Journal: bioRxiv

Article Title: Centimeter-scale quorum sensing dictates collective survival of differentiating embryonic stem cells

doi: 10.1101/2020.12.20.423651

Figure Lengend Snippet: Data for 46C cells (which have Sox1 promoter driving GFP expression) differentiating towards NE lineage in N2B27 (without any inducers such as RA) that were previously self-renewing in serum+LIF (see STAR Methods). Same RNA-Seq dataset as in . To identify secreted factors other than FGF4 that might contribute to determining a population survival, we performed RNA-Seq to detect expression of any secreted factors that are known to control cell proliferation and/or death. We performed RNA-Seq on four populations: (1) pluripotent population prior to differentiation; (2) low-density (862 cells/cm 2 ) population; (3) high-density (5172 cells/cm 2 ) population; and (4) medium-density (1931 cells/cm 2 ) population that is near the threshold density. For the three differentiating populations, we collected their cells on the first and second day after triggering differentiation. Expression levels (FPKMs) of secreted factors that are known to control proliferation and/or apoptosis in ES cells and that fall within the range of molecular weights that the membranefilter experiments identified (50 – 300 kDa with +/-50% error) . Shown are the following genes: Ctgf, Scf, Ppia, Clu, Vegfa, Vegfb, Cyr61, Fgf5, Pdgfa, Fgf4 and Hspa8 . Below each gene name is the molecule’s weight (kDa) according to two online resources: Uniprot and ExPASy. n = 3 for all plots; Error bars are s.e.m.

Article Snippet: After 2 days of culturing in N2B27, we added 500 nM of Retinoic Acid, and one or combinations of the following recombinant proteins to the medium: 200 ng/mL of recombinant mouse FGF4 (R&D Systems, #7486-F4), 200 ng/mL of recombinant human FGF5 (R&D Systems, #237-F5), 100 ng/mL of recombinant mouse PDGFA (Novus, NBP1-43148), 100 ng/mL of recombinant mouse VEGFB 186 (Novus, #767-VE), 100 ng/mL of recombinant mouse VEGFA (Novus, #493-MV), 500 ng/mL of recombinant human CYR61/CCN1 (Novus, #4055-CR), 500 ng/mL of recombinant human CTGF/CCN2 (Novus, #9190-CC), 200 ng/mL of recombinant mouse CLU (Novus, #2747-HS), 500 ng/mL of recombinant human HSPA8/HSC70 (Novus, #NBP1-30278), 1000 ng/mL of recombinant human Cyclophilin A (PPIA) (Novus, #NBC1-18425), or 2000 ng/mL of mouse recombinant SCF (STEMCELL, #78064).

Techniques: Expressing, RNA Sequencing, Control

Journal: bioRxiv

Article Title: Centimeter-scale quorum sensing dictates collective survival of differentiating embryonic stem cells

doi: 10.1101/2020.12.20.423651

Figure Lengend Snippet: The RNA-Seq revealed that 11 secreted factors that are known to control cell proliferation/and or death were highly expressed in differentiating, high-density populations. We thus reasoned that one or combinations of these factors may be the secreted molecule(s) that determine the survival-versus-extinction fate of a population. Data in (A) and (B) for 46C cells (which have Sox1 promoter driving GFP expression) differentiating towards NE lineage in N2B27+RA that were previously self-renewing in serum+LIF (see STAR Methods). (A) We tested these molecules by adding them one-by-one into the medium of a low-density population (862 cells/cm 2 ) that would ordinarily become extinct. We added the following molecules individually, each at a saturating concentration (also see STAR Methods): version of recombinant mouse FGF4 used in (200 ng/mL), recombinant human FGF5 (200 ng/mL), recombinant mouse PDGFA (100 ng/mL), recombinant mouse VEGFB 186 (100 ng/mL), recombinant mouse VEGFA (100 ng/mL), recombinant human CYR61 (500 ng/mL), recombinant human CTGF (500 ng/mL), recombinant mouse CLU (200 ng/mL), recombinant human HSPA8 (500 ng/mL), recombinant human CYPA (1000 ng/mL), and recombinant mouse SCF (2000 ng/mL). After 6 days in a medium containing one of these molecules, we measured the fold-change in density (black bars) and differentiation efficiency (green bars) of the low-density population. n = 3; error bars are s.e.m. These results show that only the recombinant mouse FGF4 causes the fold-change in population density to be higher than one. All the other factors resulted in the low-density population either approaching extinction (fold change much less than 1) or becoming extinct (indicated with an asterisk). The black dashed line marks the maximum fold-change in population density achieved when the low-density population grows in the medium of a high-density population. The green dashed line marks the maximum differentiation efficiency achieved when the low-density population grows in the medium of a high-density population. The box beneath the plot shows which signaling factors were mixed together and then given to the low-density population in (B). (B) Results obtained by giving combinations of the 11 factors together to the low-density population, with the ingredients of the mixture indicated in the box below (A). Giving all 11 factors together at once yielded the highest growth (~4-fold increase in population density; black bar), which was virtually identical to the growth obtained with a high-density population’s (5172 cells/cm 2 ) medium (black dashed line). But, with the 11 molecules added together at once, the differentiation efficiency (green) remained rather low at ~20% compared to the ~40% (green dashed line) that we get from incubating the low-density population in the medium of a high-density population. As we progressively reduced the number of signaling factors in the mixture from 11 to 2, we observed only a modest decrease in population growth, down to about ~2 fold. Importantly, recombinant FGF4 was included in all these mixtures.

Article Snippet: After 2 days of culturing in N2B27, we added 500 nM of Retinoic Acid, and one or combinations of the following recombinant proteins to the medium: 200 ng/mL of recombinant mouse FGF4 (R&D Systems, #7486-F4), 200 ng/mL of recombinant human FGF5 (R&D Systems, #237-F5), 100 ng/mL of recombinant mouse PDGFA (Novus, NBP1-43148), 100 ng/mL of recombinant mouse VEGFB 186 (Novus, #767-VE), 100 ng/mL of recombinant mouse VEGFA (Novus, #493-MV), 500 ng/mL of recombinant human CYR61/CCN1 (Novus, #4055-CR), 500 ng/mL of recombinant human CTGF/CCN2 (Novus, #9190-CC), 200 ng/mL of recombinant mouse CLU (Novus, #2747-HS), 500 ng/mL of recombinant human HSPA8/HSC70 (Novus, #NBP1-30278), 1000 ng/mL of recombinant human Cyclophilin A (PPIA) (Novus, #NBC1-18425), or 2000 ng/mL of mouse recombinant SCF (STEMCELL, #78064).

Techniques: RNA Sequencing, Control, Expressing, Concentration Assay, Recombinant

Journal: bioRxiv

Article Title: Centimeter-scale quorum sensing dictates collective survival of differentiating embryonic stem cells

doi: 10.1101/2020.12.20.423651

Figure Lengend Snippet: Data for 46C cells differentiating towards NE lineage in N2B27 (without any inducers such as RA) that were previously self-renewing in serum+LIF (see STAR Methods). We used real-time quantitative PCR (RT-qPCR) to measure the expression levels of all four receptors ( FGFR1-4 ) of Fibroblast Growth Factors (FGFs) and the expression levels of the two FGFs, FGF4 and FGF5 (primers in ). We examined a high-density population (5172 cells/cm 2 ) after two days of differentiation. We normalized the resulting expressions of a gene relative to that of the housekeeping gene, GAPDH of the same population, and then further normalized the resulting value to the pluripotent population’s normalized expression level (similar to the procedure described in the caption for ). Thus, a given gene’s expression level is compared to the pluripotent population’s expression level for that gene. Normalized expression levels of FGF4 and FGF5 (in black) and FGFR1-2 (in red). n = 3; Error bars are s.e.m. Altogether, these results show that FGF4, FGF5 and FGFR1-2 are expressed - and some more so than the pluripotent population (i.e., expression value greater than 1) - during the first 2 days in which ES cells exit pluripotency.

Article Snippet: After 2 days of culturing in N2B27, we added 500 nM of Retinoic Acid, and one or combinations of the following recombinant proteins to the medium: 200 ng/mL of recombinant mouse FGF4 (R&D Systems, #7486-F4), 200 ng/mL of recombinant human FGF5 (R&D Systems, #237-F5), 100 ng/mL of recombinant mouse PDGFA (Novus, NBP1-43148), 100 ng/mL of recombinant mouse VEGFB 186 (Novus, #767-VE), 100 ng/mL of recombinant mouse VEGFA (Novus, #493-MV), 500 ng/mL of recombinant human CYR61/CCN1 (Novus, #4055-CR), 500 ng/mL of recombinant human CTGF/CCN2 (Novus, #9190-CC), 200 ng/mL of recombinant mouse CLU (Novus, #2747-HS), 500 ng/mL of recombinant human HSPA8/HSC70 (Novus, #NBP1-30278), 1000 ng/mL of recombinant human Cyclophilin A (PPIA) (Novus, #NBC1-18425), or 2000 ng/mL of mouse recombinant SCF (STEMCELL, #78064).

Techniques: Real-time Polymerase Chain Reaction, Quantitative RT-PCR, Expressing

Journal: Cell Death & Disease

Article Title: Cancer-associated fibroblasts secrete FGF5 to inhibit ferroptosis to decrease cisplatin sensitivity in nasopharyngeal carcinoma through binding to FGFR2

doi: 10.1038/s41419-024-06671-0

Figure Lengend Snippet: Human CAFs and NFs were isolated from NPC tissues and the adjacent normal tissues, respectively. A The expression levels of α-SMA and FAP in CAFs were detected by immunofluorescence. Green, α-SMA. Red, FAP. Blue, DAPI. Scale bar: 100 μm. B The protein levels of α-SMA, vimentin and FAP in NFs and CAFs were detected by western blot. C The protein level of FGF5 in fibroblasts was detected by western blot. D The secretion of FGF5 in fibroblasts was assessed by ELISA assay. NPC/HK1 and C666-1 cells were divided into following groups: control, DDP (5 μg/mL), DDP (5 μg/mL)+CM and DDP (5 μg/mL)+anti-FGF5 antibody. E Cell viability was monitored by CCK-8 assay. F The protein levels of GPX4 and SLC7A11 were detected by western blot. G – I The levels of MDA ( G ), Fe 2+ ( H ) and GSH ( I ) in NPC cells were measured using commercial kits. J NPC cells were stained with BODIPY C11 and lipid peroxidation was assessed by flow cytometry. * P < 0.05, ** P < 0.01, and *** P < 0.001.

Article Snippet: In some experiments, mouse FGF5 recombinant protein (CSB-YP008632MO, Cusabio, Wuhan, China) were injected via tail vein on day 2.

Techniques: Isolation, Expressing, Immunofluorescence, Western Blot, Enzyme-linked Immunosorbent Assay, Control, CCK-8 Assay, Staining, Flow Cytometry

Journal: Cell Death & Disease

Article Title: Cancer-associated fibroblasts secrete FGF5 to inhibit ferroptosis to decrease cisplatin sensitivity in nasopharyngeal carcinoma through binding to FGFR2

doi: 10.1038/s41419-024-06671-0

Figure Lengend Snippet: A The mRNA level of Nrf2 in transfected NPC cells were detected by qRT-PCR. NPC/HK1 and C666-1 cells were divided into six groups: control, DDP (5 μg/mL), DDP (5 μg/mL) + Vector-CM, DDP (5 μg/mL)+FGF5-CM, DDP (5 μg/mL)+FGF5-CM+shNC and DDP (5 μg/mL) + FGF5-CM + shNrf2#2. B The protein levels of Keap1, Nrf2 and HO-1 in NPC cells were detected by western blot. C Cell viability was monitored by CCK-8 assay. D – F The levels of MDA ( D ), Fe 2+ ( E ) and GSH ( F ) in NPC cells were measured using commercial kits. G NPC cells were stained with BODIPY C11 and lipid peroxidation was assessed by flow cytometry. H The protein levels of GPX4 and SLC7A11 were detected by western blot. * P < 0.05, ** P < 0.01, and *** P < 0.001.

Article Snippet: In some experiments, mouse FGF5 recombinant protein (CSB-YP008632MO, Cusabio, Wuhan, China) were injected via tail vein on day 2.

Techniques: Transfection, Quantitative RT-PCR, Control, Plasmid Preparation, Western Blot, CCK-8 Assay, Staining, Flow Cytometry

Journal: Cell Death & Disease

Article Title: Cancer-associated fibroblasts secrete FGF5 to inhibit ferroptosis to decrease cisplatin sensitivity in nasopharyngeal carcinoma through binding to FGFR2

doi: 10.1038/s41419-024-06671-0

Figure Lengend Snippet: A The interactions between FGF5 and FGFRs in NPC tissues were detected by Co-IP assay. B The mRNA level of FGFR2 in N69, NPC/HK1 and C666-1 cells was detected by qRT-PCR. C The protein level of FGFR2 in N69, NPC/HK1 and C666-1 cells was detected by western blot. D The expression of FGFR2 in nasal polyp and NPC tissues was determined by tissue microarray. The first column: nasal polyp tissues. 2–9 columns: NPC tissues. E The immunoreactivities of FGF5 and FGFR2 in NPC tissues were detected by IHC analysis. Scale bar: 20 μm. * P < 0.05 and *** P < 0.001.

Article Snippet: In some experiments, mouse FGF5 recombinant protein (CSB-YP008632MO, Cusabio, Wuhan, China) were injected via tail vein on day 2.

Techniques: Co-Immunoprecipitation Assay, Quantitative RT-PCR, Western Blot, Expressing, Microarray

Journal: Cell Death & Disease

Article Title: Cancer-associated fibroblasts secrete FGF5 to inhibit ferroptosis to decrease cisplatin sensitivity in nasopharyngeal carcinoma through binding to FGFR2

doi: 10.1038/s41419-024-06671-0

Figure Lengend Snippet: A The mRNA level of FGFR2 in transfected NPC cells were detected by qRT-PCR. B The protein level of FGFR2 in transfected NPC cells were detected by western blot. NPC/HK1 and C666-1 cells were divided into five groups: control, DDP (5 μg/mL), DDP (5 μg/mL)+FGF5-CM, DDP (5 μg/mL) + FGF5-CM + shNC and DDP (5 μg/mL) + FGF5-CM + shFGFR2#1. C The protein levels of FGFR2, Keap1, Nrf2 and HO-1 in NPC cells were detected by western blot. D Cell viability was monitored by CCK-8 assay. E – G The levels of MDA ( E ), Fe 2+ ( F ) and GSH ( G ) in NPC cells were measured using commercial kits. H NPC cells were stained with BODIPY C11 and lipid peroxidation was assessed by flow cytometry. * P < 0.05, ** P < 0.01, and *** P < 0.001.

Article Snippet: In some experiments, mouse FGF5 recombinant protein (CSB-YP008632MO, Cusabio, Wuhan, China) were injected via tail vein on day 2.

Techniques: Transfection, Quantitative RT-PCR, Western Blot, Control, CCK-8 Assay, Staining, Flow Cytometry

Journal: Cell Death & Disease

Article Title: Cancer-associated fibroblasts secrete FGF5 to inhibit ferroptosis to decrease cisplatin sensitivity in nasopharyngeal carcinoma through binding to FGFR2

doi: 10.1038/s41419-024-06671-0

Figure Lengend Snippet: Male BALB/c nude mice were randomly divided into three groups: control, DDP (4 mg/kg) and DDP (4 mg/kg)+FGF5 recombinant protein groups. A Representative photos of xenograft tumors. B , C Quantitative analyses of tumor volume ( B ) and weight ( C ). D The immunoreactivity of 4-HNE in tumors was detected by IHC analysis. Scale bar: 5 μm. E – G The levels of MDA ( E ), Fe 2+ ( F ) and GSH ( G ) in xenograft tumors were measured using commercial kits. H The protein levels of GPX4, SLC7A11, FGFR2, Keap1, Nrf2 and HO-1 were detected by western blot. * P < 0.05, ** P < 0.01, and *** P < 0.001.

Article Snippet: In some experiments, mouse FGF5 recombinant protein (CSB-YP008632MO, Cusabio, Wuhan, China) were injected via tail vein on day 2.

Techniques: Control, Recombinant, Western Blot