Journal: bioRxiv

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

doi: 10.1101/2024.06.05.597562

Figure Lengend Snippet: A. TIRF microscopy images depicting a MIN6 cell expressing Epac-S H187 , targeted to the cilium by Smoothened. Images on top are from resting conditions, and images at the bottom are in the presence of 10 µM forskolin. B. Representative recording of Epac-S H187 FRET ratio (CFP/YFP) changes in the cilium and cytosol in response to 10 µM forskolin. Below are shown the ratio between resting ciliary and cytosolic CFP/YFP FRET ratio in Smo-EpacS H187 (n=14 cells from 4 experiments), 5HT 6 -EpacS H187 (n=24 cells from 8 experiments) and mArl13b-EpacS H188 (n=8 cells in 2 experiments) expressing MIN6 cells and mArl13b-EpacS H188 (n=39 cells in 5 independent experiments) expressing mouse islet cells. C. Pseudo-colored TIRF images of a MIN6 cell showing an increase in ciliary and cytosolic cAMP in response to 10 µM forskolin and 100 µM IBMX reported with mArl13b-RFlincA. Fluorescence changes over time are shown below. D. TIRF microscopy recordings of Smo-EpacS H187 FRET ratio in the cilium and cytosol of MIN6 cells following addition and washout of forskolin. Data presented as means for 10 cells. E. Amplitude-normalized traces from D highlighting the kinetics during forskolin-evoked cAMP increases in the cilia and cytosol Shown to the right are calculations of the time-constant for the forskolin-induced rise of cAMP. F. Amplitude-normalized traces from D highlighting the kinetics during cAMP lowering in the cilia and cytosol following forskolin washout. Shown to the right are calculations of the time-constant for the forskolin-induced rise of cAMP. Statistical significance was assessed with the two-tailed paired Student’s t-test (no difference). G. Representative confocal microscopy images of mouse islets immunostained against the GLP-1 receptor (yellow), insulin (cyan) and GABBR1 (cilia base; magenta) and treated or not with 100 nM GLP-1 for 15 min. H. Representative TIRF microscopy recordings of Smo-EpacS H187 FRET ratio changes in the cytosol (black) and cilium (magenta) of MIN6 cells exposed to 100 nM GLP-1, 10 μM forskolin, and 100 μM IBMX. I. Difference in half-maximal cAMP increases in the cytosol and cilium in response to forskolin (1 and 10 µM), IBMX (50 µM), a combination of forskolin and IBMX (F+I) or GLP-1 (100 nM) (n=11, 22, 16, 12 and 17 cells from 3-6 experiments). Statistical significance was assessed with one-sample t-test. I. Means of ± S.E.M. for the Smo-EpacS H187 FRET ratio change in the cytosol (black) and cilia (magenta) in response to Forskolin, IBMX, forskolin+IBMX or GLP-1 (two-tailed paired Student’s t-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: Microscopy, Expressing, Fluorescence, Two Tailed Test, Confocal Microscopy

Journal: The journals of gerontology. Series A, Biological sciences and medical sciences

Article Title: Preliminary Evidence of Differential Expression of Myogenic and Stress Factors in Skeletal Muscle of Older Adults With Low Muscle Strength.

doi: 10.1093/gerona/glac002

Figure Lengend Snippet: Figure 3. Expression of stress markers in skeletal muscle tissue (m. vastus lateralis) of older adults with preserved muscle strength (PMS) and low muscle strength (LMS). Bar graphs represent the average of each condition, and superimposed black dots represent individual data points. Groups were compared with an unpaired t-test (normal data: CHOP) or Mann–Whitney U test (non-normal data: p65NF-κB, ERK1/2). Representative immunoblot protein images of each target are shown for 2 participants per group. p65NF-Κb = p65 nuclear factor kappa-light-chain-enhancer of activated B cells; CHOP = C/EBP homologous protein; ERK1/2 = extracellular signal-regulated kinases 1 and 2.

Article Snippet: Next, membranes were incubated overnight at 4°C in TBS-T containing ow nloaded from https://academ ic.oup.com /biom edgerontology/article/77/6/1121/6497516 by guest on 02 M arch 2024 5% nonfat milk or 5% BSA with the following antibodies: Catabolism: phospho forkhead box O1/3a (p-FOXO1/3a; CST9464S), total FOXO3a (CST-2497S), muscle-specific RING finger protein 1 (MuRF1; sc-32920), muscle atrophy F-box (MAFbx; ECM Biosciences AP2041), microtubule-associated protein 1A/1Blight chain 3 (LC3b; CST-3868), autophagy-related protein 12 (Atg12; CST-4180S), p62 (Progen GP62-C); Oxidative capacity: citrate synthase (CST-14309S), peroxisome proliferator-activated receptor gamma coactivator 1-alpha (PGC1α; sc-517380), cytochrome c oxidase subunit 4 (COX-IV; sc-37673); Stress: phospho nuclear factor kappa B (p-p65NF-κB; sc-136548, 1/500), p65NFκB (sc-372, 1/1 000), C/EBP homologous protein (CHOP; CST2895S), phospho extracellular signal-regulated kinase 1/2 (ERK1/2; CST-4370), ERK1/2 (CST-4695); Myogenic capacity: paired box protein 7 (Pax7; DSHB), MyoD (sc-760), desmin (NBP1-97811).

Techniques: Expressing, MANN-WHITNEY, Western Blot