Fig 2: Silencing Stathmin-2 increased glucagon secretion and overexpression of stathmin-2 suppressed glucagon secretion in aTC1-6 cells. Wild type (wt; control) and stathmin-2 depleted (Stmn2-KD) aTC1-6 cells were pre-incubated 2 h in serum-free medium and then incubated with or without KCl (55 mM) for 15 min. (A) Glucagon secretion is significantly stimulated by KCL in wt cells, while in Stmn-KD cells, basal glucagon secretion is increased and does not respond to KCl. *p < 0.01 compared to basal secretion in wt cells. (B) Stmn2 mRNA levels are decreased by about 70% after siRNA-mediated depletion in aTC1-6 cells. Values are means ± SEM (n = 5), *p < 0.01. (C) Stathmin-2 protein levels are depleted after siRNA-mediated silencing of Stmn2. C (control); KD (gene silenced). Beta-actin was used as a loading control. (D) Proglucagon mRNA levels are not affected by siRNA-mediated depletion of stathmin-2. (E) Glucagon secretion is inhibited by overexpression of Stmn2. aTC1-6 cells were transfected with pcDNA3.1 (+) MAr-stmn2 or empty vector (negative control). Both basal and K+-stimulated glucagon secretion were inhibited by overexpression of Stmn2. Values are means ± SEM (n = 4). *p < 0.05; **p < 0.001 compared to unstimulated control.

Fig 3: Altered ratios of stathmin-2 and glucagon in islets of STZ-induced diabetic mice. Islets were immunostained for stathmin-2 (Stmn2) and glucagon and images were acquired as described in Methods. (A) Expression of glucagon and Stmn2 were determined in islets of non-diabetic and diabetic mice by immunofluorescence intensity analysis. (B) Ratios of glucagon: Stmn2 levels were calculated per µm2 of islets in non-diabetic and diabetic mice. (C) Linear regression analysis on binary image intensities of the Stmn2 and glucagon. Filled circles and open squares demonstrate values in non-diabetic and diabetic islets, respectively. (D) Expression of Stmn2 and Gcg mRNA levels were determined in islets of non-diabetic (n = 4) and diabetic (n = 4) mice by qRT-PCR. Gene expression levels were normalized to that of 18S rRNA. For each gene, alterations in the diabetic condition were normalized by the corresponding control group and expressed as percent of matched control. Comparison between control and STZ groups was done by t-test, α = 0.05

Fig 4: Stathmin-2 localizes to secretory granules in aTC1-6 cells. aTC1-6 cells were immunostained using primary antibodies against glucagon (GCG, green) and stathmin-2 (Stmn2, red). DAPI (blue) indicates the nucleus in the merged image. Resolution of the images was extended by applying Nyquist XY scan and then 2D- Deconvolution in NIS Elements image analysis software. Images are representative of four biological replicates with 3 technical replicates each. (A) Areas of yellow in the merged image show colocalization of glucagon and Stmn2. (B) Linear regression analysis of binary intensities of glucagon and Stmn2 predicts a significant (p < 0.001) correlation. Each value represents mean intensities of 5-7 cells. The secretion of both glucagon and Stmn2 (C) was significantly increased after KCl stimulation (KCl Stim)for 15 min. (D) Cell Stmn2 and glucagon levels show reduction following KCl stimulation (KCl Stim). Values are expressed as mean ± SEM (n = 5). *p < 0.05. Stmn2 colocalizes with the secretory granule proteins ChgA (E) and VAMP2 (F), as indicated by yellow punctate staining. (G) The extent of colocalization was analyzed by Pearson correlation coefficient for Stmn2 with ChgA or with VAMP2.

Fig 5: Stathmin-2 is present in a-cells, but not ß cells, in murine pancreatic islets. Pancreata of C57BL/6 mice (n = 7; 5 µm sections) were immunostained for glucagon (GCG), stathmin-2 (Stmn2) and insulin (INS). Images were acquired and analyzed for co-localization as described in Figure 1. (A) Both glucagon and Stmn2 localize to the mantle of the islets, and areas of yellow in the merged image demonstrate dual positive alpha cells (glucagon+ and Stmn2+). (B) Pearson's correlation coefficient for colocalization of Stmn2 and glucagon or insulin. (C) Linear regression analysis predicts a strong positive correlation between the binary intensities of glucagon and Stmn2 (p < 0.001). (D) There is no correlation between the binary intensities of insulin and Stmn2. (E,F) Colocalization of Stmn2 and the trans-Golgi marker TGN46 in murine pancreatic islets. Areas of white (arrows in the magnified panel) indicate co-localization of glucagon, Stmn2 and TGN46 in islet clusters (E) and a single islet (F).