Journal: bioRxiv

Article Title: Calcium signals tune AMPK activity and mitochondrial homeostasis in dendrites of developing neurons

doi: 10.1101/2022.12.27.521995

Figure Lengend Snippet: (A) Expression profiles of AMPK α subunits in developing hippocampal neurons in culture. Whole cell lysates were collected at indicated stages and subjected to western blotting with antibodies against AMPK α1 and/or α 2, and AMPK α1/2 phosphorylated at threonine 172 (p-AMPK α ). (B) Morphology of hippocampal neurons transfected with GFP and the following constructs: shRNA-scramble control (SCR); shRNA-AMPK α 2 #1 (AMPK α 2 KD); or shRNA-AMPK α 2 #1 with shRNA-resistant AMPK α 2 (Rescue). Cells were transfected at DIV3 and fixed at DIV5. (C, D) Quantification of total dendritic length (C) and number of branch points (D). N= 31 cells for SCR, 30 cells for AMPK α 2 KD, and 26 cells for Rescue. ***p= 4.0×10 −4 for SCR vs. AMPK α 2 KD, **p= 0.0058 for AMPK α 2 KD vs. Rescue (C); ***p= 2.0×10 −4 for SCR vs. AMPK α 2 KD, *p= 0.018 for AMPK α 2 KD vs. Rescue (D), Kruskal–Wallis test with Bonferroni multiple comparisons test. (E, F) Morphology of CA1 pyramidal neurons transfected with GFP and shRNA-scramble control (SCR) or shRNA-AMPK α 2 #1 (AMPK α 2 KD) by in utero electroporation at E14.5, followed by fixation at P5 (E) and P10 (F). (G, H) Quantification of total dendritic length (G) and number of branch points (H) in CA1 pyramidal neurons at P5 and P10. N≥ 16 cells for SCR and AMPK α 2 KD, **p= 0.0067 for P5; (n.s.) p= 0.20 for P10 (G); **p= 0.0021 for P5; (n.s.) p= 0.12 for P10 (H), Wilcoxon rank-sum test. Box plots denote the median (50th percentile) and the 25th to 75th percentiles of datasets in (C, D, G, H). Samples were collected from three independent experiments. Scale bars: 50 µm in (B, E, F).

Article Snippet: Cells were then blocked with blocking solution (PBS with 2% BSA) and incubated with rabbit anti-AMPK α 2 primary antibody (1:200, Proteintech, 18167-1-AP) at 4°C overnight.

Techniques: Expressing, Western Blot, Transfection, Construct, shRNA, Control, In Utero, Electroporation

Journal: bioRxiv

Article Title: Calcium signals tune AMPK activity and mitochondrial homeostasis in dendrites of developing neurons

doi: 10.1101/2022.12.27.521995

Figure Lengend Snippet: (A, B) Morphology of mitochondria in dendrites (A) and axons (B) in hippocampal neurons transfected with GFP and mito-DsRed together with shRNA-scramble control (SCR), shRNA-AMPKα2#1 (AMPKα2 KD), or shRNA-AMPKα2#1 with shRNA-resistant AMPKα2 (Rescue). Cells were transfected at DIV3 and fixed at DIV5. (C, D) Distribution (C) and cumulative frequency (D) of the length of dendritic mitochondria in SCR, AMPKα2 KD and Rescue cells. N= 669 mitochondria from 26 cells (SCR), 548 mitochondria from 22 cells (AMPKα2 KD), and 543 mitochondria from 22 cells (Rescue), respectively. ***p< 0.001; (n.s.) p> 0.05; Kruskal–Wallis test with Bonferroni multiple comparisons test. (E) Average length of dendritic mitochondria in individual cells. N=26 cells (SCR), 22 cells (AMPKα2 KD), and 22 cells (Rescue), respectively. ***p< 0.001; (n.s.) p> 0.05; Kruskal–Wallis test with Bonferroni multiple comparisons test. (F) Quantification of the mitochondrial fission and fusion frequencies in dendrites in SCR and AMPKα2 KD cells. N= 12 cells for SCR, and 13 cells for AMPKα2 KD. Mean ± SEM, **p= 0.0042; (n.s.) p= 0.75; unpaired two-tailed t test. (G) Morphology of dendritic mitochondria in hippocampal neurons before and 3-5 h after treatment with 1 mM AICAR (AMPK activator). Cells were labeled with tdTomato and mito-EGFP. (H, I) Distribution (H) and cumulative frequency (I) of the length of dendritic mitochondria before and after AICAR treatment. N= 230 mitochondria from 12 control cells, 228 mitochondria from 12 drug-treated cells, ***p= 1.9×10 −11 ; Wilcoxon rank-sum test. (J) Average length of dendritic mitochondria in individual cells. N= 12 cells, *** p= 4.9×10 −4 , Wilcoxon signed-rank test. (K) Morphology of dendritic mitochondria in hippocampal neurons treated with vehicle or 20 µM Compound C for 6h. Cells were labeled with GFP and mito-DsRed. (L, M) Distribution (L) and cumulative frequency (M) of the length of dendritic mitochondria treated with vehicle or Compound C. N= 575 mitochondria from 22 control cells, 483 mitochondria from 19 drug-treated cells, ***p= 8.5×10 −8 , Wilcoxon rank-sum test. (N) Average length of the dendritic mitochondria in individual cells. N=22 control cells, 19 drug-treated cells, *** p= 2.9×10 −4 , Wilcoxon rank-sum test. Box plots denote the median (50th percentile) and the 25th to 75th percentiles of datasets in (C, E, H, J, L, N). Samples were collected from three independent experiments. Scale bars: 5 µm in (A, B, G, K).

Article Snippet: Cells were then blocked with blocking solution (PBS with 2% BSA) and incubated with rabbit anti-AMPK α 2 primary antibody (1:200, Proteintech, 18167-1-AP) at 4°C overnight.

Techniques: Transfection, shRNA, Control, Two Tailed Test, Labeling

Journal: bioRxiv

Article Title: Calcium signals tune AMPK activity and mitochondrial homeostasis in dendrites of developing neurons

doi: 10.1101/2022.12.27.521995

Figure Lengend Snippet: Subcellular distribution of AMPKAR-EV FRET signals in hippocampal neurons at DIV5. Cells were transfected with AMPKAR-EV together with shRNA-scramble control (SCR) or shRNA-AMPKα2 (AMPKα2 KD). Scale bar: 50 μm. (B) Activation of AMPK induced by neuronal activity. DIV5 hippocampal neurons expressing AMPKAR-EV with shRNA-scramble control (SCR) or shRNA-AMPKα2 #1 (AMPKα2 KD) were treated with 1 µM glutamate. The YFP/CFP ratio in dendrites was monitored every 2 min for 70 min before and after glutamate treatment. N= 18 cells for control and AMPKα2 from two independent experiments. (C) Representative traces of AMPK activity and Ca 2+ influx in hippocampal neurons treated with TTX+APV. Cells were transfected with AMPKAR-EV and jRGECO1a. (D) Representative traces of AMPK activity and Ca 2+ influx in hippocampal neurons transfected with AMPKAR-EV, jRGECO1a and shRNA-AMPKα2. (E) Quantification of the frequency of AMPKAR-EV FRET peaks in hippocampal neurons expressing AMPKAR-EV before and after treatment with 1 μM Pim1 (LKB1 inhibitor) at DIV5. N= 19 cells from two independent experiments, (n.s.) p= 0.30, Wilcoxon signed-rank test. Box plots denote the median (50th percentile) and the 25th to 75th percentiles of datasets.

Article Snippet: Cells were then blocked with blocking solution (PBS with 2% BSA) and incubated with rabbit anti-AMPK α 2 primary antibody (1:200, Proteintech, 18167-1-AP) at 4°C overnight.

Techniques: Transfection, shRNA, Control, Activation Assay, Activity Assay, Expressing

Journal: bioRxiv

Article Title: Calcium signals tune AMPK activity and mitochondrial homeostasis in dendrites of developing neurons

doi: 10.1101/2022.12.27.521995

Figure Lengend Snippet: Representative traces of AMPK activity (YFP/CFP ratio) in dendrites of hippocampal neurons expressing AMPKAR-EV with shRNA-scramble control (SCR) or shRNA-AMPKα2 #1 (AMPKα2 KD). Time-lapse images were acquired every 3 sec for 3 min at DIV5 using epifluorescence microscopy. Red arrowheads indicate peaks of AMPK activity. (B) Quantification of the frequency of AMPKAR-EV FRET peaks in hippocampal neurons described in (A). N= 46 cells for SCR, 40 cells for AMPKα2 KD; ***p= 1.2×10 −8 ; Wilcoxon rank-sum test. (C) Representative traces of AMPK activity in hippocampal neurons expressing AMPKAR-EV before and after TTX+APV treatment. (D) Quantification of the frequency of AMPKAR-EV FRET peaks described in (C). N= 22 cells; ***p= 1.6×10 −4 ; Wilcoxon signed-rank test. (E, F) Representative time-lapse images and traces of AMPKAR-EV FRET signals and Ca 2+ influx in hippocampal neurons transfected with AMPKAR-EV and jRGECO1a. Scale bar: 10 μm (E). Blue and orange lines indicate AMPKAR-EV FRET and jRGECO1a signal intensity, respectively (F) (see also Supplementary Video 4). (G) The temporal cross-correlations between AMPK activity and Ca 2+ influx. Gray lines show individual cells, and the red line indicates the average temporal cross-correlation function. The arrowhead shows the highest correlation at 5 sec after Ca 2+ influx. N= 33 cells. (H) Representative traces of AMPK activity in hippocampal neurons expressing AMPKAR-EV with shRNA-scramble control (SCR) or shRNA-CaMKK2 (CaMKK2 KD). (I) Quantification of the frequency of AMPKAR-EV FRET peaks described in (H). N=32 cells for SCR, 45 cells for CaMKK2 KD; ***p= 6.8×10 −4 ; Wilcoxon rank-sum test. (J) Quantification of the frequency of AMPKAR-EV FRET peaks in hippocampal neurons expressing AMPKAR-EV before and 2h after treatment with 10 μM STO-609 (CaMKK2 inhibitor). N= 17 cells from two independent experiments; *p= 0.011; Wilcoxon signed-rank test. Box plots denote the median (50th percentile) and the 25th to 75th percentiles of datasets in (B, D, I, J). Samples were taken from three independent experiments in (B, D, G, I).

Article Snippet: Cells were then blocked with blocking solution (PBS with 2% BSA) and incubated with rabbit anti-AMPK α 2 primary antibody (1:200, Proteintech, 18167-1-AP) at 4°C overnight.

Techniques: Activity Assay, Expressing, shRNA, Control, Epifluorescence Microscopy, Transfection

Journal: bioRxiv

Article Title: Calcium signals tune AMPK activity and mitochondrial homeostasis in dendrites of developing neurons

doi: 10.1101/2022.12.27.521995

Figure Lengend Snippet: Phosphorylation of AMPKα and its downstream effectors in neurons treated with either TTX+APV (6h), 100 μΜ glutamate (5 and 10 min), 20 μΜ Compound C (CC, 6h) or 2 mM AICAR (6h) at DIV5. Blots were probed with antibodies against total and p-Thr172 AMPKα, total and p-Ser146 MFF, total and p-Ser555 ULK1 and β-actin. Similar results were obtained from three independent cultures. (B) Time-lapse images of TMRM flickering in dendritic mitochondria in hippocampal neurons. Cells were transfected with mito-GFP and SCR or AMPKα2 shRNA and treated with 20 nM TMRM. Transient loss of TMRM fluorescence in a single mitochondrion is seen (yellow arrowheads) (see also Supplementary Video 5). (C) Quantification of the frequencies of TMRM flickering in control and AMPKα2 KD cells. N= 18 cells for SCR, 20 cells for AMPKα2 KD. Samples were taken from four independent experiments. *p= 0.041, Wilcoxon rank-sum test. (D) Mito-SOX signals in dendritic mitochondria in control and AMPKα2 KD cells. Cells were transfected with mito-GFP and SCR or AMPKα2 shRNA and treated with 500 nM Mito-SOX. (E) Quantification of the Mito-SOX intensity in individual mitochondria. N= 212 mitochondria from 13 cells for SCR, 225 mitochondria from 17 cells for AMPKα2 KD. Samples were taken from two independent experiments. ***p= 6.9×10 −15 , Wilcoxon rank-sum test. Box plots denote the median (50th percentile) and the 25th to 75th percentiles of datasets in (C, E). Scale bars: 1 μm in (B, D).

Article Snippet: Cells were then blocked with blocking solution (PBS with 2% BSA) and incubated with rabbit anti-AMPK α 2 primary antibody (1:200, Proteintech, 18167-1-AP) at 4°C overnight.

Techniques: Phospho-proteomics, Transfection, shRNA, Fluorescence, Control

Journal: Scientific Reports

Article Title: Identification of extracellular matrix proteins secreted by human dermal fibroblasts cultured in 3D electrospun scaffolds

doi: 10.1038/s41598-021-85742-0

Figure Lengend Snippet: Fibronectin deposition, localization, and quantification in decellularized scaffolds of HDF and JIMT-1/HDF 3D cultures. ( A – P ) Single confocal plane images of fibronectin deposition taken in the centre of decellularized 3D cultures incubated in the absence (control) or presence of 5 ng/ml TGF-β1. The 3D cultures were decellularized, fixed in 3.7% formaldehyde and stained to visualize fibronectin (green) at 3, 7, 10, and 14 days of culturing. All images are representative of 3 independent experiments. Scale bar is 50 μm. Quantification of fluorescence intensity using ImageJ in images taken by epifluorescence microscopy in HDF decellularized scaffolds ( Q ) and JIMT-1/HDF decellularized scaffolds ( R ). The columns are represented as mean ± SEM (n = 3).

Article Snippet: For ECM staining, the fixed samples were incubated in blocking buffer (1% bovine serum albumin and 1% Tween 20 in PBS) at room temperature for 1 h. Then, the cultures were incubated with anti-fibronectin polyclonal primary antibody (ab2413, 1:200) (Abcam, Cambridge, United Kingdom), anti-laminin polyclonal primary antibody (PA5-16287, 1:250) (ThermoFisher Scientific, Carlsbad, CA, USA), or anti-collagen I polyclonal primary antibody (PA1-26204, 1:200) (ThermoFisher Scientific) for 2 h at room temperature.

Techniques: Incubation, Staining, Fluorescence, Epifluorescence Microscopy

Journal: Scientific Reports

Article Title: Identification of extracellular matrix proteins secreted by human dermal fibroblasts cultured in 3D electrospun scaffolds

doi: 10.1038/s41598-021-85742-0

Figure Lengend Snippet: Confocal images of fibronectin localization in JIMT-1/HDF 3D co-cultures in response to TGF-β1 treatment. JIMT-1/HDF 3D co-cultures were incubated in the absence (control, A – D and I – L ) or presence of 5 ng/ml TGF-β1 ( E – H and M – P ) for 7 and 14 days in 3D scaffolds. The co-cultures were fixed in 3.7% formaldehyde and stained to visualize actin filaments (red), fibronectin (green), and cell nuclei (blue) after 7 and 14 days in culture. The images were taken in the centre of 3D cultures. All images are representative of 3 independent experiments. Scale bar is 50 μm.

Article Snippet: For ECM staining, the fixed samples were incubated in blocking buffer (1% bovine serum albumin and 1% Tween 20 in PBS) at room temperature for 1 h. Then, the cultures were incubated with anti-fibronectin polyclonal primary antibody (ab2413, 1:200) (Abcam, Cambridge, United Kingdom), anti-laminin polyclonal primary antibody (PA5-16287, 1:250) (ThermoFisher Scientific, Carlsbad, CA, USA), or anti-collagen I polyclonal primary antibody (PA1-26204, 1:200) (ThermoFisher Scientific) for 2 h at room temperature.

Techniques: Incubation, Staining

Journal: Scientific Reports

Article Title: Identification of extracellular matrix proteins secreted by human dermal fibroblasts cultured in 3D electrospun scaffolds

doi: 10.1038/s41598-021-85742-0

Figure Lengend Snippet: Collagen I deposition, localization, and quantification in decellularized scaffolds of HDF and JIMT-1/HDF 3D cultures. ( A – P ) Single confocal plane images of collagen I deposition taken in the centre of decellularized 3D cultures incubated in the absence (control) or presence of 5 ng/ml TGF-β1. The 3D cultures were decellularized, fixed in 3.7% formaldehyde and stained to visualize fibronectin (green) at 3, 7, 10, and 14 days of culturing. All images are representative of 3 independent experiments. Scale bar is 50 μm. Quantification of fluorescence intensity using ImageJ in images taken by epifluorescence microscopy in HDF decellularized scaffolds ( Q ) and JIMT-1/HDF decellularized scaffolds ( R ). The columns are represented as mean ± SEM (n = 3).

Article Snippet: For ECM staining, the fixed samples were incubated in blocking buffer (1% bovine serum albumin and 1% Tween 20 in PBS) at room temperature for 1 h. Then, the cultures were incubated with anti-fibronectin polyclonal primary antibody (ab2413, 1:200) (Abcam, Cambridge, United Kingdom), anti-laminin polyclonal primary antibody (PA5-16287, 1:250) (ThermoFisher Scientific, Carlsbad, CA, USA), or anti-collagen I polyclonal primary antibody (PA1-26204, 1:200) (ThermoFisher Scientific) for 2 h at room temperature.

Techniques: Incubation, Staining, Fluorescence, Epifluorescence Microscopy

Journal: Scientific Reports

Article Title: Identification of extracellular matrix proteins secreted by human dermal fibroblasts cultured in 3D electrospun scaffolds

doi: 10.1038/s41598-021-85742-0

Figure Lengend Snippet: Laminin deposition, localization, and quantification in decellularized scaffolds of HDF and JIMT-1/HDF 3D cultures. ( A – P ) Single confocal plane images of laminin deposition taken in the centre of decellularized 3D cultures incubated in the absence (control) or presence of 5 ng/ml TGF-β1. The 3D cultures were decellularized, fixed in 3.7% formaldehyde and stained to visualize fibronectin (green) at 3, 7, 10, and 1 days of culturing. All images are representative of 3 independent experiments. Scale bar is 50 μm. Quantification of fluorescence intensity using ImageJ in images taken by epifluorescence microscopy in HDF decellularized scaffolds ( Q ) and JIMT-1/HDF decellularized scaffolds ( R ). The columns are represented as mean ± SEM (n = 3).

Article Snippet: For ECM staining, the fixed samples were incubated in blocking buffer (1% bovine serum albumin and 1% Tween 20 in PBS) at room temperature for 1 h. Then, the cultures were incubated with anti-fibronectin polyclonal primary antibody (ab2413, 1:200) (Abcam, Cambridge, United Kingdom), anti-laminin polyclonal primary antibody (PA5-16287, 1:250) (ThermoFisher Scientific, Carlsbad, CA, USA), or anti-collagen I polyclonal primary antibody (PA1-26204, 1:200) (ThermoFisher Scientific) for 2 h at room temperature.

Techniques: Incubation, Staining, Fluorescence, Epifluorescence Microscopy

Journal: Scientific Reports

Article Title: Identification of extracellular matrix proteins secreted by human dermal fibroblasts cultured in 3D electrospun scaffolds

doi: 10.1038/s41598-021-85742-0

Figure Lengend Snippet: Confocal images of fibronectin co-localization in HDF 3D mono-cultures in response to TGF-β1 treatment. HDF 3D mono-cultures were incubated in the absence (control) or presence of 5 ng/ml TGF-β1 for 7 and 14 days in 3D scaffolds. Cultures were fixed in 3.7% formaldehyde and stained to visualize actin filaments (red), fibronectin (green) and, cell nuclei (blue) after 7 and 14 days in culture. The images were taken in the centre of 3D cultures. All images are representative of 3 independent experiments. Scale bar is 50 μm.

Article Snippet: For ECM staining, the fixed samples were incubated in blocking buffer (1% bovine serum albumin and 1% Tween 20 in PBS) at room temperature for 1 h. Then, the cultures were incubated with anti-fibronectin polyclonal primary antibody (ab2413, 1:200) (Abcam, Cambridge, United Kingdom), anti-laminin polyclonal primary antibody (PA5-16287, 1:250) (ThermoFisher Scientific, Carlsbad, CA, USA), or anti-collagen I polyclonal primary antibody (PA1-26204, 1:200) (ThermoFisher Scientific) for 2 h at room temperature.

Techniques: Incubation, Staining

Journal: Scientific Reports

Article Title: Identification of extracellular matrix proteins secreted by human dermal fibroblasts cultured in 3D electrospun scaffolds

doi: 10.1038/s41598-021-85742-0

Figure Lengend Snippet: Focus stacking images obtained by confocal microscopy of fibronectin deposition and localization in 3D cultures of HDFs. Three confocal plane images were obtained with approximately 10 µm distance from each other in the 3D cultures. HDF 3D mono-cultures were incubated in the absence (control) or presence of 5 ng/ml TGF-β1 for 7 and 14 days. Cultures were fixed in 3.7% formaldehyde and labelled to visualize actin (red), fibronectin (green), and cell nuclei (blue). All images are representative of 3 independent experiments. Scale bar is 50 μm.

Article Snippet: For ECM staining, the fixed samples were incubated in blocking buffer (1% bovine serum albumin and 1% Tween 20 in PBS) at room temperature for 1 h. Then, the cultures were incubated with anti-fibronectin polyclonal primary antibody (ab2413, 1:200) (Abcam, Cambridge, United Kingdom), anti-laminin polyclonal primary antibody (PA5-16287, 1:250) (ThermoFisher Scientific, Carlsbad, CA, USA), or anti-collagen I polyclonal primary antibody (PA1-26204, 1:200) (ThermoFisher Scientific) for 2 h at room temperature.

Techniques: Confocal Microscopy, Incubation

Journal: Scientific Reports

Article Title: Identification of extracellular matrix proteins secreted by human dermal fibroblasts cultured in 3D electrospun scaffolds

doi: 10.1038/s41598-021-85742-0

Figure Lengend Snippet: Focus stacking images obtained by confocal microscopy of fibronectin deposition and localization in 3D co-cultures of JIMT-1/HDFs. Three confocal plane images were obtained with approximately 10 µm distance from each other in the 3D cultures. JIMT-1/HDF 3D co-cultures were incubated in the absence (control) or presence of 5 ng/ml TGF-β1for 7 and 14 days. Cultures were fixed in 3.7% formaldehyde and labelled to visualize actin (red), fibronectin (green), and cell nuclei (blue). All images are representative of 3 independent experiments. Scale bar in 50 μm.

Article Snippet: For ECM staining, the fixed samples were incubated in blocking buffer (1% bovine serum albumin and 1% Tween 20 in PBS) at room temperature for 1 h. Then, the cultures were incubated with anti-fibronectin polyclonal primary antibody (ab2413, 1:200) (Abcam, Cambridge, United Kingdom), anti-laminin polyclonal primary antibody (PA5-16287, 1:250) (ThermoFisher Scientific, Carlsbad, CA, USA), or anti-collagen I polyclonal primary antibody (PA1-26204, 1:200) (ThermoFisher Scientific) for 2 h at room temperature.

Techniques: Confocal Microscopy, Incubation

Journal: Scientific Reports

Article Title: Identification of extracellular matrix proteins secreted by human dermal fibroblasts cultured in 3D electrospun scaffolds

doi: 10.1038/s41598-021-85742-0

Figure Lengend Snippet: Fluorescence microscopy images of cryosectioned 3D mono-cultures of HDFs incubated in the absence or presence of TGF-β1. The HDF 3D cultures were incubated in the absence (control) or presence of TGF-β1 (5 ng/ml). After 7 and 14 days of incubation, cultures were fixed in 3.7% formaldehyde, stained to visualize actin filaments (red), fibronectin (green), and cell nuclei (blue), followed by cryosectioning, and fluorescence microscopy. All images are representative of 3 independent experiments. Scale bar is 50 μm.

Article Snippet: For ECM staining, the fixed samples were incubated in blocking buffer (1% bovine serum albumin and 1% Tween 20 in PBS) at room temperature for 1 h. Then, the cultures were incubated with anti-fibronectin polyclonal primary antibody (ab2413, 1:200) (Abcam, Cambridge, United Kingdom), anti-laminin polyclonal primary antibody (PA5-16287, 1:250) (ThermoFisher Scientific, Carlsbad, CA, USA), or anti-collagen I polyclonal primary antibody (PA1-26204, 1:200) (ThermoFisher Scientific) for 2 h at room temperature.

Techniques: Fluorescence, Microscopy, Incubation, Staining

Journal: Scientific Reports

Article Title: Identification of extracellular matrix proteins secreted by human dermal fibroblasts cultured in 3D electrospun scaffolds

doi: 10.1038/s41598-021-85742-0

Figure Lengend Snippet: Fluorescence microscopy images of cryosectioned 3D JIMT-1/HDF co-cultures incubated in the absence or presence of TGF-β1. The JIMT-1/HDF 3D cultures were incubated in the absence (control) or presence of TGF-β1 (5 ng/ml). After 7 and 14 days of incubation, cultures were fixed in 3.7% formaldehyde, stained to visualize actin filaments (red), fibronectin (green), and cell nuclei (blue), followed by cryosectioning, and fluorescence microscopy. All images are representative of 3 independent experiments. Scale bar is 50 μm.

Article Snippet: For ECM staining, the fixed samples were incubated in blocking buffer (1% bovine serum albumin and 1% Tween 20 in PBS) at room temperature for 1 h. Then, the cultures were incubated with anti-fibronectin polyclonal primary antibody (ab2413, 1:200) (Abcam, Cambridge, United Kingdom), anti-laminin polyclonal primary antibody (PA5-16287, 1:250) (ThermoFisher Scientific, Carlsbad, CA, USA), or anti-collagen I polyclonal primary antibody (PA1-26204, 1:200) (ThermoFisher Scientific) for 2 h at room temperature.

Techniques: Fluorescence, Microscopy, Incubation, Staining

Journal: The Journal of Cell Biology

Article Title: The β-cell primary cilium is an autonomous Ca 2+ compartment for paracrine GABA signaling

doi: 10.1083/jcb.202108101

Figure Lengend Snippet: GABA B1 receptors localize to the primary cilium of islet cells and are mobilized by agonist binding. (A) Immunofluorescence staining of a mouse islet showing the distribution of GABA A , GABA B1 , and GABA B2 receptors. (B) Immunofluorescence staining of a mouse islet showing γ-tubulin in green and GABA B1 receptors in magenta. (C) Immunofluorescence staining of a mouse islet showing acetylated tubulin in green and GABA B1 receptors in magenta. (D) Immunofluorescence staining of a mouse islet showing Arl13b in green and GABA B1 receptors in magenta. (E) Confocal and STED images of a mouse islet cell cilia stained for acetylated tubulin (green) and GABA B1 receptors (magenta). Boxed areas are magnified to the right with line profiles of the dotted lines shown below. Notice that the receptors localize peripheral to the microtubule core of the cilium. (F) Distribution of GABA B1 receptors along a straightened primary cilium. (G) Distribution of whole cilia lengths (determined by acetylated tubulin) and the GABA B1 receptor positive segment of the cilium in mouse islets ( n = 85 cilia from 4 islets). (H) Correlation between cilia length and the length of the GABA B1 receptor positive segment. (I) Fraction of the cilium positive for GABA B1 receptors. (J) Representative images showing the cilia localization of GABA B1 receptors (magenta) to the primary cilium (green) in MIN6 cells, MIN6 pseudo-islets, mouse islet cells, and human islet cells. (K) Quantifications of the cilia distribution of GABA B1 receptors in the different cell preparations. (L) Center-of-mass analysis of mouse islet cilia stained for acetylated tubulin and GABA B1 receptors under control conditions and following a 10-min incubation with 10 nM GABA. Notice that the addition of GABA results in a shift in the center of mass toward more distal regions of the cilium (means ± SEM; n = 39, control and n = 27, GABA cilia from at least 25 islets each, **** P < 0.0001, one-way ANOVA with Tukey’s post hoc test). (M) Immunofluorescence images of cilia from mouse islet cells immunostained for acetylated tubulin (green) and Patched (magenta) under control conditions and following 10-min stimulation with 100 nM GABA. Quantifications below show the area under the curve (AUC) of line profiles drawn along the cilia of islet cells (means ± SEM; n control = 145 cilia from 15 islets from 3 animals; n GABA = 138 cilia from 15 islets from 3 animals). *** P < 0.0001, one-way ANOVA with Tukey’s post hoc test.

Article Snippet: The following antibodies were used in the study: acetylated tubulin (T745, host: mouse, 1:500; Sigma-Aldrich), IFT88 (F41236, host: rabbit, 1:200; NSJ Bioreagents), SSTR3 (E-AB-1607, host: rabbit, 1:200; Elabscience), Patched (ab53715, host: rabbit, 1:200; Abcam), Smoothened (ab113438, host: rabbit, 1:200; Abcam), GABA-B1 receptor (NBP1-52389, host: goat, 1:200; Novus Biologicals and AGB-001, host: rabbit, 1:200; Alamone Labs), GABA-B2 receptor (ab230136, host: rabbit, 1:200; Abcam), GABA-A receptor (224-103, host: rabbit, 1:200; Synaptic Systems), CNGA3 (ab2519323002150000, host: rabbit, 1:200; Abcam), γ-tubulin (T5326, host: mouse, 1:200; Sigma-Aldrich), Arl13b (ab136648, host: mouse, 1:300; Abcam), CaV1.2 (ACC-022, host: rabbit, 1:100; Alamone labs), CaV1.3 (ACC-005, host: rabbit, 1:100; Alamone labs), and CNGB1 (bs-12093R, host: rabbit, 1:100; Bioss antibodies).

Techniques: Binding Assay, Immunofluorescence, Staining, Control, Incubation

Journal: The Journal of Cell Biology

Article Title: The β-cell primary cilium is an autonomous Ca 2+ compartment for paracrine GABA signaling

doi: 10.1083/jcb.202108101

Figure Lengend Snippet: GABA B1 receptor distribution in the cilium after stimulation. (A) Immnofluorescence staining of a mouse islet cell cilium against acetylated tubulin (green), GABA B1 receptors (Rabbit monoclonal antibody, magenta), and GABA B1 receptors (Goat polyclonal antibody, blue). (B) Quantifications of line profiles drawn along cilia of mouse islet cells immunostained for Arl13b (green) and GABA B1 receptors (magenta). An example cilium is shown to the right. (C) Quantifications of line profiles drawn along cilia of MIN6 cells immunostained for acetylated tubulin and GABA B1 receptors. The two lines are form cells treated for 48 h with control siRNA or siRNA against the GABA B1 receptor (KD). Notice how the GABA B1 immunoreactivity is reduced in cells treated with GABA B1 R siRNA ( n = 40 cells from three experiments). (D) Cilia fluorescence intensity from control and GABA B1 receptor KD MIN6 cells immunostained for acetylated tubulin (left) or GABA B1 receptors (right). One-way ANOVA with Šidák’s multiple comparison test (**** P < 0.0001; n = 40). (E) Quantifications of GABA B1 receptor fluorescence intensity and distribution along primary cilia of mouse islet cells under control conditions (black) and following 10-min stimulation with 100 nM of the GABA receptor agonist baclofen (red). Data presented as means ± SEM for the indicated number of cells. (F) Quantifications of GABA B1 receptor fluorescence intensity and distribution along primary cilia of mouse islet cells under control conditions (black) and following 10-min stimulation with 100 nM GABA (red). Data presented as means ± SEM for the indicated number of cells. (G) GABA B1 receptor distribution in mouse islet cilia under control conditions and following stimulation for 10 min with 100 nM GABA or 100 nM Baclofen. Data presented are calculations of the AUC of line profiles drawn along cilia of mouse islet cells immunostained for acetylated tubulin and GABA B1 receptors. Representative images of cilia from a control cell and a cell exposed to baclofen are shown to the right (green, acetylated tubulin; magenta, GABA B1 receptor). Bars show means ± SEM and statistical significance was assessed with ANOVA with multiple comparisons; n = 63 (control) and n = 46 (baclofen); n = 175 (control) and n = 133 (GABA). *** P < 0.001, **** P < 0.0001. (H) Quantifications of acetylated tubulin fluorescence intensity distribution in mouse islet cells under control conditions (black) and following 10-min stimulation with 100 nM GABA (green). Data presented as means ± SEM for 148 cilia from 15 islets and 3 animals. (I) Length distribution of acetylated tubulin in mouse islet cells under control conditions and following stimulation for 10 min with 100 nM GABA. Data presented are calculations of the AUC of line profiles drawn along the acetylated tubulin immunoreactivity (means ± SEM). Two-tailed Student’s unpaired t test; n = 148 (control) and n = 148 (GABA).

Article Snippet: The following antibodies were used in the study: acetylated tubulin (T745, host: mouse, 1:500; Sigma-Aldrich), IFT88 (F41236, host: rabbit, 1:200; NSJ Bioreagents), SSTR3 (E-AB-1607, host: rabbit, 1:200; Elabscience), Patched (ab53715, host: rabbit, 1:200; Abcam), Smoothened (ab113438, host: rabbit, 1:200; Abcam), GABA-B1 receptor (NBP1-52389, host: goat, 1:200; Novus Biologicals and AGB-001, host: rabbit, 1:200; Alamone Labs), GABA-B2 receptor (ab230136, host: rabbit, 1:200; Abcam), GABA-A receptor (224-103, host: rabbit, 1:200; Synaptic Systems), CNGA3 (ab2519323002150000, host: rabbit, 1:200; Abcam), γ-tubulin (T5326, host: mouse, 1:200; Sigma-Aldrich), Arl13b (ab136648, host: mouse, 1:300; Abcam), CaV1.2 (ACC-022, host: rabbit, 1:100; Alamone labs), CaV1.3 (ACC-005, host: rabbit, 1:100; Alamone labs), and CNGB1 (bs-12093R, host: rabbit, 1:100; Bioss antibodies).

Techniques: Staining, Control, Fluorescence, Comparison, Two Tailed Test

Journal: The Journal of Cell Biology

Article Title: The β-cell primary cilium is an autonomous Ca 2+ compartment for paracrine GABA signaling

doi: 10.1083/jcb.202108101

Figure Lengend Snippet: The primary cilium of intact islets of Langerhans. (A) Confocal microscopy image of a mouse islet where cilia are visualized by immunofluorescence staining against acetylated tubulin (magenta) and nuclei with Dapi staining (blue). (B) Confocal microscopy image of a small mouse islet showing insulin in green, acetylated tubulin in magenta, and nuclei in blue. (C) Maximum intensity projection of part of a mouse islet (outlined by dashed line) immunostained against acetylated tubulin. (D) Epifluorescence microscopy images of pancreas sections immunostained for acetylated tubulin (magenta), insulin (green, top panel), and glucagon (green, bottom panel). (E) 3D reconstruction of confocal micrographs of a section of a mouse islet that contains a cluster of primary cilia (pseudo-colored for clarity). (F) Quantification of the number of cilia found within a radius of 2.5 µm of another cilia compared to at random locations. *** P < 0.001 (two-tailed Student's unpaired t test). (G) Example STED images showing islet cell cilia morphologies (green) and the corresponding distribution of GABA B1 receptors (magenta). (H) Distribution of cilia morphologies in mouse islets ( n = 101 cilia from 6 islets). (I) Length of cilia and GABA B1 receptor compartment in mouse islets separated based on cilia morphology (means ± SEM; n = 101 cilia from 6 islets; two-tailed unpaired Student’s t test). (J) Immunofluorescence images showing the localization of Patched, Smoothened, IFT88, and SSTR3 (all green) to the primary cilium (acetylated tubulin; magenta) of mouse islet cells. (K) Immunofluorescence images (top) and quantification of fluorescence intensity (bottom) of acetylated tubulin (green) and Patched (magenta) in mouse islet cell cilia ( n = 146 cilia, 15 islets, 3 animals). (L) Confocal microscopy image of a large human islet showing acetylated tubulin in green.

Article Snippet: The following antibodies were used in the study: acetylated tubulin (T745, host: mouse, 1:500; Sigma-Aldrich), IFT88 (F41236, host: rabbit, 1:200; NSJ Bioreagents), SSTR3 (E-AB-1607, host: rabbit, 1:200; Elabscience), Patched (ab53715, host: rabbit, 1:200; Abcam), Smoothened (ab113438, host: rabbit, 1:200; Abcam), GABA-B1 receptor (NBP1-52389, host: goat, 1:200; Novus Biologicals and AGB-001, host: rabbit, 1:200; Alamone Labs), GABA-B2 receptor (ab230136, host: rabbit, 1:200; Abcam), GABA-A receptor (224-103, host: rabbit, 1:200; Synaptic Systems), CNGA3 (ab2519323002150000, host: rabbit, 1:200; Abcam), γ-tubulin (T5326, host: mouse, 1:200; Sigma-Aldrich), Arl13b (ab136648, host: mouse, 1:300; Abcam), CaV1.2 (ACC-022, host: rabbit, 1:100; Alamone labs), CaV1.3 (ACC-005, host: rabbit, 1:100; Alamone labs), and CNGB1 (bs-12093R, host: rabbit, 1:100; Bioss antibodies).

Techniques: Confocal Microscopy, Immunofluorescence, Staining, Epifluorescence Microscopy, Two Tailed Test, Fluorescence

Journal: The Journal of Cell Biology

Article Title: The β-cell primary cilium is an autonomous Ca 2+ compartment for paracrine GABA signaling

doi: 10.1083/jcb.202108101

Figure Lengend Snippet: Characterization of a cilia-targeted Ca 2+ sensor. (A and B) Principle for determining the enrichment of Smo-GCaMP5G-mCh within the primary cilium of islet cells imaged by TIRF microscopy. With a radius of the cilium of 100 nm and an evanescent wave penetration depth (d) of 80 nm, we estimate that a ratio of cilia-to–plasma membrane fluorescence >1.288 indicate cilia enrichment. (C) Quantifications of fluorescence intensities of Smo-GCaMP5G-mCh from the plasma membrane and cilia of islet cells. The average cilia-to–plasma membrane fluorescence ratio is 2, which indicate strong cilia enrichment (means ± SEM; n = 113 soma and 95 cilia; Student’s two-tailed unpaired t test). (D) Immunofluorescence staining of a MIN6 cells expressing Smo-GCaMP5G-mCherry using antibodies against acetylated tubulin (green) and mCherry (magenta). (E) Measurements of cilia length in islets cells from mouse pancreatic sections (green), isolated mouse islets (red), and mouse islets transduced with Smo-GCaMP5G-mCh for 48 h (black). (F) Quantification of cilia morphologies in islet cells expressing Smo-GCaMP5G-mCh (means ± SEM; n = 202 cilia from 14 islets; two-tailed unpaired Student’s t test). Images show an example of the distribution of GABA B1 receptors in a cilium expressing Smo-GCaMP5G-mCh and quantification of the distribution along the cilia of 23 islet cells.

Article Snippet: The following antibodies were used in the study: acetylated tubulin (T745, host: mouse, 1:500; Sigma-Aldrich), IFT88 (F41236, host: rabbit, 1:200; NSJ Bioreagents), SSTR3 (E-AB-1607, host: rabbit, 1:200; Elabscience), Patched (ab53715, host: rabbit, 1:200; Abcam), Smoothened (ab113438, host: rabbit, 1:200; Abcam), GABA-B1 receptor (NBP1-52389, host: goat, 1:200; Novus Biologicals and AGB-001, host: rabbit, 1:200; Alamone Labs), GABA-B2 receptor (ab230136, host: rabbit, 1:200; Abcam), GABA-A receptor (224-103, host: rabbit, 1:200; Synaptic Systems), CNGA3 (ab2519323002150000, host: rabbit, 1:200; Abcam), γ-tubulin (T5326, host: mouse, 1:200; Sigma-Aldrich), Arl13b (ab136648, host: mouse, 1:300; Abcam), CaV1.2 (ACC-022, host: rabbit, 1:100; Alamone labs), CaV1.3 (ACC-005, host: rabbit, 1:100; Alamone labs), and CNGB1 (bs-12093R, host: rabbit, 1:100; Bioss antibodies).

Techniques: Microscopy, Clinical Proteomics, Membrane, Fluorescence, Two Tailed Test, Immunofluorescence, Staining, Expressing, Isolation, Transduction

Journal: The Journal of Cell Biology

Article Title: The β-cell primary cilium is an autonomous Ca 2+ compartment for paracrine GABA signaling

doi: 10.1083/jcb.202108101

Figure Lengend Snippet: GABA triggers Ca 2+ entry into primary cilia via GABA B1 receptors. (A) TIRF microscopy recordings of GCaMP5G fluorescence in the soma and cilium of MIN6 cells exposed to 1 nM GABA. Notice that the addition of GABA induces a rise of cilia Ca 2+ without affecting the Ca 2+ concentration in the soma. (B) TIRF microscopy recordings of GCaMP5G fluorescence in the soma and cilium of MIN6 cells exposed to 10 nM GABA. Notice that the addition of GABA induces a rise of cilia Ca 2+ without affecting the Ca 2+ concentration in the soma. (C) GABA (1 and 10 nM) and the GABA B receptor agonist Baclofen (100 nM) induces increases in the cilia Ca 2+ concentration in MIN6 cells ( n = 10 cells for 1 nM GABA; n = 13 cells for 10 nM GABA; n = 7 cells for Baclofen; * P < 0.05; ** P < 0.01; **** P < 0.0001; Student’s paired t test). (D) Confocal microscopy immunofluorescence images of control (top) and GABA B1 receptor KD (bottom) MIN6 cell pseudo-islets stained for acetylated tubulin (green) and GABA B1 receptors (magenta). (E) Example TIRF microscopy image of a MIN6 pseudo-islet expressing 5HT 6 -GGECO (boxed area is magnified below). To the right are shown event counts of all cilia Ca 2+ flashes in control (black) and GABA B1 receptor knockdown MIN6 pseudo-islets following addition of baclofen. Each row represents recordings from one control and one GABA B1 receptor knockdown pseudo-islet. Quantifications are shown to the right ( n = 6 pseudo-islets for each condition). Statistical significance was assessed using Wilcoxon signed rank test. A TIRF microscopy image of a 5HT 6 -GGECO1-expressing MIN6 pseudoislet is shown to the left. (F) TIRF microscopy recordings of cilia Ca 2+ concentration changes in intact mouse islets expressing Smo-GCaMP5G-mCh. Picture on top shows the distribution of cilia within the islet, with cilia exhibiting at least one spontaneous Ca 2+ flash during the recording time highlighted in color. In the middle are shown Ca 2+ recordings from the individual cilia within the islet during addition of the GABA B receptor agonist Baclofen. The diagram at the bottom shows the overall cilia Ca 2+ activity of the whole islet. (G) Event count of all cilia Ca 2+ flashes following the addition of baclofen ( n = 7 islets). (H) Event count of all cilia Ca 2+ flashes following the addition of vigabatrin ( n = 13 islets). (I) Quantifications of the effect of baclofen and vigabatrin on cilia Ca 2+ flashes. Statistical significance was assessed using the Friedman test followed by multiple comparisons.

Article Snippet: The following antibodies were used in the study: acetylated tubulin (T745, host: mouse, 1:500; Sigma-Aldrich), IFT88 (F41236, host: rabbit, 1:200; NSJ Bioreagents), SSTR3 (E-AB-1607, host: rabbit, 1:200; Elabscience), Patched (ab53715, host: rabbit, 1:200; Abcam), Smoothened (ab113438, host: rabbit, 1:200; Abcam), GABA-B1 receptor (NBP1-52389, host: goat, 1:200; Novus Biologicals and AGB-001, host: rabbit, 1:200; Alamone Labs), GABA-B2 receptor (ab230136, host: rabbit, 1:200; Abcam), GABA-A receptor (224-103, host: rabbit, 1:200; Synaptic Systems), CNGA3 (ab2519323002150000, host: rabbit, 1:200; Abcam), γ-tubulin (T5326, host: mouse, 1:200; Sigma-Aldrich), Arl13b (ab136648, host: mouse, 1:300; Abcam), CaV1.2 (ACC-022, host: rabbit, 1:100; Alamone labs), CaV1.3 (ACC-005, host: rabbit, 1:100; Alamone labs), and CNGB1 (bs-12093R, host: rabbit, 1:100; Bioss antibodies).

Techniques: Microscopy, Fluorescence, Concentration Assay, Confocal Microscopy, Immunofluorescence, Control, Staining, Expressing, Knockdown, Activity Assay

Journal: The Journal of Cell Biology

Article Title: The β-cell primary cilium is an autonomous Ca 2+ compartment for paracrine GABA signaling

doi: 10.1083/jcb.202108101

Figure Lengend Snippet: cGMP, but not cAMP, triggers Ca 2+ influx in islet cell cilia. (A) TIRF microscopy recording of cytosolic PfPKG (cGMP sensor) FRET ratio in permeabilized MIN6 cells exposed to the indicated cAMP and cGMP concentrations. (B) Quantifications of the PfPKG FRET ratio change in response to the indicated cyclic nucleotide concentrations ( n = 14 cells). (C) Normalized PfPKG FRET ratio in response to 500 nM ANP and 100 µM IBMX in combination with 500 nM ANP (means ± SEM; n = 15 MIN6 cells). Statistical significance was assessed with paired two-tailed Student’s t test. (D) Normalized Epac-S H187 FRET ratio in response to 500 nM ANP and 100 µM 3-isobutyl-1-methylxanthine (IBMX) in combination with 500 nM ANP (means ± SEM; n = 39 MIN6 cells). Statistical significance was assessed with paired two-tailed Student’s t test. (E) Representative recording of cilium (black) and cytosolic (red) Ca 2+ concentration changes in a mouse islet cell expressing 5HT 6 -G-GECO1 and exposed to 500 nM ANP. (F) Event count of all cilia Ca 2+ flashes following the addition of 500 nM ANP or solvent (water; n = 5 islets for control; 8 islets for ANP). (G) Event count of all cilia Ca 2+ flashes following yellow light illumination of islets expressing 5HT 6 -G-GECO1 and RhGC (gray) or empty vector (black; n = 3 islets for both conditions). Red lines have been added to simplify comparison between the two test groups. (H) Event count of all cilia Ca 2+ flashes following blue light illumination of islets expressing 5HT 6 -G-GECO1 and bPAC (gray) or empty vector (black; n = 7 islets for both conditions). Red lines have been added to simplify comparison between the two test groups. (I) Immunofluorescence images of a mouse islet stained against acetylated tubulin (cilia; magenta) and cyclic nucleotide-gated Ca 2+ channel A3 (CNGA3; green). (J) STED images of a MIN6 cell cilium immunostained against acetylated tubulin (magenta) and CNGA3 (green). (K) Immunofluorescence images of a mouse islet cell cilia stained against GABA B1 receptors (GABAB1R; magenta) and CNGA3 (green). (L) Quantifications of cilia Ca 2+ responses (5HT 6 -GGECO1) from control (black) or CNGA3 KD (gray) MIN6 pseudoislets under basal conditions or following stimulation with 500 nM ANP (means ± SEM; n = 5 [control] and 4 [CNGA3 KD] pseudoislets; * P < 0.05; Mann-Whitney U test). (M) Quantifications of cilia Ca 2+ responses from mouse islet cells expressing 5HT 6 -GGECO1 and imaged with TIRF microscopy. Individual cilia Ca 2+ events were counted under basal conditions (gray) and following addition of 100 nM baclofen alone (red) or in combination with 100 µM L-cis-diltiazem (dark blue), 100 µM diltiazem (light blue), 25 µM, verapamil (light purple), 100 µM verapamil (dark purple), or 10 µM nifedipine (green). Each data point represents the number of ciliary Ca 2+ events per islet for 9 islets (baclofen), 6 islets (L-cis-diltiazem), 10 islets (diltiazem), 6 islets (25 µM verapamil) 4 islets (100 µM verapamil), and 6 islets (nifedipine). Bars show means ± SEM and statistical significance was assessed using Wilcoxon signed-rank test. * P < 0.05. (N) Single cell recordings of 5HT6-GGECO1 fluorescence in cilia of mouse islet cells cultured under control conditions (black) or in the presence of pertussis toxin (PT; blue) and stimulated with 100 nM baclofen. Quantification of the response to baclofen in pertussis toxin-treated islets is shown to the right ( n = 45 cilia from 7 islets; * P < 0.05; statistical significance assessed with Wilcoxon signed rank test).

Article Snippet: The following antibodies were used in the study: acetylated tubulin (T745, host: mouse, 1:500; Sigma-Aldrich), IFT88 (F41236, host: rabbit, 1:200; NSJ Bioreagents), SSTR3 (E-AB-1607, host: rabbit, 1:200; Elabscience), Patched (ab53715, host: rabbit, 1:200; Abcam), Smoothened (ab113438, host: rabbit, 1:200; Abcam), GABA-B1 receptor (NBP1-52389, host: goat, 1:200; Novus Biologicals and AGB-001, host: rabbit, 1:200; Alamone Labs), GABA-B2 receptor (ab230136, host: rabbit, 1:200; Abcam), GABA-A receptor (224-103, host: rabbit, 1:200; Synaptic Systems), CNGA3 (ab2519323002150000, host: rabbit, 1:200; Abcam), γ-tubulin (T5326, host: mouse, 1:200; Sigma-Aldrich), Arl13b (ab136648, host: mouse, 1:300; Abcam), CaV1.2 (ACC-022, host: rabbit, 1:100; Alamone labs), CaV1.3 (ACC-005, host: rabbit, 1:100; Alamone labs), and CNGB1 (bs-12093R, host: rabbit, 1:100; Bioss antibodies).

Techniques: Microscopy, Two Tailed Test, Concentration Assay, Expressing, Solvent, Control, Plasmid Preparation, Comparison, Immunofluorescence, Staining, MANN-WHITNEY, Fluorescence, Cell Culture

Journal: The Journal of Cell Biology

Article Title: The β-cell primary cilium is an autonomous Ca 2+ compartment for paracrine GABA signaling

doi: 10.1083/jcb.202108101

Figure Lengend Snippet: Islet cell cilia do not respond with Ca 2+ signaling following mechanical stimulation. (A) Schematic drawing of the cell press used to compress islets on the stage of a confocal microscope. (B) Confocal micrographs of a whole islet expressing Smo-GCaMP5G-mCh. Upper row shows mCherry fluorescence and bottom row shows GCaMP5G fluorescence. Notice how both mild compression and deformation is without effect on both cilia and cytosolic Ca 2+ . (C) Changes in cilia GCaMP5G/mCh fluorescence ratio during mild compression of a mouse islet ( n = 49 cells). (D) GCaMP5G/mCh ratio change following strong compression with islet deformation for the indicated number of islets and islet cells. (E) Schematic drawing of the principle of mechanical stimulation using a glass pipette. (F) Confocal microscopy images of a MIN6 cell expressing Smo-GCaMP5G-mCh following exposure to mechanical stimulation (top) and depolarization (bottom). (G) Means ± SEM ( n = 7) for the cilia and cell body GCaMP5G fluorescence change in response to mechanical stimulation and depolarization. The increase in cilia fluorescence upon depolarization is likely a contamination from the cytoplasm, which partially overlaps with the cilia in these recordings. (H) Proposed model for cilia-dependent Ca 2+ signaling in β-cells. The primary cilium is isolated against cytosolic Ca 2+ changes due to efficient Ca 2+ extrusion in the cilium. This enables the cilium to utilize Ca 2+ as an intrinsic signaling molecule. Activation of cilia-localized GABA B1 receptors results in receptor mobilization into the cilium where it triggers local Ca 2+ influx in a process that depends on L-type Ca 2+ channel activation.

Article Snippet: The following antibodies were used in the study: acetylated tubulin (T745, host: mouse, 1:500; Sigma-Aldrich), IFT88 (F41236, host: rabbit, 1:200; NSJ Bioreagents), SSTR3 (E-AB-1607, host: rabbit, 1:200; Elabscience), Patched (ab53715, host: rabbit, 1:200; Abcam), Smoothened (ab113438, host: rabbit, 1:200; Abcam), GABA-B1 receptor (NBP1-52389, host: goat, 1:200; Novus Biologicals and AGB-001, host: rabbit, 1:200; Alamone Labs), GABA-B2 receptor (ab230136, host: rabbit, 1:200; Abcam), GABA-A receptor (224-103, host: rabbit, 1:200; Synaptic Systems), CNGA3 (ab2519323002150000, host: rabbit, 1:200; Abcam), γ-tubulin (T5326, host: mouse, 1:200; Sigma-Aldrich), Arl13b (ab136648, host: mouse, 1:300; Abcam), CaV1.2 (ACC-022, host: rabbit, 1:100; Alamone labs), CaV1.3 (ACC-005, host: rabbit, 1:100; Alamone labs), and CNGB1 (bs-12093R, host: rabbit, 1:100; Bioss antibodies).

Techniques: Microscopy, Expressing, Fluorescence, Transferring, Confocal Microscopy, Isolation, Activation Assay

Journal: The Journal of Cell Biology

Article Title: The β-cell primary cilium is an autonomous Ca 2+ compartment for paracrine GABA signaling

doi: 10.1083/jcb.202108101

Figure Lengend Snippet: The primary cilium of intact islets of Langerhans. (A) Confocal microscopy image of a mouse islet where cilia are visualized by immunofluorescence staining against acetylated tubulin (magenta) and nuclei with Dapi staining (blue). (B) Confocal microscopy image of a small mouse islet showing insulin in green, acetylated tubulin in magenta, and nuclei in blue. (C) Maximum intensity projection of part of a mouse islet (outlined by dashed line) immunostained against acetylated tubulin. (D) Epifluorescence microscopy images of pancreas sections immunostained for acetylated tubulin (magenta), insulin (green, top panel), and glucagon (green, bottom panel). (E) 3D reconstruction of confocal micrographs of a section of a mouse islet that contains a cluster of primary cilia (pseudo-colored for clarity). (F) Quantification of the number of cilia found within a radius of 2.5 µm of another cilia compared to at random locations. *** P < 0.001 (two-tailed Student's unpaired t test). (G) Example STED images showing islet cell cilia morphologies (green) and the corresponding distribution of GABA B1 receptors (magenta). (H) Distribution of cilia morphologies in mouse islets ( n = 101 cilia from 6 islets). (I) Length of cilia and GABA B1 receptor compartment in mouse islets separated based on cilia morphology (means ± SEM; n = 101 cilia from 6 islets; two-tailed unpaired Student’s t test). (J) Immunofluorescence images showing the localization of Patched, Smoothened, IFT88, and SSTR3 (all green) to the primary cilium (acetylated tubulin; magenta) of mouse islet cells. (K) Immunofluorescence images (top) and quantification of fluorescence intensity (bottom) of acetylated tubulin (green) and Patched (magenta) in mouse islet cell cilia ( n = 146 cilia, 15 islets, 3 animals). (L) Confocal microscopy image of a large human islet showing acetylated tubulin in green.

Article Snippet: The following antibodies were used in the study: acetylated tubulin (T745, host: mouse, 1:500; Sigma-Aldrich), IFT88 (F41236, host: rabbit, 1:200; NSJ Bioreagents), SSTR3 (E-AB-1607, host: rabbit, 1:200; Elabscience), Patched (ab53715, host: rabbit, 1:200; Abcam), Smoothened (ab113438, host: rabbit, 1:200; Abcam), GABA-B1 receptor (NBP1-52389, host: goat, 1:200; Novus Biologicals and AGB-001, host: rabbit, 1:200; Alamone Labs), GABA-B2 receptor (ab230136, host: rabbit, 1:200; Abcam), GABA-A receptor (224-103, host: rabbit, 1:200; Synaptic Systems), CNGA3 (ab2519323002150000, host: rabbit, 1:200; Abcam), γ-tubulin (T5326, host: mouse, 1:200; Sigma-Aldrich), Arl13b (ab136648, host: mouse, 1:300; Abcam), CaV1.2 (ACC-022, host: rabbit, 1:100; Alamone labs), CaV1.3 (ACC-005, host: rabbit, 1:100; Alamone labs), and CNGB1 (bs-12093R, host: rabbit, 1:100; Bioss antibodies).

Techniques: Confocal Microscopy, Immunofluorescence, Staining, Epifluorescence Microscopy, Two Tailed Test, Fluorescence

Journal: Journal of Biological Chemistry

Article Title: Adenomatous Polyposis Coli Is Down-regulated by the Ubiquitin-Proteasome Pathway in a Process Facilitated by Axin

doi: 10.1074/jbc.m404655200

Figure Lengend Snippet: FIG. 8. Wnt3a treatment leads to ac- cumulation of APC in nuclei. A, 293T cells were incubated with either control L-CM or Wnt3a-CM for 24 h and fixed. Indirect immunofluorescence (IF) stain- ing was performed with anti-APC anti- body, and images of the central area of cells were taken by confocal microscopy. The figure shows phase-contrast (left panel) and epifluorescence microscopy (right panel). The same exposure time was used to take the two epifluorescence pictures. The results shown are repre- sentative of at least three separate exper- iments. B, the level of endogenous APC and -catenin in the nuclear fraction was enhanced upon treatment with Wnt3a- CM. Lamin A was used as a control for equal loading. The absence of a detectable signal for -tubulin suggests that there is no contamination of cytoplasmic proteins in the nuclear extracts. In B, C, and D, treatment with L-CM and Wnt3a-CM is indicated by and , respectively. C, 293T cells were grown in either control L-CM or Wnt3a-CM for 24 h. After treat- ment for 24 h, the medium was replaced with serum-free DMEM, and the cells were further incubated to the indicated time points. Nuclear extracts were pre- pared, and Western analysis was per- formed. The accumulated nuclear APC and -catenin increase until 4 h after re- moval of Wnt3a-CM and then decrease at 8 h. D, 293T cells were incubated with either L-CM or Wnt3a-CM for 24 h, and nuclear extracts were prepared. The ex- tracts were immunoprecipitated with an- ti-APC antibody (lanes 1 and 2) or anti- lamin A antibody (lanes 3 and 4), and Western blot analysis was performed with anti--catenin (top), -APC (lanes 1 and 2, bottom panel), or -lamin A antibod- ies (lanes 3 and 4, bottom panel). Lane 5 shows the level of -catenin or lamin A in nuclear extracts. The bands marked with * indicates the heavy chain of IgG. These data show that -catenin and APC can form a complex in the nucleus. IP, immunoprecipitation.

Article Snippet: Cells were incubated with anti-APC monoclonal antibody (1:200, Santa Cruz Biotechnology, Inc.) for 1 h at 37 °C and then incubated with rhodamine-conjugated horse anti-rabbit immunoglobulin (1:200, Jackson Immunoresearch Laboratories, Inc., West Grove, PA) for 1 h at 37 °C in the dark.

Techniques: Incubation, Control, Immunofluorescence, Staining, Confocal Microscopy, Epifluorescence Microscopy, Western Blot, Immunoprecipitation

Journal: Cell Reports

Article Title: Proteolytic and Opportunistic Breaching of the Basement Membrane Zone by Immune Cells during Tumor Initiation

doi: 10.1016/j.celrep.2019.05.029

Figure Lengend Snippet:

Article Snippet: For eGFP immunostaining, larvae were washed in PBST 3 times for 15 min and re-blocked with PBST containing 5% (v/v) goat serum, 3% (w/v) Bovine Serum Albumin for 2 hours at room temperature, before an over-night incubation at 4°C with rabbit monoclonal anti-GFP antibody (1:200) (2956, Cell Signaling Technology).

Techniques: Recombinant, Imaging, Plasmid Preparation, Software, Microscopy, Laser-Scanning Microscopy, Inverted Epifluorescence, Transmission Assay