Journal: iScience

Article Title: Cytoplasmic proteotoxicity regulates HRI-dependent phosphorylation of eIF2α via the Hsp70-Bag3 module

doi: 10.1016/j.isci.2022.104282

Figure Lengend Snippet: 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: ). (D) and (E) Quantification of experiment presented in Figure 2 C. 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: ). (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 .

Article Snippet: Antibodies against, Phospho-eIF2α_Ser51 (Cell Signaling Technology Cat# 3597, RRID: AB_390740 ); EIF2ΑK1/HRI- (Proteintech Cat# 20499-1-AP, RRID: AB_10697665 ) or EIF2ΑK1/HRI- (Abcam Cat# ab28530, RRID: AB_869595 ), anti-Bag3 (Proteintech Cat# 10599-1-AP, RRID: AB_2062602 ) and β-actin (Novus Cat# NB600-501, RRID: AB_10077656 ), α-K48 Ubiquitin linkage antibody (RRID: AB_2894847 , Cat#A101-050), Recombinant Human His6-Ubiquilin-1 Tandem UBA (UBE110) were from Boston Biochem.

Techniques: Transformation Assay, Western Blot, Transfection, Two Tailed Test, Imaging, Staining

Journal: iScience

Article Title: Cytoplasmic proteotoxicity regulates HRI-dependent phosphorylation of eIF2α via the Hsp70-Bag3 module

doi: 10.1016/j.isci.2022.104282

Figure Lengend Snippet:

Article Snippet: Antibodies against, Phospho-eIF2α_Ser51 (Cell Signaling Technology Cat# 3597, RRID: AB_390740 ); EIF2ΑK1/HRI- (Proteintech Cat# 20499-1-AP, RRID: AB_10697665 ) or EIF2ΑK1/HRI- (Abcam Cat# ab28530, RRID: AB_869595 ), anti-Bag3 (Proteintech Cat# 10599-1-AP, RRID: AB_2062602 ) and β-actin (Novus Cat# NB600-501, RRID: AB_10077656 ), α-K48 Ubiquitin linkage antibody (RRID: AB_2894847 , Cat#A101-050), Recombinant Human His6-Ubiquilin-1 Tandem UBA (UBE110) were from Boston Biochem.

Techniques: Recombinant, SYBR Green Assay, Software, Imaging

Journal: bioRxiv

Article Title: Cell type specific suppression of hyper-recombination by human RAD18 is linked to PCNA K164 ubiquitination

doi: 10.1101/2024.09.03.611050

Figure Lengend Snippet: (A) Western blot analysis confirming loss of MLH1 expression in two independent RPE-1 MLH1 -/- clones and two independent RAD18 -/- /MLH1 -/- clones, with β-actin as the loading control. The asterisk indicates a nonspecific band recognized by the anti-RAD18 antibody. (B) Average number of SCEs per chromosome in RPE-1 MLH1-proficient and -deficient cell lines. Black lines indicate mean values and significance was calculated using a Mann-Whitney test. No comparisons were statistically significant.

Article Snippet: Primary antibodies were diluted in 5% milk as follows: rabbit anti-RAD18 (Bethyl, A300-340A, RRID:AB_937974; 1:1000), mouse anti-PCNA (Abcam, ab29, RRID:AB_303394; 1:3000), rabbit anti-ubiquityl-PCNA (Lys164) (Cell Signaling, D5C7P, 13439, RRID:AB_2798219; 1:1000), rabbit anti-MLH1 (Abcam, ab92312, RRID:AB_2049968; 1:1000), mouse anti-FLAG (Sigma, F3165, RRID:AB_259529; 1:1000), mouse anti-α-tubulin (Millipore, T9026, clone DM1A, RRID:AB_477593; 1:10000), anti-Pol η (Abcam, ab234855, RRID:AB_3094753; 1:1000), rabbit anti-H2AX (Bethyl, A300-082A, RRID:AB_203287; 1:5000), rabbit anti-γH2AX (Bethyl, A300-081A, RRID:AB_203288; 1:2000), rabbit anti-RPA32 (phospho S4/8) (Bethyl, A300-245A, RRID:AB_210547; 1:2000), anti-β-actin (Novus, NB600-501, RRID_AB:10077656, 1:10000), anti-UBC9 (Santa Cruz, C-12, sc-271057, RRID:AB_10610674; 1:500).

Techniques: Western Blot, Expressing, Clone Assay, Control, MANN-WHITNEY