Journal: Frontiers in Pharmacology

Article Title: Coreopsis tinctoria Nutt. attenuates ultraviolet A photodamage by suppressing endoplasmic reticulum stress-induced apoptosis via Nrf2 crosstalk

doi: 10.3389/fphar.2025.1686234

Figure Lengend Snippet: Extraction from CT decreases UVA-induced ER stress in HaCaT cells. HaCaT cells were subjected to UVA (5 J/cm 2 ) radiation and then treated with the indicated concentrations of extraction from CT. (A) Western blot analysis was performed to determine the protein levels of phosphorylated IRE1 (pIRE1), ATF6, phosphorylated PERK (p-PERK), phosphorylated eIF2a (p-eIF2a) and CHOP in different treatments. (B) Immunofluorescence assay was performed to detect CHOP expression. (C) Representative images showing the ER morphology in control, UVA-treated and CT-treated cells with UVA exposure. Cells were stained with ER tracker in green and counterstained with DAPI for nuclear visualization. N = 4. * p ≤ 0.05; ** p ≤ 0.01, *** p ≤ 0.001.

Article Snippet: The membranes were blocked with 5% non-fat milk and incubated with the following primary antibodies: IRE1 (phospho S724) (Abcam, ab124945); CHOP (Proteintech,15204-1-AP); NRF2 (Proteintech, 16396-1-AP); ATF6 (Proteintech, 24169-1-AP); PERK (phospho Thr982) (Proteintech, 82534-1-RR); eIF2α(phospho Ser51) (Proteintech, 68023-1-Ig); GADD34 (Proteintech, 10449-1-AP); Caspase9 (Proteintech, 10380-1-AP); Cleaved caspase3 (Proteintech, 25128-1-AP); JNK (Proteintech, 51153-1-AP); JNK (phospho Tyr185) (Proteintech, 80024-1-RR); Bax (Proteintech, 50599-2-Ig); BCL2 (Proteintech, 60178-1-Ig); HO-1 (Proteintech, 10701-1-AP); β-Actin (Proteintech, 20536-1-AP); Horseradish peroxidase (HRP)-conjugated goat anti-rabbit and anti-mouse secondary antibodies were applied (Jackson, 111-035-003 and 115-035-003).

Techniques: Extraction, Western Blot, Immunofluorescence, Expressing, Control, Staining

Journal: Frontiers in Pharmacology

Article Title: Coreopsis tinctoria Nutt. attenuates ultraviolet A photodamage by suppressing endoplasmic reticulum stress-induced apoptosis via Nrf2 crosstalk

doi: 10.3389/fphar.2025.1686234

Figure Lengend Snippet: CT alleviates UVA-induced ER stress and apoptosis via Nrf2 in HaCaT cells. (A) HaCaT cells were subjected to UVA radiation and treated with 50 or 100 μg/mL CT and/or siNrf2. Western blot assays were performed to detect the levels of ATF6, phosphorylated elF2a (p-elF2a), CHOP, GADD34, Cleaved caspase9, Cleaved caspase3, JNK, phosphorylated JNK (p-JNK), Bax, BCL2, cytoplasmic and nuclear NRF2 and HO-1. (B) Flow cytometry analysis was performed to determine cell apoptosis. Quantification of the percentage of apoptotic cells in different treatments. (C) Heatmap generated the data from RNAseq. Genes are all related to NRF2 downstream anti-oxidant or detoxification target genes. The figure was generated by SRplot . N = 4. * p ≤ 0.05; ** p ≤ 0.01, *** p ≤ 0.001, **** p ≤ 0.0001.

Article Snippet: The membranes were blocked with 5% non-fat milk and incubated with the following primary antibodies: IRE1 (phospho S724) (Abcam, ab124945); CHOP (Proteintech,15204-1-AP); NRF2 (Proteintech, 16396-1-AP); ATF6 (Proteintech, 24169-1-AP); PERK (phospho Thr982) (Proteintech, 82534-1-RR); eIF2α(phospho Ser51) (Proteintech, 68023-1-Ig); GADD34 (Proteintech, 10449-1-AP); Caspase9 (Proteintech, 10380-1-AP); Cleaved caspase3 (Proteintech, 25128-1-AP); JNK (Proteintech, 51153-1-AP); JNK (phospho Tyr185) (Proteintech, 80024-1-RR); Bax (Proteintech, 50599-2-Ig); BCL2 (Proteintech, 60178-1-Ig); HO-1 (Proteintech, 10701-1-AP); β-Actin (Proteintech, 20536-1-AP); Horseradish peroxidase (HRP)-conjugated goat anti-rabbit and anti-mouse secondary antibodies were applied (Jackson, 111-035-003 and 115-035-003).

Techniques: Western Blot, Flow Cytometry, Generated

Journal: Scientific Reports

Article Title: PERK regulated miR-424(322)-503 cluster fine-tunes activation of IRE1 and ATF6 during Unfolded Protein Response

doi: 10.1038/srep18304

Figure Lengend Snippet: ( A ) Upper panel, shows a schematic representation of lentiviral vector used to generate miR-424 expressing clones. Lower panel, expression of GFP was monitored in 293T-control and 293T-miR-424 cells after treatment with (1.0 μg/ml) of doxycycline for 24 hours. ( B ) 293T-control and 293T-miR-424 cells were treated with (1.0 μg/ml) of doxycycline for 24 h and expression levels of miR-424 was quantified by qRT-PCR, normalizing against snoRNA. Error bars represent mean ± S.D. from two independent experiments performed in triplicate. *P < 0.05, two-tailed unpaired t-test comparing the expression in 293T-control and 293T-miR-424 cells. ( C ) 293T-control and 293T-miR-424 were transfected with the indicated UPR pathway reporter gene (ATF6, CHOP, XBP1) and 24 hours post transfection cells were treated with TG (1.0 μM). Normalized Firefly luciferase activities (Firefly/Renilla) relative to untreated control are shown. *P < 0.05, two-tailed unpaired t-test comparing the increase in luciferase activity in 293T-control and 293T-miR-424 cells. ( D ) 293T-control and 293T-miR-424 cells were either untreated or treated with (1.0 μM) TG for indicated time points and immunoblotting of total protein was performed using antibodies against ATF6, spliced XBP1, PERK, GRP78, phospho-eIF2α, total eIF2α and β-actin. ( E ) Autorads obtained after probing the membrane with GRP78 antibody were analysed using Image J. Relative intensity of band representing GRP78 in 293T-miR-424 cells relative to untreated 293T-CTRL cells after normalizing aganist actin is shown. *P < 0.05, two-tailed unpaired t-test. ( F ) Autorads obtained after probing the membrane with ATF6 antibody were analysed using Image J. Relative intensity of bands representing total ATF6 [ATF6 (F) + ATF6 (N)] in 293T-miR-424 cells relative to untreated 293T-CTRL cells after normalizing aganist actin is shown. *P < 0.05, two-tailed unpaired t-test.

Article Snippet: In ATF6 3′UTR reporter assays, 293T-CTRL and 293T-miR-424 cells were transfected with (1.0 μg) pMirTarget vector containing the ATF6 3′UTR (Cat # SC205479 Origene Technologies) along with (100 ng) Renilla luciferase vector as internal control.

Techniques: Plasmid Preparation, Expressing, Clone Assay, Control, Quantitative RT-PCR, Two Tailed Test, Transfection, Luciferase, Activity Assay, Western Blot, Membrane

Journal: Scientific Reports

Article Title: PERK regulated miR-424(322)-503 cluster fine-tunes activation of IRE1 and ATF6 during Unfolded Protein Response

doi: 10.1038/srep18304

Figure Lengend Snippet: ( A ) The panel shows miR-424 binding site in the 3′ UTR of ATF6 (TargetScan). ( B ) Alignment of human ATF6 3′UTR with several mammalian species highlights the evolutionary conservation of the miR-424 binding site. ( C ) 293T-CTRL and 293T-miR-424 cells were transfected with ATF6 3′UTR reporter plasmid containing binding sites for miR-424. 24 h after transfection cells were left either untreated or treated with (1.0 μM) TG and (1.0 μg/ml) TM for 16 h. Luciferase activity was measured 48 h after transfection using the Dual-Glo assay system and normalized luciferase activity (Firefly/Renilla) is shown. Error bars represent mean ± S.D. from three independent experiments performed in duplicate. *P < 0.05, two-tailed unpaired t-test comparing the increase in luciferase activity in 293T-control and 293T-miR-424 cells. ( D ) 293T-control and 293T-miR-424 cells were either untreated or treated with (1.0 μM) TG for indicated time points. The change in expression levels of ATF6 was quantified by qRT-PCR, normalizing against RPLP0. Error bars represent mean ± S.D. from three independent experiments performed in triplicate. *P < 0.05, two-tailed unpaired t-test comparing the mRNA expression in 293T-control and 293T-miR-424 cells.

Article Snippet: In ATF6 3′UTR reporter assays, 293T-CTRL and 293T-miR-424 cells were transfected with (1.0 μg) pMirTarget vector containing the ATF6 3′UTR (Cat # SC205479 Origene Technologies) along with (100 ng) Renilla luciferase vector as internal control.

Techniques: Binding Assay, Transfection, Plasmid Preparation, Luciferase, Activity Assay, Glo Assay, Two Tailed Test, Control, Expressing, Quantitative RT-PCR

Journal: Scientific Reports

Article Title: PERK regulated miR-424(322)-503 cluster fine-tunes activation of IRE1 and ATF6 during Unfolded Protein Response

doi: 10.1038/srep18304

Figure Lengend Snippet: In mammals, three ER transmembrane proteins IRE1, ATF6, and PERK, respond to the accumulation of unfolded proteins in the ER lumen. Our results suggest that expression of miRNAs belonging to miR-424(322)-503 is reduced during conditions of ER stress in a PERK-dependent manner. The miR-424(322) down regulates the expression of ATF6 via a miR-424 binding site in the 3′ UTR of ATF6 and represses the ER stress-mediated increase in transcriptional activity of ATF6. The miR-424(322) regulates the RIDD activity of IRE1 but has no significant effect on induction of spliced XBP1. Taken together our results suggest that PERK-mediated downregulation of miR-424(322)-503 cluster regulates optimal activation of IRE1 and ATF6 during conditions of ER stress.

Article Snippet: In ATF6 3′UTR reporter assays, 293T-CTRL and 293T-miR-424 cells were transfected with (1.0 μg) pMirTarget vector containing the ATF6 3′UTR (Cat # SC205479 Origene Technologies) along with (100 ng) Renilla luciferase vector as internal control.

Techniques: Expressing, Binding Assay, Activity Assay, Activation Assay

Journal: BMC Cancer

Article Title: Cigarette smoke induces endoplasmic reticulum stress and the unfolded protein response in normal and malignant human lung cells

doi: 10.1186/1471-2407-8-229

Figure Lengend Snippet: Effect of CS on proteolytic cleavage of ATF6 . Panel A: A549 cells were transfected with a plasmid expressing ATF6 (lanes 2 and 4, indicated by +), or a control plasmid (lanes 1 and 3), then treated with 2 mM DTT for 1 h (lanes 3 and 4) or left untreated (lanes 1 and 2). Western blots of whole cell lysates were probed with antibodies to ATF6 and α-tubulin. Panel B: All lanes show lysates from A549 cells transfected with a plasmid expressing ATF6. Cells were exposed to air (M = mock treatment) or 2R4F cigarette smoke (CS = smoke treatment) for 20 minutes with 35 cc puffs diluted in 250 cc air. Cells were then placed in fresh medium and returned to the incubator for the time periods specified. Western blots of whole cell lysates were probed with antibodies to ATF6 and α-tubulin. Gel bands were quantified as described in the Methods section. CS treatment resulted in a 23% and 26% decrease in ATF6 90 kDa protein at 2 and 4 h respectively (lane 5 compared to lane 6, lane 7 compared to lane 8).

Article Snippet: At 24 hours after the seeding, cells were transfected with ATF6 expression plasmid ATF6/pCMV6-XL5 (OriGene Technologies, Inc., Rockville MD, Catalog # SC115551).

Techniques: Transfection, Plasmid Preparation, Expressing, Control, Western Blot