Article Figures & Data
Figures
- Figure 1. OptoR2 is functional in HEK 293T cells.
(A) Schematic representation of endogenous FGFR2b and OptoR2. AB, acid box; HSPG, heparan sulphate proteoglycan (HSPG); HA, hemagglutinin epitope; Ig, immunoglobulin-like domain; LOV, light-oxygen-voltage domain; Myr, myristoylation signal; TK, tyrosine kinase domains; TM, transmembrane domain. (B, C) OptoR2 HEK 293T cells and vector-transduced control cells (ctrl) were serum-starved for 16 h. Subsequently, they were left untreated (black bar), or exposed 15 min to blue light (blue bar), or FGF7, FGF10, or EGF (10 ng/ml). (B) Western blot for phospho-ERK1/2 (p-ERK T202/Y204), total ERK1/2, HA (OptoR2), and GAPDH (loading control). (C) Immunofluorescence staining for p-ERK1/2 (green). Nuclei were counterstained with Hoechst 33342 (blue). Note that the single channels are shown in black and white; colours are only shown in the merge. Scale bar: 250 μm. (D, E) OptoR2 HEK 293T cells were serum-starved for 16 h and then illuminated for 15 min with light of different wavelengths (D) or for 1 s to 1 min with blue light (E). For (E), lysates were prepared 2 min after the onset of illumination. Western blots for p-ERK, total ERK1/2, HA (OptoR2), and GAPDH are shown. (F) OptoR2-transduced HEK 293T cells were cultured in medium with 0.5% FBS for 16 h, subjected to scratch wounding, and illuminated with blue light, or treated with FGF7, FGF10 (10 ng/ml), or 10% FBS. Bar graph shows percentage of open scratch at 48 h. (G) OptoR2-transduced HEK 293T cells were serum-starved for 16 h and then illuminated for either 1 or 24 h or treated with FGF7, FGF10 (10 ng/ml), or 10% FBS for 24 h. At 24 h they were analysed by immunofluorescence staining for Ki67. The percentage of Ki67-positive cells is shown. Bar graphs show mean ± SEM. (B, D, E) Representative of four experiments. (C): Representative pictures of two experiments. (F): n = 9–45 from three experiments. (G) n = 18–27 from two experiments. *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001, ****P ≤ 0.0001 (Mann–Whitney U test).
Source data are available for this figure.
Source Data for Figure 1[LSA-2021-01100_SdataF1.1.pdf][LSA-2021-01100_SdataF1.2.xlsx][LSA-2021-01100_SdataF1.3.xlsx]
- Figure S1. Blue light activates FGFR signaling pathways in OptoR2-expressing HEK 293T cells.
(A) RNA samples from vector-transfected HEK 293T and HaCaT cells were analysed by qRT-PCR for FGFR2 relative to RPL27. Expression levels in HaCaT cells were set to one. (B) Ctrl and OptoR2-transduced HEK 293T cells were serum-starved for 16 h and then illuminated with blue light or incubated with FGF7 or FGF10 (10 ng/ml) for 2, 5, or 10 min. Protein lysates were analysed by Western blot for OptoR2 (HA), GAPDH and total and phosphorylated forms of different signaling molecules. (C) After serum-starvation for 16 h, OptoR2 and ctrl HEK 293T cells were illuminated with blue light or incubated with FGF7 or FGF10 (10 ng/ml) for 5, 15, 30, 60, or 180 min. Protein lysates were analysed by Western blot for p-ERK1/2 (T202/Y204), total ERK1/2, HA (OptoR2), and GAPDH. (A) Expression level in HaCaT cells was set to one. One experiment, duplicate determination. (B) Representative of two experiments. (C) Representative of four experiments.
- Figure 2. OptoR2 expression is functional in murine keratinocytes, but down-regulated over time.
(A) Construct used for the generation of the transgenic mice. Functional elements include a human keratin 14 (K14) promoter, a rabbit β-globin intron, the OptoR2 cDNA and the human growth hormone poly A. (B, C) Primary keratinocytes from one newborn ctrl and two OptoR2 mice (transgenic mouse line 1) from the direct offspring of the founder mice or from progeny 1.5 yr (3 generations) later were serum-starved for 24 h and then left untreated or exposed to blue light or FGF7 or FGF10 (10 ng/ml) for 15 min. (B) Western blot for p-ERK1/2 (T202/Y204), ERK1/2, HA (OptoR2), and GAPDH. Note that different exposure times were used for the HA blots in the left and right panels. (C) Different batches of untreated primary keratinocytes were analysed by qRT-PCR for OptoR2 relative to Rps29 and by Western blot for HA (OptoR2) and GAPDH. (D) Immortalized keratinocytes from K14-OptoR2 mice and a wild-type littermate (ctrl) were serum-starved for 24 h and exposed to blue light or FGF7 or FGF10 (10 ng/ml) for 15 min, both at the beginning of the cultivation period (t = 0) and after extended cultivation and passaging (t = 6 mo). Western blots for p-ERK1/2, total ERK1/2, HA (OptoR2), and GAPDH are shown. (E) Confluent and serum-starved immortalized keratinocytes were subjected to scratch wounding and exposed to blue light or FGF7, FGF10 (10 ng/ml), or full keratinocyte growth medium. The percentage of open scratch at 24 or 48 h is shown in the bar graphs. Bar graphs show mean ± SEM. (B) Representative of four experiments. t = 0: n = 4 mice per genotype. t = 1.5 yr: n = 12–18 mice per genotype. (C) Ctrl littermate expression level was set to one. One experiment, n = 2–3 mice per genotype. (D) Representative of two experiments with three cell lines. (E) t = 0: n = 24 from two experiments. t = 6 mo: n = 20 from three experiments. **** ≤ 0.0001 (Mann-Whitney U-test).
Source data are available for this figure.
Source Data for Figure 2[LSA-2021-01100_SdataF2.1.pdf][LSA-2021-01100_SdataF2.2.xlsx][LSA-2021-01100_SdataF2.3.xlsx]
- Figure S2. Illumination of cells with low OptoR2 expression or after long cultivation time neither activates signaling nor promotes migration.
(A) Primary keratinocytes from newborn ctrl and OptoR2 mice (transgenic mouse lines 2 and 3, two different batches each) were serum-starved for 24 h and illuminated with blue light or treated with FGF7 or FGF10 (10 ng/ml) for 15 min. Protein lysates were analysed by Western blot for p-ERK1/2 (T202/Y204), ERK1/2, HA (OptoR2), and GAPDH. The line indicates a place where the gel was broken. (B) Confluent immortalized murine keratinocytes from K14-OptoR2 mice were serum-starved for 24 h, subjected to scratch wounding and illuminated with blue light or treated with FGF7, FGF10 (10 ng/ml), or full keratinocyte growth medium. The area of open scratch was measured with the “MRI wound healing tool” in ImageJ or manually in pictures taken at the same spot at 0 and 24 h and is shown as percentage of ctrl (dark) in the bar graph. Bar graphs show mean ± SEM. (A) Representative of three experiments. Transgenic mouse line 2: n = 3 mice per genotype. Transgenic mouse line 3: n = 6–8 mice per genotype. (B) OptoR2 cell line 2: n = 24 from one experiment. *P ≤ 0.05, **P ≤ 0.01 (Mann–Whitney U test).
- Figure 3. OptoR2 expression in keratinocytes of transgenic mice induces skin tumour formation, which declines over time.
(A) Left upper panel: Adult K14-OptoR2 mouse with a skin tumour. Other panels: Representative photomicrographs from skin tumour sections; hematoxylin & eosin staining (top right), Ki67 immunohistochemistry and counterstaining with hematoxylin (bottom left), or immunofluorescence staining for HA (OptoR2; green) and the endothelial cell marker CD31 (red) (bottom right). Nuclei were counterstained with Hoechst 33342 (blue). Scale bar: 100 μm. Representative pictures of n = 11 mice are shown. (B) Tumour incidence in adult K14-OptoR2 mice of both sexes during the first and second year after generation of the mouse line. Number of mice with tumours and of total mice is indicated in the graph.
Source data are available for this figure.
Source Data for Figure 3[LSA-2021-01100_SdataF3.1.pdf][LSA-2021-01100_SdataF3.2.xlsx]
- Figure S3. Activation of OptoR2 in human primary fibroblasts.
(A) Schematic representation of the construct used for the inducible overexpression of OptoR2 in HaCaT cells. Functional elements include long terminal repeats (LTR), tetracycline response element (TRE), cytomegalovirus (CMV) and ubiquitin C (UbC) promoter, reverse tetracycline transactivator (rtTA), internal ribosomal entry site (IRES), neomycin resistance gene (NeoR), and Woodchuck Hepatitis Virus (WHP) posttranscriptional regulatory element (WPRE). (B) OptoR2-transduced human primary fibroblasts were serum-starved and treated with 50 ng/ml Dox or DMSO for 48 h. Then, they were left untreated, illuminated with blue light or treated with FGF1 or FGF2 (10 ng/ml) for 2, 5, or 10 min. Western blots for OptoR2 (HA), GAPDH, and total and phosphorylated forms of different signaling molecules are shown. Similar results were obtained in an independent experiment.
- Figure 4. Activation of inducibly expressed OptoR2 in human keratinocytes mimics FGFR2 signaling.
HaCaT cells were serum-starved and treated with 50 ng/ml Dox or DMSO (vehicle, -Dox) for 24 h. (A) OptoR2- and vector-transduced (ctrl) HaCaT cells were then left untreated, illuminated or treated with FGF7 or FGF10 (10 ng/ml) for 15 min. Western blots for p-ERK1/2, total ERK1/2, HA (OptoR2), and GAPDH. (B) Immunofluorescence staining for HA (OptoR2; red) and the membrane protein E-cadherin (green), counterstained with Hoechst 33342 (blue). Single channels are shown in black and white; colours are only shown in the merge. Scale bar: 10 μm. (C, D) OptoR2-HaCaT cells were illuminated with blue light or treated with FGF7 or FGF10 (10 ng/ml) for 5, 15, 30, 60, or 180 min (C) or for 2, 5, or 10 min (D). Western blots for OptoR2 (HA), GAPDH, and total and phosphorylated forms of different signaling molecules are shown. (A) Representative of six experiments. (B) Representative confocal pictures of two experiments. (C) Representative of two experiments. (D) Representative of six experiments.
Source data are available for this figure.
Source Data for Figure 4[LSA-2021-01100_SdataF4.pdf]
- Figure 5. Activation of OptoR2 in human keratinocytes induces expression of FGF target genes and promotes cell migration.
OptoR2-transduced HaCaT cells were serum-starved and treated with 50 ng/ml Dox or DMSO for 24 h. (A) Cells were left untreated, illuminated with blue light (“1 min on/15 min off” [1/15] cycles, “1 min on/60 min off” [1/60] cycles, continuous illumination) or treated with FGF7 or FGF10 (10 ng/ml) for 6 h. RNA samples were analysed by qRT-PCR for DUSP6, HBEGF, and VEGF relative to RPL27. (B) Cells were subjected to scratch wounding and illuminated using three different illumination schemes or incubated with 10 ng/ml FGF7 or FGF10. The percentage area of open scratch at 72 h is shown in the bar graph. (C) Cells were illuminated for 15 min, 3 h or 24 h using three different illumination schemes and analysed by Western blot for HA (OptoR2) and GAPDH. Bar graphs show mean ± SEM. (A) “dark − Dox” expression level was set to one. n = 4–7 from two to five experiments. (B) n = 36 from two experiments. (C) Representative of three experiments. *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001, **** ≤ 0.0001 (Mann–Whitney U test).
Source data are available for this figure.
Source Data for Figure 5[LSA-2021-01100_SdataF5.1.xlsx][LSA-2021-01100_SdataF5.2.xlsx][LSA-2021-01100_SdataF5.3.pdf]
- Figure S4. Activation of OptoR2 in HaCaT cells promotes migration and FGF target gene expression, but also OptoR2 down-regulation.
(A) Representative pictures of the scratch wounding experiment shown in Fig 5B. (B, C) OptoR2-transduced HaCaT cells were serum-starved and treated with 50 ng/ml Dox for 24 h and then illuminated with blue light (three different illumination schemes) for 3, 6, or 24 h (B, C) or treated with FGF7 or FGF10 (C). RNA samples were analysed for expression of OptoR2 and DUSP6 relative to RPL27 by qRT-PCR with the respective “dark + Dox” values at the different time points set to one. Shown in (B) are the mean ± SEM mRNA levels of stimulated cells after illumination for different periods based on the results shown as bar graphs in Fig 5A (DUSP6 after 6 h) and (C) (DUSP6 after 3 and 24 h, OptoR2 all time points). CT values for the housekeeping gene RPL27, which was used for normalization, is shown in (C), bottom panel. Bar graphs show mean ± SEM. (A) Representative pictures of two experiments. (B, C) “dark + Dox” expression level was set to one. n = 3–7 from 3 to 5 experiments. **P ≤ 0.01 (Mann–Whitney U test).
Tables
List of primary antibodies.
Antigen Host Dilution Application Catalogue no. Manufacturer AKT Rabbit 1:1,000 Western blot 9272 Cell Signaling Technologies AKT (phospho T308) Rabbit 1:1,000 Western blot 9275 Cell Signaling Technologies AKT (phospho S473) Rabbit 1:1,000 Western blot 3787 Cell Signaling Technologies CD31 Rat 1:100 Immunofluorescence 553370 BD Biosciences E-Cadherin Mouse 1:100 Immunofluorescence 610181 BD Biosciences ERK Rabbit 1:1,000 Western Blot 9102 Cell Signaling Technologies ERK (phospho T202/Y204) Rabbit 1:100 Immunofluorescence 9101 Cell Signaling Technologies 1:1,000 Western blot FGFR2 (phospho Y769) Rabbit 1:1,000 Western blot PA5-105880 Thermo Fisher Scientific FRS2α (phospho Y196) rabbit 1:1.000 Western blot 3864 Cell Signaling Technologies FRS2α (phospho Y436) Rabbit 1:1,000 Western blot 3861 Cell Signaling Technologies GAPDH Mouse 1:5,000 Western blot 5G4cc HyTest HA (OptoR2) Rabbit 1:100 Immunofluorescence 3724 Cell Signaling Technologies 1:1,000 Western blot Ki67 Rabbit 1:500 Immunohistochemistry, Immunofluorescence ab16667 Abcam p38 Rabbit 1:1,000 Western blot 9212 Cell Signaling Technologies p38 (phospho T180/Y182) Rabbit 1:1,000 Western blot 9211 Cell Signaling Technologies PLCγ (phospho S1248) Rabbit 1:1,000 Western blot 4510 Cell Signaling Technologies List of secondary antibodies.
Antigen species Coupled with Dilution Application Catalogue no. Manufacturer Mouse Cy2 1:200 Immunofluorescence 115-225-003 Jackson ImmunoResearch Mouse HRP 1:5,000 Western blot W4021 Promega Rabbit Biotin 1:500 Immunohistochemistry 111-065-003 Jackson ImmunoResearch Rabbit Cy2 1:200 Immunofluorescence 111-225-144 Jackson ImmunoResearch Rabbit Cy3 1:200 Immunofluorescence 111-165-003 Jackson ImmunoResearch Rabbit HRP 1:5,000 Western blot W4011 Promega Rat Cy3 1:200 Immunofluorescence 712-165-153 Jackson ImmunoResearch List of PCR primers.
Gene Sequence Application DUSP6 (human) 5′-GTT CTA CCT GGA AGG TGG CT-3′ qRT-PCR 5′-AGT CCG TTG CAC TAT TGG GG-3′ FGFR2 (human) 5′-AGC TGG GGT CGT TTC ATC TG-3′ qRT-PCR 5′-TTG GTT GGT GGC TCT TCT GG-3′ HBEGF (human) 5′-TTA GTC ATG CCC AAC TTC ACT TT-3′ qRT-PCR 5′-ATC GTG GGG CTT CTC ATG TTT-3′ OptoR2 5′-TGA CCA GCA GCT TGG CAT AA-3′ Genotyping PCR 5′-GCT CTG CAA ATG GCA CAA CA-3′ 5′-GCT CTG CAA ATG GCA CAA CA-3′ qRT-PCR 5′-TGA CCA GCA GCT TGG CAT AA-3′ RPL27 (human) 5′-TCA CCT AAT GCC CAC AAG GTA-3′ qRT-PCR 5′-CCA CTT GTT CTT GCC TGT CTT-3′ Rps29 (mouse) 5′-GGT CAC CAG CAG CTC TAC TG-3′ qRT-PCR 5′-GTC CAA CTT AAT GAA GCC TAT GTC C-3′ VEGF (human) Hs_VEGFA_6_SG, QuantiTect Primer Assay (QT01682072) qRT-PCR PCR protocol for genotyping.
Cycles Time Temperature 1× 5 min 95°C 33× 35 s 95°C 35 s 60°C 50 s 72°C 1× 10 min 72°C Store at 8°C qPCR protocol.
Cycles Time Temperature 1× 10 min 95°C 50× 10 s 95°C 20 s 60°C 20 s 72°C 1× 5 s 95°C 1× 1 min 65°C 1× Slow heat 95°C Store at 40°C Protocol for H&E staining.
Cycles Solution Time 1× Mayer’s Hematoxylin solution 3 min 3× ddH2O 10 s 1× Scott water 30 s 1× ddH2O 10 s 1× 70% (v/v) ethanol/ddH2O 10 s 1× Eosin solution 1 min 2× 80% (v/v) ethanol/ddH2O 10 s 2× 95% (v/v) ethanol/ddH2O 10 s 2× Ethanol 10 s 2× Xylene 10 min Slides were finally mounted with Eukitt and air-dried
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