Fig. 3: Gem is required and sufficient for the effects of OXT on astrocytes in vitro.
From: OXTR-mediated signaling in astrocytes contributes to anxiolysis
a, b Gem siRNA knockdown prevents OXT-induced prolongation of primary processes (a; interaction: F1,405 = 10.58, p < 0.001; **p = 0.008 scRNA/Veh vs. scRNA/OXT; *p = 0.016 siRNA/OXT vs. scrRNA/Veh) and increase in primary process number (b; interaction: F1,491 = 3.204, p = 0.074; p = 0.09 for scRNA/Veh vs. scRNA/OXT; ***p < 0.001 siRNA-pretreated vs. scRNA-pretreated/OXT astrocytes). Cells were transfected with either Gem siRNA or a control oligonucleotide (scrRNA) 48 h prior to OXT (500 nM, 10 min). Representative Western blot bands of successful knockdown (scRNA/Veh vs. siRNA/Veh) are shown below. c Length of longest primary process after exposure to the ROCK-inhibitor y-27632 (1 µM, 30 min) in primary astrocytes transfected with Gem siRNA (U = 5936, **p = 0.009). d Phalloidin/F-actin immunofluorescence after Gem knockdown and subsequent OXT exposure (500 nM, 10 min; interaction: F1,37 = 0.588, p = 0.448; **p = 0.003 scrRNA/Veh vs. scrRNA/OXT, p = 0.063 siRNA/Veh vs. siRNA/OXT). e Relative phosphorylation of MYPT at Thr696 following Gem knockdown and subsequent OXT stimulation (interaction: F1,12 = 15.42, p = 0.006; **p = 0.003 siRNA/Veh vs. siRNA/OXT). Representative Western blot bands are shown below. f Absolute area of lucifer yellow diffusion (dye spread experiments) after Gem knockdown and subsequent OXT exposure (interaction: F1,59 = 9.424, p = 0.003; *p = 0.041 scrRNA/Veh vs. scrRNA/OXT). g Gem protein levels in primary astrocytes transfected with either EGFP control plasmid or Gem overexpression (OE) plasmid indicating successful Gem OE after 72 h (t8 = 3.137, *p = 0.014). h–j Effects of Gem OE on the cytoskeleton of astrocytes reflected by increased process length (h; interaction: F1,354 = 23.64, p < 0.001; ***p < 0.001 EGFP/Veh vs. Gem OE/Veh and vs. EGFP/OXT) and process number (J; U = 1881, **p = 0.002). Representative images taken from EGFP-expressing control or Gem OE cells stained for DAPI (blue) and GFAP (red) (I; scale bar = 50 µm). k Representative images of phalloidin staining in EGFP vs. Gem OE -transfected cells. Scale bar = 20 µm. l Phalloidin/F-actin immunofluorescence intensity in EGFP vs. Gem OE -transfected cells (t14 = 4.467, ***p < 0.001). m pMYPT/Thr696 phosphorylation levels reflecting ROCK activity in EGFP vs. Gem OE -expressing cells (t8 = 2.792, *p = 0.026). n Representative images of scrape-loading dye transfer experiments in EGFP vs. Gem OE -transfected cells. Scale bar = 100 µm; biocytin-AlexaFluor594 streptavidin staining pseudocolored in green. o Degree of GJIC in EGFP vs. Gem OE -transfected cells followed by stimulation with OXT (500 nM, 10 min) (interaction: F1,44 = 7.450, p = 0.009; ***p < 0.001 EGFP/Veh vs. Gem OE/Veh and vs. Gem OE/OXT; *p = 0.018 EGFP/Veh vs. EGFP/OXT). Data represent mean relative or absolute values +/−SEM for normally distributed data and median +/−min/max values for non-normally distributed data.
