Journal: The EMBO Journal

Article Title: Somatostatin triggers local cAMP and Ca 2+ signaling in primary cilia to modulate pancreatic β-cell function

doi: 10.1038/s44318-025-00383-7

Figure Lengend Snippet: ( A ) Confocal microscopy images showing ciliary localization of SSTR3 (green) in both insulin-positive and -negative cells in a human islet. ( B ) Confocal microscopy images from a mouse islet (top) and MIN6 pseudoislet (bottom) showing the localization of SSTR3 to primary cilia (visualized by anti-acetylated tubulin immunostaining and shown in magenta). ( C ) Quantifications of the effect of 18 h somatostatin treatment (100 nM) on cilia length (determined as AUC for normalized acetylated tubulin immunoreactivity) in MIN6 cells (means ± SEM; nctrl = 111, nSST = 87 cilia from three different preparations, change in cilia length P = 0.003 assessed by unpaired Kolmogorov–Smirnov test). ( D ) Quantifications of the effect of 18 h ( G ) and 15 min ( H ) somatostatin treatment on cilia SSTR3 immunoreactivity in MIN6 pseudoislets (means ± SEM; for 18 h SST treatments; n ctrl = 111, n SST = 87 cilia from three different preparations, change in SSTR3 not significant; for 15 min SST treatment n ctrl = 340, n SST = 295 cilia from three different preparations, P = 0.0146. Significance is assessed by unpaired Kolmogorov–Smirnov test). ( E ) Quantification of fraction of SSTR3-positive cilia in MIN6 cells exposed to 100 nM somatostatin for 18 h (means ± SEM; n ctrl = 111, n SST = 87 cilia from three different preparations). ( F ) STED microscopy images showing the distribution of SSTR3 (magenta) in mouse islet cells under resting condition and after 15 min SST stimulation. ( G – I ) Quantifications of the effect of 18 h somatostatin treatment on cilia length ( G ), cilia SSTR3 immunoreactivity ( H ) and fraction of SSTR3-positive cilia ( I ) in mouse islet cells (means ± SEM; n ctrl = 170, n SST = 171 cilia from two different animals, P AcT = 0.0055, P SSTR3 = 0.0030, assessed by unpaired Kolmogorov–Smirnov test).

Article Snippet: The following primary antibodies were used in the study; acetylated tubulin (T745, host: mouse, 1:500; Sigma-Aldrich), Acetyl-α-Tubulin (K40) (5335, host: rabbit, 1:500; BioNordika), SSTR3 (E-AB-16077, host: rabbit, 1:200; Elabscience), Arl13b (ab136648, host: mouse, 1:300; Abcam), SST (A0566, host: rabbit, 1:500, Dako), Insulin (A0564, host: guinea pig, 1:500; Dako), SSTR5 (66772-1-Ig, host: mouse, 1:200, Proteintech), SSTR2 (ab134152, host: rabbit, 1:200, Abcam), Pericentrin (ab4448, Abcam, host: rabbit, 1:200), GABBR1 (AGB001AN102, Alomone labs, host: rabbit, 1:200), GLP-1R (Mab 7F38-s, Developmental Studies Hybridoma Bank, host: mouse, 1:10) and GFP (AB16901, Abcam, host: chicken, 1:500).

Techniques: Confocal Microscopy, Immunostaining, Microscopy

Journal: The EMBO Journal

Article Title: Somatostatin triggers local cAMP and Ca 2+ signaling in primary cilia to modulate pancreatic β-cell function

doi: 10.1038/s44318-025-00383-7

Figure Lengend Snippet: ( A ) Confocal microscopy images of human islets from a non-diabetic and a type-2 diabetic (T2D) organ donor immunostained against insulin (cyan) and acetylated tubulin (magenta) (white arrowheads point to cilia). ( B ) Quantifications of cilia length in insulin-positive and negative cells of non-diabetic and type-2 diabetic islets (means ± SD; ND: N = 7 donors; 10–20 islets per donor; 946 cilia. T2D: N = 5 donors; 10–20 islets per donor; 505 cilia; Student’s two-tailed unpaired t test). ( C ) Number of cilia per area in islets from non-diabetic and type-2 diabetic islets (means ± SD; ND: n = 7 donors; T2D: n = 5 donors; Mann–Whitney U test). ( D ) Length of cilia with a swollen tip morphology in human islets from non-diabetic and typ-2 diabetic donors (means ± SD; ND: n = 6 donors; T2D: n = 5 donors; Mann–Whitney U test). ( E ) Length of cilia with rod-like morphology in human islets from non-diabetic and typ-2 diabetic donors (means ± SD; ND: n = 7 donors; T2D: n = 5 donors; Mann–Whitney U test). ( F ) Fraction of cilia with a swollen tip morphology in human islets from non-diabetic and typ-2 diabetic donors (means ± SD; ND: n = 7 donors; T2D: n = 5 donors; Mann–Whitney U test). ( G ) Correlation between cilium length and BMI in insulin-positive (Ins + , top) and insulin-negative (Ins-, bottom) cells in human islets from non-diabetic (black) and type-2 diabetic (red) donors (ND: n = 6 donors; T2D: n = 5 donors). ( H ) Correlation between cilium length and donor age in insulin-positive (Ins + , top) and insulin-negative (Ins-, bottom) cells in human islets from non-diabetic (black) and type-2 diabetic (red) donors (ND: n = 6 donors; T2D: n = 5 donors). ( I ) Primary cilia length in human islets cultured for 7 days in the absence (Ctrl) or presence (PA) of 0.5 mM palmitic acid (means ± SEM; Ctrl: 30 islets; PA: 22 islets; Student’s unpaired t test; 2 donors). ( J ) Confocal microscopy images of human islet from non-diabetic and type-2 diabetic donors immunostained against acetylated tubulin (green) and somatostatin (magenta). ( K ) Number of δ-cells per islet area (means ± SEM; ND: 31 islets, 3 donors; T2D: 32 islets, 2 donors; Student’s unpaired two-tailed t test). ( L ) Shortest distance between primary cilia and δ-cells (means ± SEM; ND: 31 islets, 3 donors; T2D: 32 islets, 2 donors; Student’s unpaired two-tailed t test). ( M ) Correlation between number of δ-cells and shortest distance between δ-cells and primary cilia (ND: 31 islets, 3 donors; T2D: 32 islets, 2 donors). ( N ) Confocal microscopy images of human islets from a non-diabetic and a type-2 diabetic donor immunostained against acetylated tubulin (magenta) and SSTR3 (yellow).

Article Snippet: The following primary antibodies were used in the study; acetylated tubulin (T745, host: mouse, 1:500; Sigma-Aldrich), Acetyl-α-Tubulin (K40) (5335, host: rabbit, 1:500; BioNordika), SSTR3 (E-AB-16077, host: rabbit, 1:200; Elabscience), Arl13b (ab136648, host: mouse, 1:300; Abcam), SST (A0566, host: rabbit, 1:500, Dako), Insulin (A0564, host: guinea pig, 1:500; Dako), SSTR5 (66772-1-Ig, host: mouse, 1:200, Proteintech), SSTR2 (ab134152, host: rabbit, 1:200, Abcam), Pericentrin (ab4448, Abcam, host: rabbit, 1:200), GABBR1 (AGB001AN102, Alomone labs, host: rabbit, 1:200), GLP-1R (Mab 7F38-s, Developmental Studies Hybridoma Bank, host: mouse, 1:10) and GFP (AB16901, Abcam, host: chicken, 1:500).

Techniques: Confocal Microscopy, Two Tailed Test, MANN-WHITNEY, Cell Culture

Journal: The EMBO Journal

Article Title: Somatostatin triggers local cAMP and Ca 2+ signaling in primary cilia to modulate pancreatic β-cell function

doi: 10.1038/s44318-025-00383-7

Figure Lengend Snippet: ( A ) Confocal microscopy image of a mouse islet immunostained for somatostatin and acetylated tubulin. ( B ) Quantification of the number of cilia in close proximity to somatostatin-positive δ-cells and non-δ-cells in mouse islets (means ± SEM; n = 35; N = 7; P = 1.0E-5, Student’s paired t test). ( C ) Confocal microscopy images of mouse islets with transgenic expression of tdtomato in somatostatin-positive δ-cells and immunostained for acetylated tubulin to visualize cilia. ( D ) Quantification of the number of cilia in close proximity to tdtomato-positive or negative cells in mouse islets (means±SEM; n = 64; N = 10; P = 1.1E-12, Student’s paired t test). ( E ) Confocal microscopy image of a mouse islet with transgenic expression of tdtomato in δ-cells (magenta) immunostained for acetylated tubulin (cyan) and SSTR3 (yellow). Arrowheads show SSTR3-positive cilia. ( F ) Confocal microscopy images from two human islets immunostained against somatostatin (magenta), acetylated tubulin (cyan/green) and insulin (yellow). ( G ) Schematic cartoon of SST1.0 (left) and a confocal microscopy image (right) of a mouse islet expressing SST1.0. ( H ) Confocal microscopy image from a mouse islet with transgenic expression of both tdtomato (magenta) and SST1.0 (yellow) in δ-cells and immunostained against acetylated tubulin (cyan). ( I ) Confocal micrograph of a mouse islet with transgenic expression of tdtomato in δ-cells (magenta) and infected with adenovirus to express SST1.0 (green). Boxed region is magnified to the right. Traces are from cilia and cilia-adjacent plasma membrane regions and show SST1.0 fluorescence changes in response to 10 mM glucose. The regions corresponding to traces “C” and “PM” are indicated in the confocal micrograph. ( J ) SST1.0 fluorescence change in a mouse islet stimulated with 1 µM somatostatin. Traces are means ± SEM for 19 cilia and 17 cilia-adjacent plasma membrane regions. ( K ) SST1.0 fluorescence change in the cilium (magenta) and cilium-adjacent plasma membrane (black) within mouse islets exposed to 10 mM glucose (means ± SEM; N = 4 islets; n = 25 cells; two-tailed Student’s paired t test). ( L ) Frequency of SST1.0 fluorescence changes in the cilium (magenta) and cilium-adjacent plasma membrane (black) within mouse islets exposed to 10 mM glucose (means ± SEM; N = 4 islets; n = 25 cells; two-tailed Student’s paired t test). ( M ) Correlation between SST1.0 response amplitude in the plasma membrane (black) and cilium (magenta) and distance to nearest δ-cell. Notice that response amplitude is reduced with increased distance to δ-cells.

Article Snippet: The following primary antibodies were used in the study; acetylated tubulin (T745, host: mouse, 1:500; Sigma-Aldrich), Acetyl-α-Tubulin (K40) (5335, host: rabbit, 1:500; BioNordika), SSTR3 (E-AB-16077, host: rabbit, 1:200; Elabscience), Arl13b (ab136648, host: mouse, 1:300; Abcam), SST (A0566, host: rabbit, 1:500, Dako), Insulin (A0564, host: guinea pig, 1:500; Dako), SSTR5 (66772-1-Ig, host: mouse, 1:200, Proteintech), SSTR2 (ab134152, host: rabbit, 1:200, Abcam), Pericentrin (ab4448, Abcam, host: rabbit, 1:200), GABBR1 (AGB001AN102, Alomone labs, host: rabbit, 1:200), GLP-1R (Mab 7F38-s, Developmental Studies Hybridoma Bank, host: mouse, 1:10) and GFP (AB16901, Abcam, host: chicken, 1:500).

Techniques: Confocal Microscopy, Transgenic Assay, Expressing, Infection, Clinical Proteomics, Membrane, Fluorescence, Two Tailed Test

Journal: The EMBO Journal

Article Title: Somatostatin triggers local cAMP and Ca 2+ signaling in primary cilia to modulate pancreatic β-cell function

doi: 10.1038/s44318-025-00383-7

Figure Lengend Snippet: ( A ) Confocal microscopy image of a primary cilium (acetylated tubulin; magenta) in a human islet that expresses SSTR5 (green). ( B ) Confocal microscopy image of primary cilia (acetylated tubulin; magenta) in a mouse islet that expresses SSTR5 (green). ( C ) Confocal microscopy image of a primary cilium (acetylated tubulin; magenta) in a MIN6 pseudoislet that lack expression of SSTR5 (green). ( D ) An illustration shows the membrane localization of SSTRs, with IC3 loop indicated by the purple box. Sequences of mSSTR2, mSSTR3 and mSSTR5. Conserved motif RxRxxR is highlighted. ( E ) Sequences of SSTR5 and SSTR3 showing the evolutionary conservation of a stretch of amino acids in purple.

Article Snippet: The following primary antibodies were used in the study; acetylated tubulin (T745, host: mouse, 1:500; Sigma-Aldrich), Acetyl-α-Tubulin (K40) (5335, host: rabbit, 1:500; BioNordika), SSTR3 (E-AB-16077, host: rabbit, 1:200; Elabscience), Arl13b (ab136648, host: mouse, 1:300; Abcam), SST (A0566, host: rabbit, 1:500, Dako), Insulin (A0564, host: guinea pig, 1:500; Dako), SSTR5 (66772-1-Ig, host: mouse, 1:200, Proteintech), SSTR2 (ab134152, host: rabbit, 1:200, Abcam), Pericentrin (ab4448, Abcam, host: rabbit, 1:200), GABBR1 (AGB001AN102, Alomone labs, host: rabbit, 1:200), GLP-1R (Mab 7F38-s, Developmental Studies Hybridoma Bank, host: mouse, 1:10) and GFP (AB16901, Abcam, host: chicken, 1:500).

Techniques: Confocal Microscopy, Expressing, Membrane

Journal: The EMBO Journal

Article Title: Somatostatin triggers local cAMP and Ca 2+ signaling in primary cilia to modulate pancreatic β-cell function

doi: 10.1038/s44318-025-00383-7

Figure Lengend Snippet: ( A ) Representative TIRF microscopy recordings of cytosolic (black) and ciliary (magenta) FRET ratio changes in an islet cell expressing mArl13b-EpacS H188 following exposure to 1 µM forskolin and 100 nM somatostatin. ( B ) Means of ±SEM for the mArl13b-EpacS H188 FRET ratio change in mouse islet of cilia (magenta) and cytosol (black) in response to 1 μM forskolin, followed by the addition of 100 nM somatostatin. n cytosol = 23 cells, n cilia = 27 cilia, from three different animals. cilia basal-forskolin P = 0.00000024, cilia forskolin-SST P = 0.00000107, cytosol basal-forskolin P = 0.00000342, cytosol forskolin-SST P = 0.00000446; Sidak’s multiple comparison test. Bottom panel shows means ± SEM for the mArl13b-EpacS H188 FRET ratio change in MIN6 pseudoislet cilia (magenta) and cytosol (black) in response to 1 μM forskolin, followed by the addition of 100 nM somatostatin. n cytosol = 11 cells, n cilia = 18 cilia, from three different preparations. cilia basal-forskolin P = 0.000012, cilia forskolin-SST P = 0.000012, cytosol basal-forskolin P = 0.009468, cytosol forskolin-SST P = 0.013005, assessed with two-way ANOVA, Sidak’s multiple comparison test. ( C ) Means of ±SEM for the mArl13b-EpacS H188 FRET ratio change in MIN6 pseudoislet cilia (magenta) and cytosol (black) in response to TUG-891 (100 μM) followed by the addition of 100 nM somatostatin, and after addition of 10 μM forskolin. n cytosol = 33 cells, n cilia = 41 cilia, from three different preparations. Cilia; basal-TUG P = 0.0000002, TUG-SST P = 0.0000000000005, basal-forskolin P = 0.00002, cytosol; basal-TUG P = 0.0000009, TUG-SST P = 0.00000002, basal-forskolin P = 0.000007. Statistics was assessed with two-way ANOVA, Tukey’s multiple comparison test. ( D ) Quantitative RT-PCR determination of the relative reduction in SSTR3 mRNA following shRNA-mediated knockdown in MIN6 cells (six different experiments; Mann–Whitney U test). ( E ) Confocal microscopy images of primary cilia from MIN6 pseudoislets transfected with control (left) or SSTR3 (right) siRNA and immunostained against acetylated tubulin (green) and SSTR3 (magenta). The SSTR3 immunoreactivity in the cilium is quantified below (means ± SEM; n siCtrl = 83, n siSSTR3 = 86 cilia from three separate experiments; Mann–Whitney U test). ( F ) TIRF microscopy recordings of Arl13b-EpacS H188 FRET ratio in control (left) and SSTR3 KD (right) MIN6 pseudoislets exposed to 1 µM forskolin and 100 nM somatostatin. Magenta color indicates recordings from the cilium and black from the cell body (means ± SEM for 53 cells for control, 46 cells for knockdown). ( G ) Quantifications of the Arl13b-EpacS H188 FRET ratio from siSSTR3 transfected MIN6 pseudoislets in the presence of forskolin and somatostatin (normalized to pre-stimulatory level) (means ± SEM; 25 cilia-cytosol pairs for control and 22 cilia-cytosol pairs for knockdown. **** P = 0.000064; two-way ANOVA, Uncorrected Fisher´s LSD.). ( H ) Quantifications of the Arl13b-EpacS H188 FRET ratio following shRNA-mediated knockdown of SSTR3 in MIN6 pseudoislets exposed to forskolin and somatostatin (normalized to pre-stimulatory level) (means ± SEM; WT cilia = 50, WTcytosol = 44, KD cilia = 89, KD cytosol = 106. **** P = 0.000058; two-way ANOVA, Uncorrected Fisher´s LSD).

Article Snippet: The following primary antibodies were used in the study; acetylated tubulin (T745, host: mouse, 1:500; Sigma-Aldrich), Acetyl-α-Tubulin (K40) (5335, host: rabbit, 1:500; BioNordika), SSTR3 (E-AB-16077, host: rabbit, 1:200; Elabscience), Arl13b (ab136648, host: mouse, 1:300; Abcam), SST (A0566, host: rabbit, 1:500, Dako), Insulin (A0564, host: guinea pig, 1:500; Dako), SSTR5 (66772-1-Ig, host: mouse, 1:200, Proteintech), SSTR2 (ab134152, host: rabbit, 1:200, Abcam), Pericentrin (ab4448, Abcam, host: rabbit, 1:200), GABBR1 (AGB001AN102, Alomone labs, host: rabbit, 1:200), GLP-1R (Mab 7F38-s, Developmental Studies Hybridoma Bank, host: mouse, 1:10) and GFP (AB16901, Abcam, host: chicken, 1:500).

Techniques: Microscopy, Expressing, Comparison, Quantitative RT-PCR, shRNA, Knockdown, MANN-WHITNEY, Confocal Microscopy, Transfection, Control

Journal: The EMBO Journal

Article Title: Somatostatin triggers local cAMP and Ca 2+ signaling in primary cilia to modulate pancreatic β-cell function

doi: 10.1038/s44318-025-00383-7

Figure Lengend Snippet: ( A ) Quantification of cytosolic area selected in MIN6 pseudoislets. Fluorescence intensity is from control (black) and SSTR3 KD (gray) cells immunostained for SSTR3. (means ± SEM; n ctrl = 50 and n K D = 48, 3 different preparations, no statistical difference by Mann–Whitney U test, unpaired.). ( B ) Quantifications of line profiles drawn along cilia of MIN6 pseudoislets for acetylated tubulin. Acetylated tubulin signal is unaffected on the left. (means ± SEM; n ctrl = 83 and n KD = 86, three different preparations, no statistical difference by Mann–hitney U test, unpaired.). ( C ) Quantifications of line profiles drawn along cilia of MIN6 pseudoislets positive for SSTR3. Ciliary SSTR3 signal is significantly reduced in shSSTR3 expressing cells (means ± SEM; n ctrl = 44 and n KD = 40 cilia, 1 preparation; Mann–Whitney U test, unpaired).

Article Snippet: The following primary antibodies were used in the study; acetylated tubulin (T745, host: mouse, 1:500; Sigma-Aldrich), Acetyl-α-Tubulin (K40) (5335, host: rabbit, 1:500; BioNordika), SSTR3 (E-AB-16077, host: rabbit, 1:200; Elabscience), Arl13b (ab136648, host: mouse, 1:300; Abcam), SST (A0566, host: rabbit, 1:500, Dako), Insulin (A0564, host: guinea pig, 1:500; Dako), SSTR5 (66772-1-Ig, host: mouse, 1:200, Proteintech), SSTR2 (ab134152, host: rabbit, 1:200, Abcam), Pericentrin (ab4448, Abcam, host: rabbit, 1:200), GABBR1 (AGB001AN102, Alomone labs, host: rabbit, 1:200), GLP-1R (Mab 7F38-s, Developmental Studies Hybridoma Bank, host: mouse, 1:10) and GFP (AB16901, Abcam, host: chicken, 1:500).

Techniques: Fluorescence, Control, MANN-WHITNEY, Expressing

Journal: The EMBO Journal

Article Title: Somatostatin triggers local cAMP and Ca 2+ signaling in primary cilia to modulate pancreatic β-cell function

doi: 10.1038/s44318-025-00383-7

Figure Lengend Snippet: ( A ) TIRF microscopy image of a mouse islet expressing 5HT 6 -GGECO1. Traces below show the GGECO1 fluorescence change in cilia (magenta) and cytosol (green) in response to 100 nM somatostatin. ( B ) TIRF microscopy image of a MIN6 pseudoislet expressing 5HT 6 -GGECO1. Traces below show the GGECO1 fluorescence change in cilia (magenta) and cytosol (green) in response to 100 nM somatostatin. ( C ) Event count of all cilia Ca 2+ changes in mouse islet cells under resting conditions and following addition of 100 nM somatostatin (means ± SEM; n = 6 islets, 72 cilia; P = 0.0312 Wilcoxon test). ( D ) Event count of all cilia Ca 2+ flashes in MIN6 pseudoislet cells under resting conditions and following addition of 100 nM somatostatin (means ± SEM; n = 17 islets, 95 cilia; P = 0.0008 Wilcoxon test). ( E ) Event count of ciliary Ca 2+ changes of MIN6 pseudoislets following the addition of 100 nM somatostatin in control cells (black) and SSTR3 KD cells (magenta) (means ± SEM; n ctrl = 10 islets and 40 cilia, n KD = 11 islets and 30 cilia from three different preparations, P sictrl = 0.003, no change in siSSTR3, assessed by Sidak´s multiple comparison test). ( F ) Event count of all ciliary Ca 2+ changes in mouse islet cells that were either cultured under control condition or in the presence of pertussis toxin for 18 h and exposed to 100 nM somatostatin (means ± SEM; n ctrl = 6, 72 cilia, n PT = 7, 65 cilia; islets from four different preparations, SST response in control P = 0.0212, SST response in Pertussis-toxin treated islets not significant, assessed by Sidak´s multiple comparison). ( G ) Event count of ciliary Ca 2+ changes in mouse islet cells in response to 100 nM somatostatin, followed by the addition of 100 μM TUG-891. Means ± SEM for 25 cilia, from three different animals. *** P = 0.0004, ** P = 0.0021; two-way ANOVA, Dunn’s multiple comparison test. ( H ) Traces show the 5HT 6 -GGECO1 fluorescence change in cilia (purple) and cytosol (green) of mouse islet cells. Event count of ciliary Ca 2+ changes from mouse islet cells in response to 10 μM H89 are shown to the right (means ± SEM; 8 islets; 82 cilia; 3 different preparations. **** P = 0.000000001, Wilcoxon-matched pair t test). ( I ) Event count of ciliary Ca 2+ changes from mouse islets in response to 100 nM ghrelin (means ± SEM; 13 islets; 131 cilia from seven different preparations. *** P = 0.0008, Wilcoxon-matched pair t test). To the right is shown event count of ciliary Ca 2+ flashes from mouse islet in response to 100 nM ghrelin, followed by 100 nM SST (means ± SEM; 8 islets; 72 cilia from five different preparation. Dunn’s multiple comparisons test; basal-ghrelin P = 0.0035, basal-SST P = 0.0000000005, ghrelin-SST P = 0.0053). ( J ) Percentage of cilia in mouse islets that exhibit Ca 2+ changes in response to SST ( n = 72) or Ghrelin ( n = 46). ( K ) TIRF microscopy image of a mouse islet expressing 5HT 6 -GGECO1. Highlighted areas are: yellow and orange (δ-cells), green (β-cells), light purple (cilia close to δ-cells), purple (cilia far from δ-cells). ( L ) Ca 2+ concentration changes within the shaded areas in ( K ) in response to 100 nM ghrelin and 100 nM somatostatin. Scale bars: 5 µm.

Article Snippet: The following primary antibodies were used in the study; acetylated tubulin (T745, host: mouse, 1:500; Sigma-Aldrich), Acetyl-α-Tubulin (K40) (5335, host: rabbit, 1:500; BioNordika), SSTR3 (E-AB-16077, host: rabbit, 1:200; Elabscience), Arl13b (ab136648, host: mouse, 1:300; Abcam), SST (A0566, host: rabbit, 1:500, Dako), Insulin (A0564, host: guinea pig, 1:500; Dako), SSTR5 (66772-1-Ig, host: mouse, 1:200, Proteintech), SSTR2 (ab134152, host: rabbit, 1:200, Abcam), Pericentrin (ab4448, Abcam, host: rabbit, 1:200), GABBR1 (AGB001AN102, Alomone labs, host: rabbit, 1:200), GLP-1R (Mab 7F38-s, Developmental Studies Hybridoma Bank, host: mouse, 1:10) and GFP (AB16901, Abcam, host: chicken, 1:500).

Techniques: Microscopy, Expressing, Fluorescence, Control, Comparison, Cell Culture, Concentration Assay

Journal: The EMBO Journal

Article Title: Somatostatin triggers local cAMP and Ca 2+ signaling in primary cilia to modulate pancreatic β-cell function

doi: 10.1038/s44318-025-00383-7

Figure Lengend Snippet: ( A ) Confocal microscopy images of mouse islets expressing Gli2-Halo JFX650 (yellow) and immunostained against acetylated tubulin (magenta) and insulin (cyan). Eighteen hours of treatment with 100 nM SAG or 100 nM SST induced translocation of Gli2 to the nucleus. ( B ) Fraction of β-cell nuclei positive for Gli2- Halo JFX650 in control mouse islets and in islets treated for 18 h with 100 nM SAG or 100 nM somatostatin (means ± SEM; n control = 34, n SAG = 40, n SST = 39 islets from two experiments; Student’s unpaired two-tailed t test). ( C ) Quantifications of cilia length (acetylated tubulin staining) from control (black), 18 h SST-treated (yellow) and 18 h SAG-treated (blue) mouse islet cells (means ± SEM; n ctrl = 111, n SST = 87, and n SAG = 105 cilia from two different preparations; Student’s unpaired two-tailed t test). ( D ) Quantifications of ciliary Ptch1 intensity from control (black), SST-treated (100 nM, 15 min; yellow) and SAG-treated (100 nM, 15 min; blue) mouse islet cells (means ± SEM; n = 79, 27 and 77 cilia; Student’s unpaired two-tailed t test). ( E ) Means ± SEM for the nucleus/cilia Gli2 ratio change in shSSTR3 transfected MIN6 cells following 18 h exposure to 100 nM somatostatin ( P ctrl =0.0418, P shSSTR3 > 0.999, assessed with Sidak´s multiple comparison test). ( F ) TIRF microscopy recordings of cilia Ca 2+ from mouse islets expressing 5HT 6 -GGECO1 and stimulated with 100 nM somatostatin. The islets were either cultured under control condition (black) or in the presence of somatostatin (purple) for 18 h followed by a 60 min recovery period. ( G ) Histogram showing ciliary Ca 2+ changes following 18 h incubation with (blue) or without (black) somatostatin followed by 30 min in the absence of somatostatin and acute re-application of 100 nM somatostatin ( n ctrl =16 cilia, n SST = 27 cilia; ciliary Ca 2+ changes response to SST is quantified; P Ctrl = 0.0016, P SST = 0.00011; Wilcoxon test). ( H ) Quantification of the effect of the effect of long-term somatostatin treatment on somatostatin-induced cilia Ca 2+ changes (means ± SEM; n ctrl = 3 islets, n SST = 4 islets). ( I ) Confocal image of a MIN6 cell expressing Gli2-GFP (green) and Arl13b-Parvalbumin (cyan) and immunostained against acetylated tubulin (magenta). Means ± SEM for the nucleus/cilia Gli2 ratio change in MIN6 cells expressing Arl13b-Parvalbumin and exposed to somatostatin for 18 h ( n ctrl =132, n SST = 132 cells from 4 independent experiments, Sidak´s multiple comparison test). ( J ) Changes in SSTR3 expression in MIN6 cells exposed to 100 nM somatostatin or 100 nM SAG for 18 h (means ± SEM; n = 2–3 experiments). ( K ) Changes in GLI2 expression in control or SSTR3 knockdown MIN6 cells exposed to 100 nM somatostatin or 100 nM SAG for 18 h (means ± SEM; n = 3–4 experiments; * P = 0.0376; Tukey´s multiple comparison test). ( L ) Changes in GLI3 expression in control or SSTR3 knockdown MIN6 cells exposed to 100 nM somatostatin or 100 nM SAG for 18 h (means ± SEM; n = 3–4 experiments; ** P = 0.0047; Tukey´s multiple comparison test). ( M ) Pseudo-colored TIRF microscopy images showing the increase cytosolic cAMP in response to 100 nM GLP-1 reported with EpacS H187 . Means ± SEM for the FRET ratio changes from MIN6 pseuodo-islets treated for 18 h with SAG (yellow), SST (purple) and SAG + SST (blue) are shown to the right (control-SAG P = 0.000008531, control-SST P = 0.000000004, control-SAG + SST P = 0.000002794; assessed by Dunn´s multiple comparison test; n ctrl = 143, n SAG = 161, n SST = 132 and n SAG+SST = 131 cells from three independent experiments). ( N , O ) Traces (means ± SEM) and scatter plot from EpacS H187 -expressing control or SSTR3 knockdown MIN6 pseudoislets exposed or not to somatostatin for 18 h, followed by acute stimulation with 100 nM GLP-1 (WT-WT_SST P < 0.000000000000001, WT_SST-KD_SST P = 0.0000002, WT-KD P = 0.00070; Sidak´s multiple comparison test; n WT = 240, n WT-SST = 200, n shSSTR3 = 259, n shSSTR3-SST = 148 from three different preparations). ( P ) A model of somatostatin signaling in the β-cell primary cilium.

Article Snippet: The following primary antibodies were used in the study; acetylated tubulin (T745, host: mouse, 1:500; Sigma-Aldrich), Acetyl-α-Tubulin (K40) (5335, host: rabbit, 1:500; BioNordika), SSTR3 (E-AB-16077, host: rabbit, 1:200; Elabscience), Arl13b (ab136648, host: mouse, 1:300; Abcam), SST (A0566, host: rabbit, 1:500, Dako), Insulin (A0564, host: guinea pig, 1:500; Dako), SSTR5 (66772-1-Ig, host: mouse, 1:200, Proteintech), SSTR2 (ab134152, host: rabbit, 1:200, Abcam), Pericentrin (ab4448, Abcam, host: rabbit, 1:200), GABBR1 (AGB001AN102, Alomone labs, host: rabbit, 1:200), GLP-1R (Mab 7F38-s, Developmental Studies Hybridoma Bank, host: mouse, 1:10) and GFP (AB16901, Abcam, host: chicken, 1:500).

Techniques: Confocal Microscopy, Expressing, Translocation Assay, Control, Two Tailed Test, Staining, Transfection, Comparison, Microscopy, Cell Culture, Incubation, Knockdown

Journal: The EMBO Journal

Article Title: Somatostatin triggers local cAMP and Ca 2+ signaling in primary cilia to modulate pancreatic β-cell function

doi: 10.1038/s44318-025-00383-7

Figure Lengend Snippet: ( A ) Confocal microscopy images of MIN6 cells expressing Gli2-GFP (green) and immunostained against acetylated tubulin (magenta). 18 h treatment with 100 nM SAG and 100 nM SST induced translocation of Gli2 to the nucleus. ( B ) Means ± SEM for the nucleus/cilia Gli2 ratio change in MIN6 cells ( P SAG = 0.0345, P SST = 0.0266, P SAG+SST = 0.0186 all compared to control and assessed by Sidak´s multiple comparison test). ( C , D ) Islet averages ( C ) and example recordings ( D ) of glucose-induced R-GECO1 fluorescence changes in MIN6 pseudoislets treated for 18 h with DMSO (control), 100 nM SAG, 100 nM somatostatin or 100 nM SAG in combination with 100 nM somatostatin ( n = 3 replicates). ( E , F ) Islet averages ( E ) and example recordings ( F ) of glucose-induced R-GECO1 fluorescence changes in control and SSTR3 knockdown MIN6 pseudoislets treated for 18 h with DMSO (control) or 100 nM somatostatin ( n = 3 replicates).

Article Snippet: The following primary antibodies were used in the study; acetylated tubulin (T745, host: mouse, 1:500; Sigma-Aldrich), Acetyl-α-Tubulin (K40) (5335, host: rabbit, 1:500; BioNordika), SSTR3 (E-AB-16077, host: rabbit, 1:200; Elabscience), Arl13b (ab136648, host: mouse, 1:300; Abcam), SST (A0566, host: rabbit, 1:500, Dako), Insulin (A0564, host: guinea pig, 1:500; Dako), SSTR5 (66772-1-Ig, host: mouse, 1:200, Proteintech), SSTR2 (ab134152, host: rabbit, 1:200, Abcam), Pericentrin (ab4448, Abcam, host: rabbit, 1:200), GABBR1 (AGB001AN102, Alomone labs, host: rabbit, 1:200), GLP-1R (Mab 7F38-s, Developmental Studies Hybridoma Bank, host: mouse, 1:10) and GFP (AB16901, Abcam, host: chicken, 1:500).

Techniques: Confocal Microscopy, Expressing, Translocation Assay, Control, Comparison, Fluorescence, Knockdown

Journal: The EMBO Journal

Article Title: Somatostatin triggers local cAMP and Ca 2+ signaling in primary cilia to modulate pancreatic β-cell function

doi: 10.1038/s44318-025-00383-7

Figure Lengend Snippet: Reagents and tools table

Article Snippet: The following primary antibodies were used in the study; acetylated tubulin (T745, host: mouse, 1:500; Sigma-Aldrich), Acetyl-α-Tubulin (K40) (5335, host: rabbit, 1:500; BioNordika), SSTR3 (E-AB-16077, host: rabbit, 1:200; Elabscience), Arl13b (ab136648, host: mouse, 1:300; Abcam), SST (A0566, host: rabbit, 1:500, Dako), Insulin (A0564, host: guinea pig, 1:500; Dako), SSTR5 (66772-1-Ig, host: mouse, 1:200, Proteintech), SSTR2 (ab134152, host: rabbit, 1:200, Abcam), Pericentrin (ab4448, Abcam, host: rabbit, 1:200), GABBR1 (AGB001AN102, Alomone labs, host: rabbit, 1:200), GLP-1R (Mab 7F38-s, Developmental Studies Hybridoma Bank, host: mouse, 1:10) and GFP (AB16901, Abcam, host: chicken, 1:500).

Techniques: Recombinant, Plasmid Preparation, Sequencing, Virus, Cloning, Modification, Software, Microscopy, Real-time Polymerase Chain Reaction, SYBR Green Assay, Ligation

Journal: The Journal of Biological Chemistry

Article Title: Golgi Phosphoprotein 3 Determines Cell Binding Properties under Dynamic Flow by Controlling Golgi Localization of Core 2 N -Acetylglucosaminyltransferase 1 *

doi: 10.1074/jbc.M112.346528

Figure Lengend Snippet: Interaction of GOLPH3 with C2GnT1 CT in vitro. A, GOLPH3 Western blot of the pulldown from KG1a cell lysate with biotinylated control or C2GnT1 1–20-aa peptide. B, GOLPH3 Western blot analysis of the pulldown from the lysate of BL21 bacteria transformed with GST-GOLPH3 cDNA and from purified recombinant GOLPH3 using biotinylated control or C2GnT1(1–20 aa) peptide. C, C2GnT1 Western blot analysis of the pulldown from KG1a cell lysate using GST (control) or GOLPH3-GST immobilized on glutathione-resin.

Article Snippet: The pGEX-6P-2 vector and the construct containing the 897-bp GOLPH3 cDNA was then transformed into E. coli strain BL21(DE3) host cells (New England Biolabs).

Techniques: In Vitro, Western Blot, Transformation Assay, Purification, Recombinant

Journal:

Article Title: Nocardia sp. Carboxylic Acid Reductase: Cloning, Expression, and Characterization of a New Aldehyde Oxidoreductase Family

doi: 10.1128/AEM.70.3.1874-1881.2004

Figure Lengend Snippet: Strains and plasmids used in this study

Article Snippet: Ampicillin (100 μg/ml) was incorporated into LB medium to select for recombinants, and isopropyl-β- d -thiogalactopyranoside (IPTG) (1 mM) and/or 5-bromo-4-chloro-3-indolyl-β- d -galactopyranoside (X-Gal) (80 μg/ml) were included for recombinant identification. table ft1 table-wrap mode="anchored" t5 TABLE 1. caption a7 Strain or plasmid Relevant properties a Source or reference Strains Nocardia sp. strain NRRL 5646 Wild type 24 E. coli JM 109 RecA − , recombinant vector host strain Promega E. coli BL21 (DE3) Inducible T7 RNA polymerase, Amp r Stratagene E. coli BL21-CodonPlus (DE3)-RP Has argU and proL tRNA genes to provide tRNA for rare codons found in many GC-rich bacteria Stratagene Plasmids pGEM-T easy T/A PCR cloning vector, Amp r Promega pHAT10 Cloning vector for in-frame addition of HAT tag (MTMITPSLKDHLIHNVH KEEHAHAHNKIDDDDKVDGS) to the N terminus of CAR, pLac for expression, Amp r Clontech pHAT-305 pHAT-10 with car insert This study pHAT-DHFR Positive control expression vector with dihydrofolate reductase gene tagged with HAT at the N terminus Clontech Open in a separate window a Amp r , ampicillin resistance.

Techniques: Plasmid Preparation, Recombinant, PCR Cloning, Clone Assay, Expressing, Positive Control

Journal:

Article Title: Nocardia sp. Carboxylic Acid Reductase: Cloning, Expression, and Characterization of a New Aldehyde Oxidoreductase Family

doi: 10.1128/AEM.70.3.1874-1881.2004

Figure Lengend Snippet: SDS-PAGE and Western blot analysis of Nocardia CAR expression in E. coli carrying pHAT10-based vectors. Samples were taken from the lysates of E. coli cells carrying different vectors and were separated in duplicate by SDS-10% PAGE and either stained with 0.1% Coomassie blue R-250 (A) or subjected to Western blotting using a HAT-specific antibody (B). Lane 1, molecular mass markers for myosin (209 kDa), β-galactosidase (124 kDa), bovine serum albumin (80 kDa), ovalbumin (49.1 kDa), carbonic anhydrase (34.8 kDa), soybean trypsin inhibitor (21.5 kDa), lysozyme (20.6 kDa), and aprotinin (7.1 kDa). Lane 2, E. coli CodonPlus cells carrying pHAT-DHFR. Lane 3, E. coli BL21(DE3) cells carrying pHAT-305 (induced); lane 4, E. coli CodonPlus cells carrying pHAT-305 (induced). Lane 5, purified HAT-CAR. Lane 6, E. coli CodonPlus cells carrying pHAT10.

Article Snippet: Ampicillin (100 μg/ml) was incorporated into LB medium to select for recombinants, and isopropyl-β- d -thiogalactopyranoside (IPTG) (1 mM) and/or 5-bromo-4-chloro-3-indolyl-β- d -galactopyranoside (X-Gal) (80 μg/ml) were included for recombinant identification. table ft1 table-wrap mode="anchored" t5 TABLE 1. caption a7 Strain or plasmid Relevant properties a Source or reference Strains Nocardia sp. strain NRRL 5646 Wild type 24 E. coli JM 109 RecA − , recombinant vector host strain Promega E. coli BL21 (DE3) Inducible T7 RNA polymerase, Amp r Stratagene E. coli BL21-CodonPlus (DE3)-RP Has argU and proL tRNA genes to provide tRNA for rare codons found in many GC-rich bacteria Stratagene Plasmids pGEM-T easy T/A PCR cloning vector, Amp r Promega pHAT10 Cloning vector for in-frame addition of HAT tag (MTMITPSLKDHLIHNVH KEEHAHAHNKIDDDDKVDGS) to the N terminus of CAR, pLac for expression, Amp r Clontech pHAT-305 pHAT-10 with car insert This study pHAT-DHFR Positive control expression vector with dihydrofolate reductase gene tagged with HAT at the N terminus Clontech Open in a separate window a Amp r , ampicillin resistance.

Techniques: SDS Page, Western Blot, Expressing, Staining, Purification

Journal: Frontiers in Microbiology

Article Title: Human Single-chain Variable Fragments Neutralize Pseudomonas aeruginosa Quorum Sensing Molecule, 3O-C12-HSL, and Prevent Cells From the HSL-mediated Apoptosis

doi: 10.3389/fmicb.2020.01172

Figure Lengend Snippet: Production and characterization of HuscFvs to 3O-C12-HSL. (A) Schematic diagram of the inserted DNA construct in pLATE52 where the DNA sequence coding for HuscFv ( vh-linker-vl ) was flanked with DNA sequences of 6 × His at the 5′ end and E-tag at the 3′ end. (B) Amplicons of huscfv -LIC fragments (∼ 850 bp) for sub-cloning into pLATE52 vector. M, 100 bp-plus DNA ladder; 1–3, huscfv -LIC amplicons of three representatives transformed NiCo21(DE3) E. coli clones. Numbers at the left are DNA sizes in bp. (C) Stained SDS-PAGE-separated purified recombinant HuscFvs. M, protein standard; 1–3, purified HuscFv-E44, HuscFv-F15 and HuscFv-F19, respectively. Numbers at the left are protein masses in kDa. (D) CD spectra of the refolded HuscFv-E44, HuscFv-F15, and HuscFv-F19.

Article Snippet: After PCR screening and DNA sequencing, the recombinant plasmids were introduced into an expression host, NiCo21(DE3) E. coli (New England Biolabs, St. Albans, Herts, United Kingdom), and the transformed bacteria were grown at 37°C for 16 h on LB agar containing 100 μg/ml of ampicillin.

Techniques: Construct, Sequencing, Subcloning, Plasmid Preparation, Transformation Assay, Clone Assay, Staining, SDS Page, Purification, Recombinant