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. Author manuscript; available in PMC: 2021 Nov 17.
Published in final edited form as: J Mol Cell Cardiol. 2020 Apr 25;143:132–144. doi: 10.1016/j.yjmcc.2020.04.012

Fig. 5.

Fig. 5.

Effects of GRP78 on Cardiac Myocyte Viability and AKT/SMAD2 Signaling- Panels A, B- NRVMs were plated at 4 × 105 cells/well on 12-well plates. Sixteen hours after plating, NRVM culture medium was replaced with DMEM/F-12 supplemented with 0.5% FBS without antibiotics, 120 nM siRNA, and 1.25 μl HiPerfect / 1 μl Grp78 or control siRNA. After 16 h the culture medium was replaced with DMEM/F-12 with BSA (1 mg/ml) for 48 h, after which cultures were treated for 24 h ± TG (2 μM) in low (0.4 ml) or high (2 ml) volume. Cell extracts and media were examined by immunoblotting. Panel C– NRVMs were plated at 1.25 × 105 cells/well on 48-well plates. Sixteen hours after plating, cultures were transfected with siRNA as described in Panel A. Forty-eight hours after transfection, cultures were treated without or with TG (2 μM) in serum- and BSA-free DMEM/F-12 in low (0.125 ml) or high (0.75 ml) volume. After 48 h, culture viabilities were determined by MTT assay. Shown are the mean optical density (O.D.) values ± S.E.M. *, #, p ≤ .05 different from all other values. Panel D- NRVMs were plated at 1.25 × 105 cells/well on 48-well plates. Sixteen hours after plating, media were then replaced with either high (0.75 ml) or low (0.125 ml) volume of serum- and BSA-free DMEM/F-12 ± TG (2 μM), ± 4 μg/ml anti-GRP78 or 4 μg/ml non-immune goat IgG. After 48 h, cell viability was determined by MTT assay; n = 4 cultures/treatment. *, #, $, p ≤ .05 different from all other values, as determined by ANOVA followed by Newman Keul’s post hoc analysis. Panel E- NRVMs were plated as described in Panel C. Media were then replaced with either high volume (0.75 ml) or low volume (0.125 ml) of serum- and BSA-free DMEM/F-12 without or with TG (2 μM), in combination with recombinant GRP78 (2 μg/ml) or bovine serum albumin (2 μg/ml). After 48 h, cell viability was determined by MTT assay; n = 4 cultures/treatment. After 48 h, culture viabilities were determined by MTT assay. *, #, p ≤ .05 different from all other values, as determined by ANOVA followed by Newman Keul’s post hoc analysis.