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: ( A ) Main graph: fold-change in initial population density, after 6 days in N2B27 supplemented with any of the 11 recombinant molecules (indicated on horizontal axis) or all of 11 combined (“All”) at day 0. All data are for initially low-density population (862 cells / cm 2 ). Data for 46C cells differentiating in N2B27+RA that were previously self-renewing in serum with LIF. Also see STAR Methods and for full details of concentrations added. Horizontal dashed line shows the maximum fold-change in density obtained in our study (nearly 4 fold), which occurs when the same low-density population is rescued by a high-density population’s (5172 cells / cm 2 initially) medium. Middle inset: fold-change in initial population density after adding various concentrations of recombinant FGF4 (horizontal axis). Same procedure as described for main graph. Right inset: foldchange in initial population density (black bar) and percentage of cells entering NE lineage (Sox1-GFP expressing cells) (green bar), both measured 10 days after differentiation begins in presence of 200 ngImL FGF4 that we added at the start of differentiation. Error bars are s.e.m.; n = 3. ( B ) ELISA measurements of concentrations of extracellular FGF4 in the medium of a high-density population (8620 cells / cm 2 initially) during unguided differentiation in N2B27 during 2 days (blue points) (previously self-renewing in serum with LIF) (see STAR Methods). Vertical axis shows FGF4 concentration relative to that of a ~80% confluent pluripotent population (denoted “1x” and marked with yellow horizontal line). 80% confluency equals i~8 x 10 6 cells in 10-cm diameter dish. Lower detection limit of the ELISA assay is indicated (in grey). See for ELISA standard curves. Error bars are s.e.m.; n = 3. ( C ) Cartoon shows PD173074, a well-characterized smallmolecule inhibitor of FGF receptors ( , ) (see STAR Methods). Fold-change in initial population density (bottom graph) and percentage of populations that enter NE lineage (top graph), both measured 6 days after differentiation began and as a function of initial population density. Data for 46C cells differentiating in N2B27+RA that were previously self-renewing in serum with LIF. Red data points in both graphs are for populations that were incubated with 2 μM (1056 ngImL) of PD173074 from the start of differentiation. PD173074 was dissolved in DMSO. Thus, as a control, black and green points are for populations without PD173074 but with the same amount of DMSO (volume per volume) as the populations represented by red data points. Blue shade indicates population expansion and red shade indicates population extinction. Error bars are s.e.m.; n = 3. ( D ) Heat map showing transcriptome-wide changes in unguided differentiation (N2B27) of 46C cells (previously self-renewing in serum with LIF) of low-density population (862 cells / cm 2 ; enclosed in pink box), near-threshold (medium-density) population (1931 cells / cm 2 ; enclosed in grey box), and high-density population (5172 cells / cm 2 ; enclosed in blue box) (see STAR Methods). Leftmost column shows data for self-renewal (pluripotent) population before differentiation begins (labeled “All” since every population starts as this population before differentiation). Each column of differentiating population shows data for 1 day after (labeled “1”) or 2 days after (labeled “2”) starting differentiation. Each row shows a different gene, each of which are either activated (21 genes) or repressed (19 genes) by YAP1, either directly or indirectly. lists all genes. Color represents row Z-score: a measure of by how much a gene’s expression level for a given condition deviates from that gene’s expression level averaged across all different conditions (i.e., different populations and days). Purple represents a positive row Z-score (more expressed than average). Orange represents a negative row Z-score (less expressed than average). Data based on 3 biological replicates. ( E ) Cartoon shows YAP1 which exists as either phosphorylated (labeled “P”) or dephosphorylated. Verteporfin (VP) inhibits active (dephosphorylated) YAP1 from entering the nucleus and regulating target gene expression. Fold-change in population density for high-density population (5172 cells / cm 2 initially, in blue box) and low-density population that was rescued with medium of 2-days-old high-density population (862 cells / cm 2 initially, in green box) after 6 days of differentiation towards NE-lineage. Data for 46C cells differentiating in N2B27+RA that were previously self-renewing in serum with LIF. Black bar: Verteporfin (VP) was always absent. Red bar in middle of each box: VP was added to medium after the first two days. Third column of each box shows absence of cells (extinction) when VP was present from the start of differentiation. Also see for full data. Error bars are s.e.m.; n = 3. ( F ) ELISA measurements showing amounts of YAP1 protein phosphorylated at Ser397 (inactive YAP1) (see STAR Methods and also ). Vertical axis shows the relative amount of inactive YAP1: the level of inactive YAP1 for a differentiating population divided by the amount of inactive YAP1 for a pluripotent population (in serum with LIF medium) that has the same cell numbers as the differentiating population at the time of lysing the cells for ELISA. Values are for 46C cells previously propagated in serum with LIF medium, 3 days after starting differentiation towards NE lineage with N2B27+RA medium. Pink: low-density population (862 cells / cm 2 initially). Blue: high-density population (5172 cells / cm 2 initially). Grey: low-density population rescued after two days by medium from a 2-day-old high-density population. Green: low-density population rescued by adding 200 ngImL FGF4 to its medium on day 0 (see (A)). Error bars are s.e.m.; n = 3. ( G ) Bcl2 (anti-apoptotic gene) and Cyr61 (YAP1-specific target) expression levels over time after initiating differentiation. Data obtained with RT-qPCR for 46C cells differentiating in N2B27+RA that were previously self-renewing in serum with LIF (see STAR Methods). Same color scheme as shown in (F). Also see for other genes. On each day, we first normalized a population’s gene ( Bcl2 or Cyr61 ) expression level by that population’s Gapdh level (housekeeping gene). Afterwards, plotted on the vertical axis, we divided each population’s Gapdh -normalized gene ( Bcl2 and Cyr61 ) expression level on a given day by the Gapdh -normalized value for one-day-old low-density population (whose value is thus “1x” here). Error bars are s.e.m.; n = 3.

Article Snippet: We used Mouse FGF4 ELISA Kit (ELISAGenie / Westburg, MOES00755) and followed the manufacturer’s protocol.

Techniques: Recombinant, Expressing, Enzyme-linked Immunosorbent Assay, Concentration Assay, Incubation, Labeling, Quantitative RT-PCR

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: ( A ) Spreading a relatively small number of cells (~5000 cells / mL of N2B27) on a 10-cm diameter dish yields small colonies (size ~ 400 μm 2 ) at the start of differentiation which eventually become extinct over time. Images are for 46C cells differentiating in N2B27+RA that were previously self-renewing in serum with LIF. Scale bars = 200 μm. Microscopy images are taken after 24 hours and 120 hours. ( B ) A same number of cells as in shown in (A) confined within a small area with a droplet on a 10-cm diameter dish yields macroscopic colony (size ~ 28 mm 2 ) at the start of differentiation which eventually survives, expands and differentiates over time. Images are for 46C cells differentiating in N2B27+RA that were previously self-renewing in serum with LIF. Scale bar = 2 mm. Microscopy images are taken after 24 hours and 120 hours. In each of the two images, we stitched together multiple fields of view, with each field of view being 1.40 mm x 0.99 mm (same field size as in ). The stitching creates the checkered outline in both images. ( C ) Macroscopic colony, according to procedure described in (B), is visible to the naked eye after several days of differentiation (shown here is after 10 days of differentiation in N2B27+RA). Image taken after 10 days. In the greyscale image at the bottom, we stitched together multiple fields of view, with each field of view being 1.40 mm x 0.99 mm (same field size as in ). The stitching creates the checkered outline in this image. ( D ) Summary of strategies for inducing collective survival of differentiating ES cells. Spreading a relatively low number of cells (5000 cells / mL of N2B27) on a 10-cm diameter dish (shown here: 58 cm 2 surface area (862 cells / cm 2 ) and 10-mL N2B27) results in all cells becoming extinct during differentiation. Adding recombinant FGF4 (200 ngImL) to supernatant rescues spread cells from extinction (~1.3 fold-growth and ~19% Sox1-GFP positive). Lowering the height of liquid medium over millimetres rescues spread cells from extinction (~2.9 fold-growth and ~71% Sox1-GFP positive). Transplanting spread cells into a high-density population’s medium on day 2 rescues cells from extinction (~4.3 fold-growth and ~39% Sox1-GFP positive). Clustering a same number of cells, also used for spreading, into a macroscopic colony at the start of differentiation rescues cells from extinction (~20 fold-growth and ~92% Sox1-GFP positive). Data for 46C cells differentiating in N2B27+RA that were previously self-renewing in serum with LIF.

Article Snippet: We used Mouse FGF4 ELISA Kit (ELISAGenie / Westburg, MOES00755) and followed the manufacturer’s protocol.

Techniques: Microscopy, 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 (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: We used Mouse FGF4 ELISA Kit (ELISAGenie / Westburg, MOES00755) and followed the manufacturer’s protocol.

Techniques: Expressing, RNA Sequencing Assay

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 selfrenewing in serum+LIF (see STAR Methods). Same RNA-Seq dataset as in . We performed RNA-Seq to measure the expression levels of all 22 FGF ligands (A) and their receptors (FGFRs) (B). 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. (A) Expression levels of all FGF ligands. Shown are the following genes: Fgf1-8, Fgf20-21 and Fgf23 . Below each gene name is the corresponding molecular weight in kDa. Note that FGF4 expression prominently stands out among all the FGFs. n = 3 for all plots; Error bars are s.e.m. (B) Expression levels of all FGF receptors (FGFRs). Shown are the following genes: Fgfr1-4 . Below each gene name is the corresponding molecular weight in kDa, according to two online resources: Uniprot and ExPASy. n = 3 for all plots; Error bars are s.e.m. (C) Expression levels of Wnt ligands. Shown are the following genes: Wnt6, Wnt10a, Wnt9a, Wnt3, Wnt3a, Wnt9b, Wnt5a, Wnt1, Wnt8a, Wnt8b, Wnt2b, Wnt4, Wnt16, Wnt7a, Wnt5b and Wnt11 . None of the Wnt genes prominently stand out, except for Wnt4 which still has an order of magnitude lower expression relative to FGF4 expression. n = 3 for all plots; Error bars are s.e.m.

Article Snippet: We used Mouse FGF4 ELISA Kit (ELISAGenie / Westburg, MOES00755) and followed the manufacturer’s protocol.

Techniques: Expressing, RNA Sequencing Assay, Molecular Weight

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: We used Mouse FGF4 ELISA Kit (ELISAGenie / Westburg, MOES00755) and followed the manufacturer’s protocol.

Techniques: RNA Sequencing Assay, 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: We used Mouse FGF4 ELISA Kit (ELISAGenie / Westburg, MOES00755) and followed the manufacturer’s protocol.

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

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: We performed ELISA that detects mouse FGF4 (see STAR Methods). 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). (A) Standard curve based on a recombinant mouse FGF4 that came with the commercial ELISA kit. Note that this recombinant FGF4 is not necessarily the same version as the FGF4 that our cells secrete. Each measurement (absorbance at 450 nm) was done in duplicate (black data points). Then, we performed a logistic regression on the data by fitting a 4-parameter logistic function (red curve): , where A, B, C and D are constant coefficients and x is the known concentration of the recombinant mouse FGF4 that we added. We found: A = 0.0084, B = 1.313, C = 1312 and D = 6.59. (B) Standard curve based on the version of FGF4 that pluripotent cells secrete into their medium. We first concentrated the medium taken from a highly confluent (~80% confluent) pluripotent population with a 3-kDa filter and then performed ELISA on serially diluted fractions of this concentrated medium. Each measurement (absorbance at 450 nm) was done in duplicate (black points). Then, we performed a logistic regression on the black data points by fitting a 4-parameter logistic function (red curve): , where A, B, C and D as constant coefficients and x as amount of lysed, pluripotent cells (day 0). We found: A = 0.07123, B = 1.184, C = 1.407 and D = 4.069. The standard curve shows that pluripotent ES cells secrete a version of FGF4 that our ELISA can detect. Moreover, it also shows a limitation of our ELISA: the assay can only detect sufficiently high concentration of FGF4 as seen by the fact that it cannot detect any FGF4 in a 1:100 dilution of a concentrated medium taken from a highly confluent ES cells. (C) Standard curve based on recombinant mouse FGF4 from a different manufacturer (not from the ELISA kit) that we could add to the cell-culture medium to rescue low-density populations (also see STAR Methods). Each measurement (absorbance at 450 nm) was done in duplicates (black points). As seen here, the ELISA cannot detect any amounts of this version of FGF4, even when its concentration is 100-folds higher than the highest concentration - of the version supplied by the ELISA kit - that we used in (A). The three standard curves (A-C) show that ELISA is highly sensitive to the form of FGF4 - we used three different forms in each of (A-C). The two versions of FGF4 that are not supplied by the ELISA kit (B-C) are detected with lower efficiency than the version supplied by the kit (A). (D) ELISA measurements of secreted FGF4 (in pg/mL) in various conditions (indicated with labels on the horizontal axis). We detected abundant FGF4 in the pluripotency medium (~500 pg/mL). In the medium of the high-density population (8620 cells/cm 2 ) after two days of differentiation, we detected ~50 pg/mL of FGF4. After 1 day of differentiation, the medium of the high-density population did not contain any detectable amounts of FGF4. Hence, high-density populations take 2 days to accumulate appreciable (detectable) amounts of FGF4.

Article Snippet: We used Mouse FGF4 ELISA Kit (ELISAGenie / Westburg, MOES00755) and followed the manufacturer’s protocol.

Techniques: Enzyme-linked Immunosorbent Assay, Recombinant, Concentration Assay, Cell Culture

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: Determining the degradation rate of FGF4 that cells secrete tells us whether FGF4 can diffuse by millimeters or not, through the Stokes-Einstein equation (see STAR Methods). We performed ELISA that targets FGF4 (see STAR Methods) to determine the concentrations of secreted FGF4 when incubated without cells in liquid medium. For this, we used a 3-kDa filter to concentration the pluripotency medium (serum+LIF) taken from a confluent population of 46C cells. Then we incubated the medium without any cells in a 37°C incubator for the hours indicated on the horizontal axis. We then took it out of the incubator and performed ELISA on it to measure the remaining [FGF4]. We observed that after incubating for 72 hours (3 days), the initial concentration of the secreted FGF4 was not appreciably degraded. As a control, we confirmed that no ingredient of a 3-kDa-concentrated pluripotency medium (without cells) interferes with our ELISA measurement to produce a non-zero concentration. Thus, this control showed no such signal (see ). Altogether, this result suggests that the concentration of secreted FGF4, by itself and in the absence of cells, is stable over at least 3 days and thus – according to the Stokes-Einstein equation (see STAR Methods) – can diffuse over millimeters (see STAR Methods).

Article Snippet: We used Mouse FGF4 ELISA Kit (ELISAGenie / Westburg, MOES00755) and followed the manufacturer’s protocol.

Techniques: Enzyme-linked Immunosorbent Assay, Incubation, Concentration Assay

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: To further support the idea that diffusion alone spreads the cell-secreted factors in our experiments, we determined how fast a droplet of a dye molecule of a known weight spreads in a differentiation medium without any cells, under the same incubation conditions as our cell cultures. (A) We used a gel loading dye (DNA Gel Loading Dye 6X, Thermo Scientific, #R0611) which consists of two molecules: bromophenol blue (669.96 Da) and xylene cyanol (538.61 Da). For simplicity, our calculations below will assume that the dye consists of only the heavier molecule, bromophenol blue. We injected a single, 0.5-μL droplet of the dye at the center of a 6-cm diameter plate that contained 5-mL of transparent N2B27 medium, either at room temperature (20°C) or pre-warmed at 37°C. We used a wide-field microscope to make a time-lapse movie with a bird’s eye view and snapshots every 10-seconds. Three snapshots (at 0, 30 and 60 seconds; all done at 37°C) of a single droplet of the dye shows the droplet expanding. Scale bar = 200 μm. (B) We determined the diffusion constant D of the dye in two ways: using the time-lapse movie and from theory. In the plots, three different colors represent three independent experiments. To determine D from the movies, we tracked the visible droplet boundary over time in a movie to plot the droplet area over time (shown in the two plots here at 37°C and 20°C). As shown, the droplet area linearly increased over time, which is consistent with pure diffusion (pure Brownian motion) since the area of a droplet is proportional to the mean squared displacement of a particle. Specifically, for a particle that undergoes a pure three-dimensional diffusion (Brownian motion), its mean squared displacement 〈 R 2 〉 at time t is: 〈 R 2 〉 = 6 Dt . Let A be the 2-dimensionally projected area of the droplet. Then, and hence, , where A slope is the slope of the linear fits to the droplet area as shown in the two plots here. From these fits, the experimentally determined diffusion constants D exp at 37°C and 20°C are 51.5 ± 17.8 μm 2 /s and 23.5 ± 3.9 μm 2 /s respectively ( n = 3; error bars are s.e.m.). As a comparison, we determined the diffusion constant D from theory - via the Stokes-Einstein equation which states, where k is the Boltzmann constant, T is temperature, η is the medium’s dynamic viscosity, and r dye is the radius of the dye molecule. For water, η = 0.000692 kg/m·s at 37°C and η = 0.001003 kg/m·s at 20°C (from BioNumbers (74)). We conservatively estimated r dye by noting that bromophenol blue consists of ~10 carbon-carbon bonds which would mean that the dye molecule’s diameter is 10 x 0.126 nm. For simplicity, we assume that r dye = 1 nm . The Stokes-Einstein equation then states that the dye’s diffusion constants D theory at 37°C and 20°C are 328.1 μm 2 /s and 214.0 μm 2 /s respectively. Hence, D exp < D theory . The fact that our analysis relies on the visible (by eye) boundary of the expanding droplet would underestimate the D exp since the dye must be spreading at least as fast as the boundary does. More importantly, if there were significant convection currents in the liquid medium, then D exp would be much larger than the measured value. This argues against there being any significant liquid convection in our cell-culture media. In other words, the dye-based experiment strongly indicates that cell-secreted survival-promoting factors spread out by pure diffusion rather than by convection currents which, according to the dye, are negligible in our cell-culture conditions. Furthermore, note that and ; the experimental and theoretical values for pure diffusion closely match (proportional to a factor on the order of one). (C) Based on Brownian motion in 3 dimensions with the experimentally determined diffusion constant, the dye molecule has a mean squared displacement of 〈 R 2 〉 = 1 mm 2 after (with D = D exp ( dye , 37° C) = 51.5 μm 2 /s). The same calculation, but now based on the Stokes-Einstein estimate of the diffusion constant would yield t dye = 8.5 minutes for a 1 mm 2 mean squared displacement (with D theory ( dye , 37°C) = 328.1 μm 2 /s). Hence the theory predicts a faster spreading of dye than experimentally observed - again, arguing against liquid convection or any other mechanism besides diffusion helping to spread the dye. A secreted molecule of 100 kDa would have a mean squared displacement of 1 mm 2 after time, , where we estimate the radius of the molecule to be r secreted = 20 nm (see STAR Methods). Hence the two days taken to observe appreciable amount of FGF4 and other survivalpromoting factor(s) traveling millimeters and accumulating is consistent with the t secreted calculated here (i.e., if t secreted were much larger than two days, then we should not be observing the survival-factors travelling by millimeters within two days).

Article Snippet: We used Mouse FGF4 ELISA Kit (ELISAGenie / Westburg, MOES00755) and followed the manufacturer’s protocol.

Techniques: Diffusion-based Assay, Incubation, Injection, Microscopy, Convection, Cell Culture

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+RA that were previously self-renewing in serum+LIF (see STAR Methods). With realtime quantitative PCR (primers in ), we measured anti-apoptotic, pro-apoptotic, and YAP1-mediated cell-signaling genes over the course of differentiation. Normalization of expression values: for each gene g , we first divided its expression level by the expression level of Gapdh , resulting in a value N g . For each population, we divided its N g by the low-density population’s N g on day 1 to get the final, normalized expression level μ which is plotted in here in all graphs. Thus, “1x” is the expression level of the low-density population on the first day after starting differentiation. We examined four populations: (1) high-density population (5172 cells/cm 2 ); (2) low-density population (862 cells/cm 2 ); (3) low-density population that we rescued from extinction by transplanting it, after two days, into the high-density population’s medium; and (4) low-density population that we rescued after adding 200 ng/mL recombinant mouse FGF4 to its differentiation medium on day 0. (A) Expression levels of two anti-apoptotic genes, Bcl2 (left graph) and Mdm2 (right graph). Data for Bcl2 is a replicate of the data shown in which we show here for comparison with Mdm2 . Both Bcl2 and Mdm2 show increased expressions (more anti-apoptotic) for high-density population (blue) and low-density population that was rescued by the medium of the high-density population after the 2nd day (black) or with FGF4 (green). Low-density population that goes extinct (red) shows nearly constant, low expression level of both genes. No data for 4th day is shown for the low-density population because it becomes extinct after the 3rd day (there were already barely any cells left for the 3rd day data shown here). (B) Expression levels of two pro-apoptotic genes - Bax (left graph) and Bbc3 (right graph). Color scheme is the same as in (A). The high-density population initially has a higher Bbc3 expression than the low-density population but eventually down-regulates and has lower Bbc3 expression than the low-density population. The low-density population, in turn, gradually increases its Bbc3 expression over time, up to the moment of extinction (~ day 3). Note that the rescued low-density population keeps its Bbc3 expression level low, past day 2 (which is when it receives the medium from a high-density population) and has nearly same low Bbc3 expression as the high-density population after being rescued. Note that differentiation is known to increase expression of apoptotic genes. (C) Expression levels of two cell-signaling genes that are upregulated by Yap1, Cyr61 (left graph) and Amotl2 (right graph). Data for Cyr61 is a replicate of the data shown in . Only the high-density and the rescued low-density populations gradually increase the expression levels of both genes whereas the low-density population that heads towards extinction (red) maintains a nearly constant, low expression of both genes (consistent with our findings in that secreted factors that are abundant for high-density populations increase YAP1 activity (and thus upregulate expression of Cyr61 and Amotl2 ). In all the plots, n = 3; Error bars are s.e.m.

Article Snippet: We used Mouse FGF4 ELISA Kit (ELISAGenie / Westburg, MOES00755) and followed the manufacturer’s protocol.

Techniques: Real-time Polymerase Chain Reaction, Expressing, Recombinant, Activity Assay

Journal: Science translational medicine

Article Title: Steroid-induced fibroblast growth factors drive an epithelial-mesenchymal inflammatory axis in severe asthma

doi: 10.1126/scitranslmed.abl8146

Figure Lengend Snippet: (A) Secreted FGFs and (B) IL-8 were measured by Luminex (A to I) or ELISA (J), respectively, in basal supernatants of primary human BAECs grown at air-liquid interface (BAEC-ALI) after exposure to F.p. for 24 hours. Each line represents a different donor. * denotes P < 0.05 using a one-way analysis of variance (ANOVA) with Dunnett’s multiple comparisons test; unless the control group was zero, # denotes P < 0.05 using Wilcoxon paired t tests. (C) Secreted FGF1, FGF2, FGF4, FGF18, (D) IL-8, and (E) G-CSF were measured in supernatants from F.p.-stimulated precision-cut lung slices. Three precision-cut lung slices were incubated per well per donor and exposed to F.p. (10−7 M) for 24 hours, with three replicates per experiment. One of three experiments is shown. *P < 0.05 with a paired t test.

Article Snippet: Enzyme-linked immunosorbent assay (ELISA) kits were used to determine concentrations of FGF3 (US Biological), FGF4 (Thomas Scientific), FGF7 (Thomas Scientific), FGF8 (Thomas Scientific), FGF9 (Abcam), FGF16 (Thomas Scientific), and FGF18 (US Biological).

Techniques: Luminex, Enzyme-linked Immunosorbent Assay, Control, Incubation

Journal: Science translational medicine

Article Title: Steroid-induced fibroblast growth factors drive an epithelial-mesenchymal inflammatory axis in severe asthma

doi: 10.1126/scitranslmed.abl8146

Figure Lengend Snippet: (A) Normal human lung fibroblasts were cultured in 3D collagen-rich rafts, as described in Methods. Cells from five different donors were stimulated with a cocktail of FGF1, FGF2, FGF4, and FGF18, all at 10 ng/ml, for 24 hours before RNA isolation for RNA-seq. Differentially expressed genes (DEGs) where P < 0.05 are displayed. nRPKM, normalized reads per kilobase of transcript per million mapped reads. (B) Expression, in RPKM, of select DEGs is shown. *P < 0.05 by a paired t test. (C) The experimental setup depicts BAEC-ALI stimulation with F.p. (10−7 M) or diluent control for 24 hours and transfer of basal supernatant, mixed 50:50 with fresh medium, to fibroblast cultures for 24 hours. (D and E) Expression of CSF3 (D) and HAS2 (E) was measured in fibroblasts after exposure to supernatant from BAEC-ALI, as described above. Media, 100% fresh medium; Cont. Sup., 50% medium from diluent-exposed BAEC-ALI with 50% fresh medium; F.p. Sup., 50% medium from F.p.-exposed (10−7 M) BAEC-ALI with 50% fresh medium. Erdafitinib (Erd; 100 nM) was added, as indicated, to the fibroblast cultures. Data are expressed as fold change (FC) from fibroblasts cultured with medium alone. (F to H) F.p.-exposed (10−7 M) precision-cut lung slices were incubated in the presence (100 nM) or absence (diluent control) of erdafitinib. Three precision-cut lung slices were incubated per well for 24 hours, with three technical replicates per experiment. One of three experiments is shown. IL-8 (F), G-CSF (G), and hyaluronan (H) concentrations were measured by Luminex or ELISA. Error bars show SEM. *P < 0.05 with an unpaired t test.

Article Snippet: Enzyme-linked immunosorbent assay (ELISA) kits were used to determine concentrations of FGF3 (US Biological), FGF4 (Thomas Scientific), FGF7 (Thomas Scientific), FGF8 (Thomas Scientific), FGF9 (Abcam), FGF16 (Thomas Scientific), and FGF18 (US Biological).

Techniques: Cell Culture, Isolation, RNA Sequencing, Expressing, Control, Incubation, Luminex, Enzyme-linked Immunosorbent Assay

Journal: Science translational medicine

Article Title: Steroid-induced fibroblast growth factors drive an epithelial-mesenchymal inflammatory axis in severe asthma

doi: 10.1126/scitranslmed.abl8146

Figure Lengend Snippet: C57BL/6 mice were treated with 10 μg of HDM in 25 μl by the intratracheal route on days 0, 2, and 4. On days 14, 16, 18, 28, and 30, mice were given 10 μg of HDM + F.p. (1 mg/kg) with erdafitinib (30 mg/kg) or diluent control (−, HP-β-CD), as indicated. (A) Total and differential cell analysis of airway infiltrates in BAL. (B) BAL cytokines and hyaluronan were measured by Luminex and ELISA, respectively. Error bars show SEM. *P < 0.05 with Mann-Whitney test. (C and D) Lung inflammation severity (C) and airway epithelial goblet cell hyperplasia (GCH) severity (D) scores were determined by an anatomic pathologist, in a blinded manner, from hematoxylin and eosin– and Alcian blue/periodic acid–Schiff–stained tissue sections, respectively. Scale bars, 100 μm. Severity scores were compared by Kruskal-Wallis with Dunn’s multiple comparisons test. (E) Serum cytokines were measured by Luminex. A minimum of four mice was used per group, with one of two experiments shown. Error bars show SEM. *P < 0.05 with Mann-Whitney test.

Article Snippet: Enzyme-linked immunosorbent assay (ELISA) kits were used to determine concentrations of FGF3 (US Biological), FGF4 (Thomas Scientific), FGF7 (Thomas Scientific), FGF8 (Thomas Scientific), FGF9 (Abcam), FGF16 (Thomas Scientific), and FGF18 (US Biological).

Techniques: Control, Cell Analysis, Luminex, Enzyme-linked Immunosorbent Assay, MANN-WHITNEY, Staining

Journal: Molecular Medicine Reports

Article Title: Pretreatment of BMSCs with TZD solution decreases the proliferation rate of MCF-7 cells by reducing FGF4 protein expression

doi: 10.3892/mmr.2016.4959

Figure Lengend Snippet: Levels of (A) FGF4, (B) CCL2, (C) CCL5, (D) IL-6, (E) VEGF and (F) TGF-β in the conditioned medium of BMSCs pretreated with pioglitazone and/or rosiglitazone. The experimental conditions were: conditioned medium of BMSCs, conditioned medium of BMSCs pretreated with DMSO, conditioned medium of BMSCs pretreated with 40 µ M pioglitazone, conditioned medium of BMSCs pretreated with 20 µ M pioglitazone + 20 µ M rosiglitazone, and conditioned medium of BMSCs pretreated with 40 µ M rosiglitazone. The levels of these soluble growth factors in the conditioned medium were assayed by enzyme-linked immunosorbent assay. The levels of soluble growth factors in the conditioned medium of BMSCs pretreated with DMSO were defined as 100%. Data are expressed as the means ± standard deviation from three separate experiments. One-way analysis of variance was used to compare the levels of soluble growth factor in BMSCs pretreated with DMSO only (control) with the BMSCs pretreated with the thiazolidinedione(s). * P<0.05, *** P<0.001 compared with the control. FGF4, fibroblast growth factor 4; CCL2/5, chemokine (C-C motif) ligand-2/5; IL-6, interleukin-6; VEGF, vascular endothelial growth factor; TGF-β, transforming growth factor-β; BMSCs, bone marrow-derived mesenchymal stem cells; DMSO, dimethylsulfoxide.

Article Snippet: The levels of FGF4, CCL2, CCL5, IL-6, vascular endothelial growth factor (VEGF) and transforming growth factor β (TGFβ) in the conditioned medium of BMSCs pretreated with pioglitazone and/or rosiglitazone were determined using RayBio ® enzyme-linked immunosorbent assay (ELISA) kits (RayBiotech, Inc., Norcross, GA, USA).

Techniques: Enzyme-linked Immunosorbent Assay, Standard Deviation, Derivative Assay