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MDA-MB-231 cells were treated for the indicated times with 0.5 μM Tg after which cells where harvested. RNA was extracted and qPCR used to assess the relative expression of PERK ( A ), IRE1 ( B ) and ATF6 ( C ) transcripts. Mean relative expression ± SD, reference gene GAPDH , N = 4. D Cell lysates were treated with <t>calf-intestinal</t> alkaline <t>phosphatase</t> (to dephosphorylate PERK and IRE1) and analyzed via immunoblotting for IRE1, XBP1s, PERK, ATF4 and ATF6 with ( E–G ) relative changes compared to 0 h determined by densitometry (mean relative expression ± SEM). Actin was used as a loading control. Blots are representative of N = 3. Statistical significance was determined using one-way ANOVA followed by TUKEY HSD post-hoc analysis (qPCR). * p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001 vs 0 h Tg.
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MDA-MB-231 cells were treated for the indicated times with 0.5 μM Tg after which cells where harvested. RNA was extracted and qPCR used to assess the relative expression of PERK ( A ), IRE1 ( B ) and ATF6 ( C ) transcripts. Mean relative expression ± SD, reference gene GAPDH , N = 4. D Cell lysates were treated with <t>calf-intestinal</t> alkaline <t>phosphatase</t> (to dephosphorylate PERK and IRE1) and analyzed via immunoblotting for IRE1, XBP1s, PERK, ATF4 and ATF6 with ( E–G ) relative changes compared to 0 h determined by densitometry (mean relative expression ± SEM). Actin was used as a loading control. Blots are representative of N = 3. Statistical significance was determined using one-way ANOVA followed by TUKEY HSD post-hoc analysis (qPCR). * p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001 vs 0 h Tg.
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MDA-MB-231 cells were treated for the indicated times with 0.5 μM Tg after which cells where harvested. RNA was extracted and qPCR used to assess the relative expression of PERK ( A ), IRE1 ( B ) and ATF6 ( C ) transcripts. Mean relative expression ± SD, reference gene GAPDH , N = 4. D Cell lysates were treated with <t>calf-intestinal</t> alkaline <t>phosphatase</t> (to dephosphorylate PERK and IRE1) and analyzed via immunoblotting for IRE1, XBP1s, PERK, ATF4 and ATF6 with ( E–G ) relative changes compared to 0 h determined by densitometry (mean relative expression ± SEM). Actin was used as a loading control. Blots are representative of N = 3. Statistical significance was determined using one-way ANOVA followed by TUKEY HSD post-hoc analysis (qPCR). * p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001 vs 0 h Tg.
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MDA-MB-231 cells were treated for the indicated times with 0.5 μM Tg after which cells where harvested. RNA was extracted and qPCR used to assess the relative expression of PERK ( A ), IRE1 ( B ) and ATF6 ( C ) transcripts. Mean relative expression ± SD, reference gene GAPDH , N = 4. D Cell lysates were treated with <t>calf-intestinal</t> alkaline <t>phosphatase</t> (to dephosphorylate PERK and IRE1) and analyzed via immunoblotting for IRE1, XBP1s, PERK, ATF4 and ATF6 with ( E–G ) relative changes compared to 0 h determined by densitometry (mean relative expression ± SEM). Actin was used as a loading control. Blots are representative of N = 3. Statistical significance was determined using one-way ANOVA followed by TUKEY HSD post-hoc analysis (qPCR). * p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001 vs 0 h Tg.
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MDA-MB-231 cells were treated for the indicated times with 0.5 μM Tg after which cells where harvested. RNA was extracted and qPCR used to assess the relative expression of PERK ( A ), IRE1 ( B ) and ATF6 ( C ) transcripts. Mean relative expression ± SD, reference gene GAPDH , N = 4. D Cell lysates were treated with <t>calf-intestinal</t> alkaline <t>phosphatase</t> (to dephosphorylate PERK and IRE1) and analyzed via immunoblotting for IRE1, XBP1s, PERK, ATF4 and ATF6 with ( E–G ) relative changes compared to 0 h determined by densitometry (mean relative expression ± SEM). Actin was used as a loading control. Blots are representative of N = 3. Statistical significance was determined using one-way ANOVA followed by TUKEY HSD post-hoc analysis (qPCR). * p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001 vs 0 h Tg.
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MDA-MB-231 cells were treated for the indicated times with 0.5 μM Tg after which cells where harvested. RNA was extracted and qPCR used to assess the relative expression of PERK ( A ), IRE1 ( B ) and ATF6 ( C ) transcripts. Mean relative expression ± SD, reference gene GAPDH , N = 4. D Cell lysates were treated with calf-intestinal alkaline phosphatase (to dephosphorylate PERK and IRE1) and analyzed via immunoblotting for IRE1, XBP1s, PERK, ATF4 and ATF6 with ( E–G ) relative changes compared to 0 h determined by densitometry (mean relative expression ± SEM). Actin was used as a loading control. Blots are representative of N = 3. Statistical significance was determined using one-way ANOVA followed by TUKEY HSD post-hoc analysis (qPCR). * p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001 vs 0 h Tg.

Journal: Cell Death & Disease

Article Title: IRE1 signaling increases PERK expression during chronic ER stress

doi: 10.1038/s41419-024-06663-0

Figure Lengend Snippet: MDA-MB-231 cells were treated for the indicated times with 0.5 μM Tg after which cells where harvested. RNA was extracted and qPCR used to assess the relative expression of PERK ( A ), IRE1 ( B ) and ATF6 ( C ) transcripts. Mean relative expression ± SD, reference gene GAPDH , N = 4. D Cell lysates were treated with calf-intestinal alkaline phosphatase (to dephosphorylate PERK and IRE1) and analyzed via immunoblotting for IRE1, XBP1s, PERK, ATF4 and ATF6 with ( E–G ) relative changes compared to 0 h determined by densitometry (mean relative expression ± SEM). Actin was used as a loading control. Blots are representative of N = 3. Statistical significance was determined using one-way ANOVA followed by TUKEY HSD post-hoc analysis (qPCR). * p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001 vs 0 h Tg.

Article Snippet: Calf Intestinal Alkaline Phosphatase (18009-019) was obtained from ThermoFisher.

Techniques: Expressing, Western Blot, Control