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. 2022 Apr 22;25(5):104282. doi: 10.1016/j.isci.2022.104282

Figure 2.

Figure 2

Phosphorylation of translation initiation factor eIF2⍺ in response to JG-98 is mediated by Hsp70-Bag3-HRI complex

(A) Treatment by JG-98 strongly stimulates phosphorylation of eIF2⍺ in Her2-transformed cells compared to untransformed cells. Cells were treated with 1 μM JG-98 for 12 h or left untreated. Levels of phospho-eIF2⍺ were determined in cell lysates by immunoblotting with the corresponding antibody, independent experiments were performed three times (n = 3).

(B) Quantification of experiment presented in (A). Quantification of the relative band intensity was performed using ImageJ where β-actin served as loading controls. Scatterplot showing individual data points for n = 3 as means ± SEM, Statistical analysis was performed using two-way ANOVA.

(C) Depletion of Bag3 significantly reduced phosphorylation of eIF2⍺ in the presence of JG-98. Untransformed cells were transfected with siBag3 or siControl and further treated by JG-98 (1uM, 12 h) or left untreated. Levels of phospho-eIF2⍺, Bag3, and total-eIF2⍺ were determined in cell lysates by immunoblotting with corresponding antibody, independent experiments were performed seven times (n = 7), Raw data for corresponding cropped images are provided separately as a supplement (PAGE #3&4, file: Data S1).

(D) and (E) Quantification of experiment presented in Figure 2C. Quantification of the relative band intensity for (D) p-eIF2⍺ and (E) Bag3 was performed using ImageJ where β-actin served as loading controls. Scatter plot showing individual data points for n = 7 as means ± SEM, Statistical analysis was performed using two-way ANOVA.

(F) GSEA analysis showing upregulation of the Heme metabolism pathway in response to JG-98 treatment.

(G) Efficiency of HRI depletion following siRNA treatment. Levels of HRI mRNA were quantified by qPCR, experiments were performed in biological replicates (n = 3) shown as means ± SEM, statistics was performed using unpaired Welch’s correction, two-tailed t-test.(H) Protein levels for HRI was also checked using immunoblotting, statistics was performed unpaired Welch’s correction, two-tailed t-test (n = 7) plotted as means ± SEM, representative images are available in supplement section.

(I) Depletion of HRI led to significant suppression of eIF2⍺ phosphorylation in presence of JG-98. Untransformed cells were transfected with siHRI or siControl and further treated by JG-98 (1uM, 12 h) or left untreated. Levels of phospho-eIF2⍺, HRI, and total-eIF2⍺ were determined in cell lysates by immunoblotting with the corresponding antibody, experiment was performed six times (n = 6). siControl of siBag3 and siHRI is same as experiment was conducted together and were immunoblotted on the same membrane, Raw data for corresponding cropped images are provided separately as a supplement (Page #5&6, file: Data S1).

(J) Quantification of the relative band intensity for p-eIF2⍺ was performed. Scatter plot showing individual data points for n = 6 as means ± SEM, Statistical analysis was performed using two-way ANOVA.

(K) HRI depletion reduces cell death in response to JG-98. Untransformed cells were transfected with siControl and siHRI to silence HRI followed by 1 μM JG-98 treatment for 24 h or left untreated. Cell survival was evaluated by Hermes Imaging of DAPI-stained cells.

(L) Statistical analysis was performed by two-way ANOVA, n = 3 shown as means ± SEM Quantification of the blot was performed using ImageJ and OD ratios for each protein compared to the reference after normalization is added below each blot. Specifically, phospho-eIF2⍺ was normalized additionally by total-eIF2⍺. All statistical analysis was performed using Graphpad (v9), level of significance was taken as (∗p < 0.0332, ∗∗p < 0.0021, ∗∗∗p < 0.0002, ∗∗∗∗p < 0.0001). See also Figures S3–S7.