) or BMP7 (50 ng mL −1 ) for 24 h, before RNA or proteins were extracted for downstream experiments.

Techniques: Expressing, Marker, Control, Comparison, Mutagenesis, Western Blot

Journal: Advanced Science

Article Title: Targeting Tumor Heterogeneity by Breaking a Stem Cell and Epithelial Niche Interaction Loop

doi: 10.1002/advs.202307452

Figure Lengend Snippet: Targeting BMP7‐INHBA signaling loop inhibits progression of luminal breast cancer subtype. A) Percentage distribution of basal and luminal cells in subtypes of human breast cancer. B) Percentage distribution of basal and luminal cells in MMTV‐PyMT tumor at the ages indicated. C,D) Relative mRNA expression of C) Bmp7 and D) Inhba in luminal and basal cells of MMTV‐PyMT mice at the ages indicated. E) Gating strategy for sorting PyMT MECs based on CD24 and Itga6 (CD49f) by FACS. Note that an identical strategy was used to separate luminal cells (CD24 hi CD49f low ) from basal cells (CD24 med CD49f hi ) (see Figure ), but it failed to do so here. F–J) in vitro culture of PyMT MECs transfected with a lentiviral construct expressing either F,F’) a control scrambled shRNA or G,G’) individually sh‐ Bmp7 , H,H’) sh‐ Inhba , or in combination I,I’) by which Bmp7 and Inhba expression were knocked down using the shRNA technique. MEC outgrowths, as measured by area fold‐change, were quantified (J), ( n >10). K,L) Tumor growths of PyMT cells 8 weeks post‐surgery in nude mice with reduced expression of Bmp7 and Inhba via shRNA knockdown (K), with tumor volume quantified in (L). Values shown are the mean ± SD for each data point: * P < 0.05; ** P < 0.01; **** P < 0.0001. unpaired, two‐tailed Student's t tests. M) Schematic diagram of a model depicting the interactions among mammary stem cells and their stromal fibroblastic and luminal epithelial niches. Fibroblast production of FGF ligands, including FGF2, FGF7, and FGF10 are essential for stem cell pool expansion and luminal epithelial morphogenesis. Stromal FGFs are also essential for BMP7 production by MSCs via FGFR2 activation, which activates BMPR1a/2 signaling of luminal niche cells. This in turn upregulates luminal production of INHBA to promote stem cell expansion and completes a positive feedback signaling loop between MSCs and the luminal niche.

Article Snippet: After the cells were cultured as spheres in the stem cell medium, they were placed in a sphere containing 2% Growth factor, starved for 24 h in the basic medium of reduced Matrigel, treated with FGF2, FGF7 (GenScript, Z03154), or FGF10 (GenScript, Z03155) (50 ng mL −1 ) or BMP7 (50 ng mL −1 ) for 24 h, before RNA or proteins were extracted for downstream experiments.

Techniques: Expressing, In Vitro, Transfection, Construct, Control, shRNA, Knockdown, Two Tailed Test, Activation Assay