Journal: bioRxiv

Article Title: Non-canonical Wnt signaling triggered by WNT2B drives adrenal aldosterone production

doi: 10.1101/2024.08.23.609423

Figure Lengend Snippet: a. HEK293 cells stably expressing NL-WNT2B were incubated with 1μM of purified SFRP2, WIF1, GPC4 or GPC6 ectodomains in serum-free media. NL-WNT2B release was measured at various time points by NanoLuc luciferase (NL) luminescence. Bovine serum albumin (BSA) served as negative control. WNT2B is released mainly by SFRP2, GPC4 and GPC6. Data represent the mean of two biological replicates, normalized to total NL-WNT in lysates, and error bars show SD. b. R-Spondin 3 (RSPO3; 0, 25, 100, 200 and 400ng/ml) or purified WNT3A-GPC4 complex (0, 0.01, 0.03, 0.1, 0.3 and 1μM with respect to WNT3A) with or without RSPO3 (400ng/ml) was added to Wnt reporter cells. After 24h, Wnt pathway activity was measured by luciferase assay. Incubation with BSA served as negative control. RSPO3 does not potentiate WNT3A-GPC4 activity. Points represent average activation for two biological replicates, normalized to untreated cells, and error bars represent SD. See also for protein purification and activity of WNT5A-GPC4 complex and WNT3A-carrier or WNT2B-carrier conditioned media. c. As in (b), but with purified WNT2B-GPC4 complex. WNT2B-GPC4 complex is unable to activate canonical Wnt signaling, even with RSPO3. d. As in (b), but purified WNT3A-SFRP2 complex (1μM) was mixed with varying amounts of GPC4 alone or in complex with WNT3A, WNT5A or WNT2B (0.1, 0.3 and 1μM). Both WNT5A-GPC4 and WNT2B-GPC4 complexes abolish WNT3A-SFRP2 activity, in contrast to GPC4 alone or in complex with WNT3A. See for a similar experiment using WNT3A-GPC4 complex. e. NL-WNT2B-SFRP2 complex was covalently captured on HaloLink beads from conditioned media, via HT7 fused to the C-terminus of SFRP2. The beads were then incubated with purified FZD-CRDs (5µM) and NL-WNT2B release was measured at different time points by NL luminescence. Incubation with BSA (5µM) served as negative control. WNT2B is preferentially transferred to FZD3-CRD and FZD6-CRD more than FZD8-CRD. Points represent average for two biological replicates, normalized by total NL-WNT on beads, and error bars represent SD. f. As in (e), but with NL-WNT2B-GPC4 on beads. g. Purified WNT2B-SFRP2 (5μM) was incubated with the extracellular domain (ECD) of ROR2 (2.5μM), followed by immunoprecipitation with antibodies against the FLAG tag attached to ROR. Samples were analyzed by SDS-PAGE and immunoblotting. WNT2B-SFRP2 complex interacts with ROR2-ECD. See also for protein purification and a similar experiment using purified SFRP2. h. As in (g), but with purified WNT5A-SFRP2 complex. WNT5A-SFRP2 complex binds to ROR2-ECD. i. As in (g), but with purified WNT3A-SFRP2 complex. WNT3A-SFRP2 complex does not bind to ROR2-ECD. j. As in (g), but WNT2B-SFRP2 complex (5μM) was incubated with ROR1-ECD (2.5μM). WNT2B-SFRP2 does not bind to ROR1-ECD. k. As in (j), but with WNT5A-SFRP2 complex. WNT5A-SFRP2 does not bind to ROR1-ECD. l. As in (j), but with WNT3A-SFRP2 complex. WNT3A-SFRP2 does not bind to ROR1-ECD. m. As in (g) but using SFRP2 alone. SFRP2 is unable to interact with ROR2-ECD. n. Activity of RhoA in FZD( – ) KO cells expressing FZD3, FZD6 or FZD7 was assessed by Rhotekin-RBD pull-down assay after 6h of treatment with GPC4 alone or in complex with WNT2B (2μM). RhoA endogenous levels are shown in the lysates. RhoA activity by WNT2B-GPC4, in contrast to GPC4 alone, is rescued in cells expressing FZD3 or FZD6, but not the canonical FZD7. Blotting for α-tubulin served as loading control. o. As in (n), but measuring activity of RhoA in ROR( – ) KO cells expressing ROR1 or ROR2. WNT2B-GPC4, in contrast to GPC4 alone, activates RhoA only when ROR2 expression is rescued, not ROR1. Smoothened (SMO) transfection served as negative control.

Article Snippet: After washing the beads three times with 2mM CaCl and 0.2% DDM, bound proteins were eluted in elution buffer (20mM HEPES, pH 7.5; 200mM NaCl; 5mM EDTA; 100μg/mL FLAG or HPC peptide) and were analyzed by SDS-PAGE followed by immunoblotting using rabbit monoclonal or polyclonal antibodies against WNT3A (Cell Signaling, #2721S), WNT5A/B (Cell Signaling, #2530S) or, WNT2B (Abcam, #ab203225), and anti-mouse monoclonals against FLAG-M1 and anti-HPC, a generous gift from Andrew C Kruse (Harvard Medical School).

Techniques: Stable Transfection, Expressing, Incubation, Purification, Luciferase, Negative Control, Activity Assay, Activation Assay, Protein Purification, Immunoprecipitation, FLAG-tag, SDS Page, Western Blot, Pull Down Assay, Control, Transfection

Journal: bioRxiv

Article Title: Non-canonical Wnt signaling triggered by WNT2B drives adrenal aldosterone production

doi: 10.1101/2024.08.23.609423

Figure Lengend Snippet: a. WNT2B-GPC4 ectodomain, C-terminally tagged with HaloTag7 (HT7) and HPC tag, was affinity purified from conditioned media on an anti-HPC antibody matrix, and analyzed by SDS-PAGE, followed by Coomassie staining or anti-WNT2B immunoblotting (WB). Arrowhead indicates unmodified GPC4, bracket indicates glycosaminoglycan (GAG)-modified species, and asterisks indicate WNT2B protein. b. As in (a), but with WNT5A in complex with GPC4, and with anti-WNT5A immunoblotting. c. R-Spondin 3 (RSPO3, 0, 25, 100, 200 and 400ng/ml) or purified WNT5A-GPC4 complex (0.01, 0.03, 0.1, 0.3 and 1μM with respect to WNT3A) with or without RSPO3 (400ng/ml) was added to Wnt reporter cells. After 24h, Wnt pathway activity was measured by luciferase assay. Incubation with BSA served as negative control. WNT5A-GPC4 does not activate canonical Wnt signaling, even when incubated with RSPO3. Points represent average activation for two biological replicates, normalized to untreated cells, and error bars represent SD. d. SFRP2 (1μM) was added in serum-free media in WNT3A- or WNT2B-expressing HEK293 cells. Serial dilutions of the conditioned media were then added to Wnt reporter cells, and Wnt pathway activity was measured by Dual-Glo luciferase 24h later. BSA (1μM) served as negative control. WNT2B released by SFRP2 is unable to activate canonical Wnt signaling, in contrast to WNT3A-SFRP2 conditioned media. Points represent average activation for two biological replicates, normalized to the negative control, and error bars represent SD. e. As in (d), but WNT-expressing cells were incubated with 1μM of GPC4. f. As in , but purified WNT3A-GPC4 complex (1μM) was mixed with the indicated concentrations of GPC4 alone or in complex with WNT3A, WNT5A or WNT2B. WNT3A-SFRP2 activity is abolished by WNT5A-GPC4 and WNT2B-GPC4 complexes in a dose-dependent manner, which contrasts GPC4 alone or WNT3A-GPC4 complex. g. Extracellular domains (ECD) of ROR1 and ROR2, N-terminally tagged with a FLAG tag, were affinity purified from conditioned media on an anti-FLAG antibody matrix. Purified proteins were analyzed by SDS-PAGE and Coomassie staining. h. As in (a), but with WNT2B in complex with SFRP2, C-terminally tagged with 8x-His tag and HPC taI. i. As in (b), but with WNT5A in complex with SFRP2. j. Purified SFRP2 (5μM) was incubated with FLAG-tagged ROR1-ECD (2.5μM), followed by immunoprecipitation with antibodies against the FLAG tag. Samples were analyzed by SDS-PAGE and immunoblotting. SFRP2 does not interact with ROR1-EcD. k. Activity of RhoA in cell lysates of HEK293 cells treated for 6h with GPC4 alone or in complex with WNT3A, WNT5A or WNT2B (2μM) was assessed by Rhotekin-RBD pull-down assay. RhoA endogenous levels are shown in the lysates. Both WNT5A-GPC4 and WNT2B-GPC4 complexes induce activity of RhoA, in contrast to GPC4 alone or in complex with WNT3A. Blotting for α-tubulin served as loading control. l. HEK293 cells were co-transfected with the firefly luciferase reporter (pGL4.34) and the renilla luciferase thymidine kinase reporter (pRL-TK). They were then used to assay RhoA activation by purified GPC4 alone or in complex with WNT3A, WNT5A, and WNT2B (1 μM). We found that the activity of RhoA is induced by WNT2B-GPC4 or WNT5A-GPC complexes, but not by WNT3A-GPC4 or GPC4 alone. The bars represent the average from three independent experiments performed in duplicate, normalized to untreated cells. Statistical significance was determined using one-way ANOVA with Tukey’s post-test (ns, not significant; **p < 0.01; ***p < 0.001). Data are represented as mean ± SEM.

Article Snippet: After washing the beads three times with 2mM CaCl and 0.2% DDM, bound proteins were eluted in elution buffer (20mM HEPES, pH 7.5; 200mM NaCl; 5mM EDTA; 100μg/mL FLAG or HPC peptide) and were analyzed by SDS-PAGE followed by immunoblotting using rabbit monoclonal or polyclonal antibodies against WNT3A (Cell Signaling, #2721S), WNT5A/B (Cell Signaling, #2530S) or, WNT2B (Abcam, #ab203225), and anti-mouse monoclonals against FLAG-M1 and anti-HPC, a generous gift from Andrew C Kruse (Harvard Medical School).

Techniques: Affinity Purification, SDS Page, Staining, Western Blot, Modification, Purification, Activity Assay, Luciferase, Incubation, Negative Control, Activation Assay, Expressing, FLAG-tag, Immunoprecipitation, Pull Down Assay, Control, Transfection

Journal: Advanced Functional Materials

Article Title: A Bio‐Functional Wnt3a Gradient Microarray for Cell Niche Studies

doi: 10.1002/adfm.202301941

Figure Lengend Snippet: Figure 1. Scheme of the fabrication of Wnt3a gradient using multiphoton microfabrication and micropatterning (MMM) technology for reconstituting human hair dermal papilla niche.

Article Snippet: Subsequently, the rabbit polyclonal anti-Wnt3a antibody (ab28472, Abcam) solution (diluted at 1:100 with diluting buffer (PBS (1×) supplemented with 1% (w/v) BSA and 0.1% (v/v) Tween-20) was applied and allowed for incubating at room temperature for 1 h, followed by incubating with the Alexa Fluor Plus 647 conjugated goat-anti rabbit secondary antibody (A32733, Invitrogen, Waltham, MA, USA) (diluted at a 1:2500 with diluting buffer) in darkness at room temperature for another 1 h. Last, the immunofluorescence signal was measured by the CLSM (TCS-SP8, Leica Microsystems) using a diode laser and a 40× lens at an excitation wavelength of 638 nm and a detection wavelength range of 649–689 nm.

Techniques:

Journal: Advanced Functional Materials

Article Title: A Bio‐Functional Wnt3a Gradient Microarray for Cell Niche Studies

doi: 10.1002/adfm.202301941

Figure Lengend Snippet: Figure 3. Characterization, optimization, and bioactivity verification of biotinylated Wnt3a. A) Workflow of biotinylating Wnt3a and characterization. B) Measurement of the level of biotin labeling (LOL) of the biotinylated Wnt3a showing a linear (R2 = 0.992) controllability of molar ratio of NHS- PEG4-Biotin to Wnt3a in the biotinylation reaction over the level of biotin conjugated on the Wnt3a molecules. n = 2 in two independent experiments. C) Verification of the successfulness of biotinylation of Wnt3a by measuring the optical density (OD) value of the NA surface bound biotinylated Wnt3a (bWnt3a) with different LOLs via in-direct ELISA performed on the commercialized NA-coated microwell plate. n = 2 in two independent experiments. D) Verification of the bioactivity of Wnt3a after biotinylation by evaluating the nuclear translocation of β-catenin in mouse L cells upon addition of either non-biotinylated Wnt3a (fWnt3a) or bWnt3a with LOL of 12 at a serial of ascending concentrations (0, 100, 200, 400, 800, and 1000 ng mL−1) in the culture medium for 24 h by immunofluorescence staining. Representative immunofluorescence images of β-catenin nuclear translocation (denoted by white arrow heads) in mouse L cells triggered by fWnt3a i) or bWnt3a ii) (scale bar: 50 µm and 10 µm for large images and close-up images, respec- tively), and quantitative image analysis of the fluorescence intensity of the nuclear β-catenin upon treatment with fWnt3a or bWnt3a iii). n ≧ 100 cells in each group in two independent experiments. The differences of means of the logarithm of intensity of nuclear β-catenin among multiple groups were analyzed by two-way ANOVA with Bonferroni’s post-hoc tests. ****p < 0.0001; ns: not significant (p > 0.05).

Article Snippet: Subsequently, the rabbit polyclonal anti-Wnt3a antibody (ab28472, Abcam) solution (diluted at 1:100 with diluting buffer (PBS (1×) supplemented with 1% (w/v) BSA and 0.1% (v/v) Tween-20) was applied and allowed for incubating at room temperature for 1 h, followed by incubating with the Alexa Fluor Plus 647 conjugated goat-anti rabbit secondary antibody (A32733, Invitrogen, Waltham, MA, USA) (diluted at a 1:2500 with diluting buffer) in darkness at room temperature for another 1 h. Last, the immunofluorescence signal was measured by the CLSM (TCS-SP8, Leica Microsystems) using a diode laser and a 40× lens at an excitation wavelength of 638 nm and a detection wavelength range of 649–689 nm.

Techniques: Labeling, Direct ELISA, Translocation Assay, Immunofluorescence, Staining, Fluorescence

Journal: Advanced Functional Materials

Article Title: A Bio‐Functional Wnt3a Gradient Microarray for Cell Niche Studies

doi: 10.1002/adfm.202301941

Figure Lengend Snippet: Figure 4. Characterization of Wnt3a gradient. A) Scheme of Wnt3a gradient formation and characterization. B) Immunofluorescence images of arma- dillo shaped Wnt3a gradient generated by applying biotinylated Wnt3a (bWnt3a) (LOL = 12, 50 ng, upper row), non-biotinylated Wnt3a (fWnt3a) (50 ng, middle row) and PBS (lower row) to the NA gradient fabricated by increasing laser power (18, 27, 36, and 45 Mw) with constant number (5) of scan cycle. Scale bar: 20 µm. C) Quantification of the fluorescence intensity of NA-bound bWnt3a (LOL = 12) squares i) and NA-bound fWnt3a squares ii) showing a non-linear controllability of amount of bWnt3a iii) or fWnt3a iv) input over the fluorescence intensity of NA-bound Wnt3a at a fixed laser power used for micropatterning NA. Scale bar: 10 µm. n = 4 in two independent experiments. D) Estimation of the maximal surface density of Wnt3a gradient. A calibration curve ii) was derived from a plot of immunofluorescence intensity of NA-bound bWnt3a (LOL = 12) against a serial of known ascending maximal density of NA-bound bWnt3a (i). Measurement of the immunofluorescence intensity of bWnt3a (LOL = 12, 100 ng of input) bound to the NA gradient with a surface area of 600 × 600 µm per gradient fabricated by the laser power gradient (18, 27, 36, and 45 Mw) (iii), the value of which was interpolated into the calibration curve (ii) to estimate the surface density of Wnt3a gradient (iv).

Article Snippet: Subsequently, the rabbit polyclonal anti-Wnt3a antibody (ab28472, Abcam) solution (diluted at 1:100 with diluting buffer (PBS (1×) supplemented with 1% (w/v) BSA and 0.1% (v/v) Tween-20) was applied and allowed for incubating at room temperature for 1 h, followed by incubating with the Alexa Fluor Plus 647 conjugated goat-anti rabbit secondary antibody (A32733, Invitrogen, Waltham, MA, USA) (diluted at a 1:2500 with diluting buffer) in darkness at room temperature for another 1 h. Last, the immunofluorescence signal was measured by the CLSM (TCS-SP8, Leica Microsystems) using a diode laser and a 40× lens at an excitation wavelength of 638 nm and a detection wavelength range of 649–689 nm.

Techniques: Immunofluorescence, Generated, Fluorescence, Derivative Assay

Journal: Advanced Functional Materials

Article Title: A Bio‐Functional Wnt3a Gradient Microarray for Cell Niche Studies

doi: 10.1002/adfm.202301941

Figure Lengend Snippet: Figure 5. Dose- and time-dependent Wnt/β-catenin signaling activated by NA-bound Wnt3a. A) Study design. B) Representative immunofluorescence images of Wnt/β-catenin signaling activation in terms of nuclear translocation of β-catenin in mouse L cells after cultured on NA patterns in the cul- ture medium only (1st column in (i)), on NA patterns in the culture medium supplemented with non-biotinylated Wnt3a (fWnt3a) at 400 ng mL−1 (2nd column in (i)), and on NA patterns bound with different inputs of bWnt3a (LOL = 12) at 125 (3rd column in (i)), 250 (4th column in (i)), and 500 ng (5th column in (i)), for 24 h. Scale bar: 50 µm. Quantification of the immunofluorescence intensity of nuclear β-catenin in mouse L cells after cultured under different conditions as indicated above ii). n ≧ 50 cells in each group in two independent experiments. The differences of means of the loga- rithm of intensity of nuclear β-catenin among multiple groups were analyzed by Welch’s ANOVA with Games-Howell’s post-hoc tests. ****p < 0.0001. C) Representative immunofluorescence images of temporal changes of nuclear β-catenin in mouse L cells after cultured on NA patterns only, on NA patterns with fWnt3a supplemented in the medium at 400 ng mL−1, and on NA patterns bound with bWnt3a (LOL = 12, 500 ng of input) for i) 24, ii) 48, iii) and 72 h. Quantification of immunofluorescence intensity of nuclear β-catenin in mouse L cells after cultured under different conditions as indicated above iv–vi). n ≧ 84 cells in each group in two independent experiments. The differences of means of the logarithm of intensity of nuclear β-catenin among multiple groups were analyzed by Welch’s ANOVA with Games-Howell’s post-hoc tests. *p = 0.0102; ****p < 0.0001. (vii) shows the temporal changes of nuclear β-catenin in different groups that were grouped by the incubation time. Error bar: standard deviation (SD).

Article Snippet: Subsequently, the rabbit polyclonal anti-Wnt3a antibody (ab28472, Abcam) solution (diluted at 1:100 with diluting buffer (PBS (1×) supplemented with 1% (w/v) BSA and 0.1% (v/v) Tween-20) was applied and allowed for incubating at room temperature for 1 h, followed by incubating with the Alexa Fluor Plus 647 conjugated goat-anti rabbit secondary antibody (A32733, Invitrogen, Waltham, MA, USA) (diluted at a 1:2500 with diluting buffer) in darkness at room temperature for another 1 h. Last, the immunofluorescence signal was measured by the CLSM (TCS-SP8, Leica Microsystems) using a diode laser and a 40× lens at an excitation wavelength of 638 nm and a detection wavelength range of 649–689 nm.

Techniques: Immunofluorescence, Activation Assay, Translocation Assay, Cell Culture, Incubation, Standard Deviation

Journal: Advanced Functional Materials

Article Title: A Bio‐Functional Wnt3a Gradient Microarray for Cell Niche Studies

doi: 10.1002/adfm.202301941

Figure Lengend Snippet: Figure 6. Dose- and time-dependent cell proliferation activated by NA-bound Wnt3a. A) Workflow. B) Representative immunofluorescence images of cell proliferation in terms of PCNA expression in mouse L cells after cultured on NA patterns in the culture medium only (1st column in (i)), on NA patterns in the culture medium supplemented with non-biotinylated Wnt3a (fWnt3a) at 400 ng mL−1 (2nd column in (i)), and on NA patterns bound with different inputs of bWnt3a (LOL = 12) at 125 (3rd column in (i)), 250 (4th column in (i)), and 500 ng (5th column in (i)), for 24 h. Scale bar: 100 µm. Quantification of the number of PCNA positive mouse L cells after cultured under different conditions as indicated above ii). n = 4 in two independent experiments. The differences of means of the PCNA positive cell count among multiple groups were analyzed by one-way ANOVA with Bonferroni’s post-hoc test. *p = 0.035; **p = 0.006; ***p < 0.001; ns: not significant (p > 0.05). C) Representative immunofluorescence images of temporal changes of PCNA positive cell count in mouse L cells after cultured on NA patterns only, on NA patterns with fWnt3a supplemented in the medium at 400 ng mL−1, and on NA patterns bound with bWnt3a (LOL = 12, 500 ng of input) for i) 24, ii) 48, iii) and 72 h. Quantification of the number of PCNA positive mouse L cells after cultured under different conditions as indicated above iv–vi). n = 4 in two independent experiments. The differ- ences of means of the PCNA positive cell count among multiple groups were analyzed by one-way ANOVA with Bonferroni’s post-hoc test. *p < 0.04; **p < 0.009; ***p < 0.001; ns: not significant (p > 0.05). vii) shows the temporal changes of PCNA positive mouse L cell count in different groups that were grouped by the incubation time. Error bar: standard deviation (SD).

Article Snippet: Subsequently, the rabbit polyclonal anti-Wnt3a antibody (ab28472, Abcam) solution (diluted at 1:100 with diluting buffer (PBS (1×) supplemented with 1% (w/v) BSA and 0.1% (v/v) Tween-20) was applied and allowed for incubating at room temperature for 1 h, followed by incubating with the Alexa Fluor Plus 647 conjugated goat-anti rabbit secondary antibody (A32733, Invitrogen, Waltham, MA, USA) (diluted at a 1:2500 with diluting buffer) in darkness at room temperature for another 1 h. Last, the immunofluorescence signal was measured by the CLSM (TCS-SP8, Leica Microsystems) using a diode laser and a 40× lens at an excitation wavelength of 638 nm and a detection wavelength range of 649–689 nm.

Techniques: Immunofluorescence, Expressing, Cell Culture, Cell Counting, Incubation, Standard Deviation

Journal: Advanced Functional Materials

Article Title: A Bio‐Functional Wnt3a Gradient Microarray for Cell Niche Studies

doi: 10.1002/adfm.202301941

Figure Lengend Snippet: Figure 7. Wnt3a-gradient-dependent proliferation of human hair dermal papilla cells (HHDPCs). A) Study design. B) Light microscope images of cell attachment on the Wnt3a gradient after 1 h post cell seeding i) and quantitative analysis of cell number on each Wnt3a concentration gradient ii). The differences of means of cell count among multiple groups were analyzed by one-way ANOVA with Bonferroni’s post-hoc test. ns: not significant (p > 0.05). n = 8 in two independent experiments. C) Evaluation of the proliferation of HHDPCs on the Wnt3a or NA gradient by immunofluorescence staining of PCNA i) and further regression analysis of the relationship between the PCNA positive cell count and the Wnt3a or NA gradient controlled by the laser power ii). n = 4 in two independent experiments. Scale bar: 100 µm. D) Representative fluorescence images of DAPI labeled HHDPCs after cultured on the Wnt3a gradient, Wnt3a gradient incubated with different concentrations of DTT (20, 100, and 200 mmol), and NA gradient, for 24 h i) and quantitative cell numbers in different groups ii). Scale bar: 100 µm. The differences of means of cell number among multiple groups were analyzed by one-way ANOVA with Bonferroni’s multiple comparisons. *p < 0.003; ns: not significant (p > 0.05). n = 3 in three independent experi- ments. E) Immunofluorescence staining of PCNA in the cells cultured on the DTT-treated Wnt3a gradients for 24 h i). Quantification of the number of PCNA positive cells cultured on the DTT-treated Wnt3a gradients ii). The differences of means of PCNA positive cell count among multiple groups were analyzed by one-way ANOVA with Bonferroni’s multiple comparisons. ***p = 0.0002; ****p < 0.0001; ns: not significant (p > 0.05). n = 4 in two independent experiments.

Article Snippet: Subsequently, the rabbit polyclonal anti-Wnt3a antibody (ab28472, Abcam) solution (diluted at 1:100 with diluting buffer (PBS (1×) supplemented with 1% (w/v) BSA and 0.1% (v/v) Tween-20) was applied and allowed for incubating at room temperature for 1 h, followed by incubating with the Alexa Fluor Plus 647 conjugated goat-anti rabbit secondary antibody (A32733, Invitrogen, Waltham, MA, USA) (diluted at a 1:2500 with diluting buffer) in darkness at room temperature for another 1 h. Last, the immunofluorescence signal was measured by the CLSM (TCS-SP8, Leica Microsystems) using a diode laser and a 40× lens at an excitation wavelength of 638 nm and a detection wavelength range of 649–689 nm.

Techniques: Light Microscopy, Cell Attachment Assay, Concentration Assay, Cell Counting, Immunofluorescence, Staining, Fluorescence, Labeling, Cell Culture, Incubation

Journal: Advanced Functional Materials

Article Title: A Bio‐Functional Wnt3a Gradient Microarray for Cell Niche Studies

doi: 10.1002/adfm.202301941

Figure Lengend Snippet: Figure 8. Maintenance of human hair dermal papilla cells (HHDPCs) on the Wnt3a gradient. A) Study design and workflow of characterizing the fibronectin meshwork deposited by the HHDPCs cultured on Wnt3a gradient. B) Representative immunofluorescence images of fibronectin deposited by the HHDPCs cultured on the NA gradient bound with bWnt3a (LOL = 12, 100 ng of input) i) and on the NA gradient only in the culture medium ii), for 24 h. Scale bar: 100 µm. Linear regression analysis of the relationship between the area of fibronectin matrix iii), the total length of fibronectin matrix iv), the percentage (%) of the high-density fibronectin matrix v) and the lacunarity of the fibronectin matrix vi) on the Wnt3a or NA gradient. Evaluation of β-catenin nuclear accumulation in HHDPCs cultured on the Wnt3a gradient by counting the number of cells positive in the nuclear β-catenin vii). n = 4 in two independent experiments. The differences of means among groups were analyzed by one-way ANOVA with Bonferroni’s post-hoc tests. *p < 0.05, **p < 0.01. C) Representative immunofluorescence images of ALP expression in the HHDPCs maintained on the NA gradient bound with bWnt3a (LOL = 12, 100 ng of input) for 24 h i) and the quantification of the area ii) and the level iii) of ALP expression. Scale bar: 100 µm. The differ- ences of means among groups were analyzed by Welch’s ANOVA with Dunnett’s T3 post-hoc tests. *p < 0.05, ***p = 0.0002, ****p < 0.0001. ns: not significant (p > 0.05). n = 9, 26, 32, and 30 cells in 18, 27, 36, and 45 mW group, respectively. D) Representative immunofluorescence images of BMP2 expression in the HHDPCs maintained on the NA gradient bound with bWnt3a (LOL = 12, 100 ng of input) for 24 h i) and the quantification of the area ii) and the level iii) of BMP2 expression. Scale bar: 100 µm. The differences of means among groups were analyzed by Welch’s ANOVA with Dunnett’s T3 post-hoc tests. ns: not significant (p > 0.05). n = 21, 20, 17, and 24 cells in 18, 27, 36, and 45 mW group, respectively. E) Representative immunofluo- rescence images of versican expression in the HHDPCs maintained on the NA gradient bound with bWnt3a (LOL = 12, 100 ng of input) for 24 h i) and the quantification of the area ii) and the level iii) of versican expression. Scale bar: 100 µm. The differences of means among groups were analyzed by Welch’s ANOVA with Dunnett’s T3 post-hoc tests. ns: not significant (p > 0.05). n = 12, 15, 39, and 38 cells in 18, 27, 36, and 45 mW group, respectively.

Article Snippet: Subsequently, the rabbit polyclonal anti-Wnt3a antibody (ab28472, Abcam) solution (diluted at 1:100 with diluting buffer (PBS (1×) supplemented with 1% (w/v) BSA and 0.1% (v/v) Tween-20) was applied and allowed for incubating at room temperature for 1 h, followed by incubating with the Alexa Fluor Plus 647 conjugated goat-anti rabbit secondary antibody (A32733, Invitrogen, Waltham, MA, USA) (diluted at a 1:2500 with diluting buffer) in darkness at room temperature for another 1 h. Last, the immunofluorescence signal was measured by the CLSM (TCS-SP8, Leica Microsystems) using a diode laser and a 40× lens at an excitation wavelength of 638 nm and a detection wavelength range of 649–689 nm.

Techniques: Cell Culture, Immunofluorescence, Expressing