Journal: Biochimica et biophysica acta. Molecular basis of disease

Article Title: Involvement of kisspeptin in androgen-induced hypothalamic endoplasmic reticulum stress and its rescuing effect in PCOS rats.

doi: 10.1016/j.bbadis.2021.166242

Figure Lengend Snippet: Fig. 2. DHT suppresses kiss1 expression and promotes unfolded protein response (UPR) in DHT-induced PCOS rats. (a) Quantitative Real-time PCR (qPCR) analysis of kiss1 in the hypothalamus of DHT-induced PCOS (PCOS_DHT) and control (ctrl) rats after DHT exposure for 1 or 3 weeks. n = 9. ***P < 0.001, t-test. (b) Immunofluorescence labeling for kiss1 (green) in the hypothalamus of PCOS_DHT and ctrl rats after DHT exposure for 3 weeks. DAPI (Blue) was used for nuclear label. Scale bar, 20 μm. (c) qPCR of UPR pathway factors (ATF4, BIP, CHOP, PDI) in DHT and ctrl rats DHT exposure for 3 weeks. n = 3. * P < 0.05, ** P < 0.01, *** P < 0.001, t-test. (d-e) Western blots for (d) and qPCR analysis of (e) kiss1 and PDI in DHT and ctrl rats after DHT exposure for 9 weeks. n = 3–6. ** P < 0.01, *** P < 0.001, t-test. qPCR data are relative mRNA levels normalized with that of GAPDH for kiss1(a) and PDI (e) or β-actin for other genes of interest (c and e). β-actin was used as a loading control for all western blots.

Article Snippet: Antibodies used are: IRE1a (1:500, catalog 3294, Cell Signaling Technology [CST], USA), Perk (1:500, catalog 5683, CST), BIP (1:500, catalog 3177, CST), PDI (1:500, catalog 3501,CST), Phospho-eIF2α (1:500, catalog 9721, CST), eIF2α (1:500, catalog 9722, CST), Bax (1:500, catalog 2772, CST), ATF6 (1:500, catalog 15,794–1-AP, ProteinTech, USA), ATF4 (1:500, catalog 10,835–1-AP, ProteinTech), KISS1 (1:500, catalog H-048-46PA5-106920, Thermo Fisher, USA), β-actin (1:5000, catalog A8481, Sigma-Aldrich), Xbp1 (1:500, catalog ab37152, Abcam, USA), Phospho-IRS (1:500, catalog 3203, CST), PI3K (1:500 catalog 4249, CST), Phospho-GSK-3β (1:500,catalog 5558,CST)GSK-3β (1:500, catalog 9315, CST) and GAPDH (1:5000, catalog sc-47,724, Santa Cruz, USA).

Techniques: Expressing, Real-time Polymerase Chain Reaction, Control, Immunofluorescence, Labeling, Western Blot

Journal: Biochimica et biophysica acta. Molecular basis of disease

Article Title: Involvement of kisspeptin in androgen-induced hypothalamic endoplasmic reticulum stress and its rescuing effect in PCOS rats.

doi: 10.1016/j.bbadis.2021.166242

Figure Lengend Snippet: Fig. 3. Kisspeptin mediates DHT's effects on endoplasmic reticulum (ER) stress in GT1–7 cells. (a) qPCR of kiss1 and UPR pathway factors (BIP, PDI, CHOP, ATF4, GADD34 and Xbp1s) in GT1–9 cells treated with DHT (10 nM), kp234 (a kiss1 inhibitor, 1 nM) or DMSO as the control (Ctrl) for 24 h. n = 3. ** P < 0.01, *** P < 0.001, t-test. (b-c) qPCR analysis of Kiss1, BIP, PDI, CHOP, ATF4, GADD34 and Xbp1s (b) and western blots for ATF4 and Bax (c) in GT1–9 cells transfected with siRNAs against kiss1 (two designs, sikiss1_1 or sikiss1_2) or non-silencing control siRNAs (NC) for 24 h. n = 3. * P < 0.05, ** P < 0.01, *** P < 0.001, t-test. qPCR data are relative mRNA levels normalized with that of GAPDH. GAPDH was used as a loading control for all western blots. Error bars are smaller than symbol size except where shown.

Article Snippet: Antibodies used are: IRE1a (1:500, catalog 3294, Cell Signaling Technology [CST], USA), Perk (1:500, catalog 5683, CST), BIP (1:500, catalog 3177, CST), PDI (1:500, catalog 3501,CST), Phospho-eIF2α (1:500, catalog 9721, CST), eIF2α (1:500, catalog 9722, CST), Bax (1:500, catalog 2772, CST), ATF6 (1:500, catalog 15,794–1-AP, ProteinTech, USA), ATF4 (1:500, catalog 10,835–1-AP, ProteinTech), KISS1 (1:500, catalog H-048-46PA5-106920, Thermo Fisher, USA), β-actin (1:5000, catalog A8481, Sigma-Aldrich), Xbp1 (1:500, catalog ab37152, Abcam, USA), Phospho-IRS (1:500, catalog 3203, CST), PI3K (1:500 catalog 4249, CST), Phospho-GSK-3β (1:500,catalog 5558,CST)GSK-3β (1:500, catalog 9315, CST) and GAPDH (1:5000, catalog sc-47,724, Santa Cruz, USA).

Techniques: Control, Western Blot, Transfection

Journal: Biochimica et biophysica acta. Molecular basis of disease

Article Title: Involvement of kisspeptin in androgen-induced hypothalamic endoplasmic reticulum stress and its rescuing effect in PCOS rats.

doi: 10.1016/j.bbadis.2021.166242

Figure Lengend Snippet: Fig. 4. Kisspeptin rescues DHT-induced ER stress in GT1–7 cells. (a-b) Western blots for ATF6, IRE1α, Perk, BIP p-eIF2α and eIF2α (a) and qPCR analysis of GADD34 and Xbp1s/Xbp1 (b) in GT1–7 cells 30 min after treatment with DHT (10 nM), Kp10 (a kiss1 peptide, 10 nM) or DMSO as control (ctrl). n = 3. ** P < 0.01, ***P < 0.001, t-test. (c-d) Western blots (c) or qPCR analysis (d) for ATF4, CHOP, PDI and XBP1 in GT1–7 cells 24 h after treatment with DHT (10 nM), Kp10 (10 nM), or DMSO as Ctrl. n = 3. ** P < 0.01, *** P < 0.001, t-test. (e) qPCR analysis of ATF4, BIP, PDI and CHOP in GT1–7 cells after treatment with thapsigargin (TG, an ER stress inducer, 20 nM), Kp10 (10 nM) or vehicle control (Ctrl). n = 3. *** P < 0.001, t-test. GAPDH was used as a loading control for all western blots. Error bars are smaller than symbol size except where shown.

Article Snippet: Antibodies used are: IRE1a (1:500, catalog 3294, Cell Signaling Technology [CST], USA), Perk (1:500, catalog 5683, CST), BIP (1:500, catalog 3177, CST), PDI (1:500, catalog 3501,CST), Phospho-eIF2α (1:500, catalog 9721, CST), eIF2α (1:500, catalog 9722, CST), Bax (1:500, catalog 2772, CST), ATF6 (1:500, catalog 15,794–1-AP, ProteinTech, USA), ATF4 (1:500, catalog 10,835–1-AP, ProteinTech), KISS1 (1:500, catalog H-048-46PA5-106920, Thermo Fisher, USA), β-actin (1:5000, catalog A8481, Sigma-Aldrich), Xbp1 (1:500, catalog ab37152, Abcam, USA), Phospho-IRS (1:500, catalog 3203, CST), PI3K (1:500 catalog 4249, CST), Phospho-GSK-3β (1:500,catalog 5558,CST)GSK-3β (1:500, catalog 9315, CST) and GAPDH (1:5000, catalog sc-47,724, Santa Cruz, USA).

Techniques: Western Blot, Control

Journal: Life sciences in space research

Article Title: Comparison of signaling profiles in the low dose range following low and high LET radiation.

doi: 10.1016/j.lssr.2020.02.002

Figure Lengend Snippet: Fig. 1. Average total fold intensity of phospho-protein signal as compared to median control value. Colored bars (dark blue = 0.5 Gy, light blue = 0.1 Gy and green = 0.05 Gy) indicate values significantly different from controls (yellow). Significance bars are shown for all significant differences between doses and 0 (p ≤0.05). Average fold intensity levels are shown at 2 h post radiation for γH2AX (A), pATF2 (B) and pSMC1 (C). Persistent effects are shown at 24 h for γH2AX (D), pATF2 (E) and pSMC1 (F). (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

Article Snippet: For staining, 0.5 × 106 fixed cells were washed in phosphate buffered saline (PBS), resuspended in blocking buffer (2% FBS/PBS) and incubated for 1 h with primary antibody on ice, with resuspension of the pellet every 15 min. Primary antibodies used in flow cytometry include mouse monoclonal γH2AXSer139 (1:800 dilution) and pSMC1Ser957 (1:800 dilution) from Millipore (Temecula, CA) and rabbit polyclonal pATF2 Ser490/498 (1:1000 dilution) from Rockland, Inc. (Gilbertsville, PA).

Techniques: Control

Journal: Life sciences in space research

Article Title: Comparison of signaling profiles in the low dose range following low and high LET radiation.

doi: 10.1016/j.lssr.2020.02.002

Figure Lengend Snippet: Fig. 3. Average total fold pATF2 intensity over median control level versus fluence for various radiation qualities. Average fold intensity levels are shown at 2 h post radiation for Si ions (A),Fe ions (C) and Ti ions (E). Persistent effects are shown at 24 h for Si ions (B), Fe ions (D) and Ti ions (F).

Article Snippet: For staining, 0.5 × 106 fixed cells were washed in phosphate buffered saline (PBS), resuspended in blocking buffer (2% FBS/PBS) and incubated for 1 h with primary antibody on ice, with resuspension of the pellet every 15 min. Primary antibodies used in flow cytometry include mouse monoclonal γH2AXSer139 (1:800 dilution) and pSMC1Ser957 (1:800 dilution) from Millipore (Temecula, CA) and rabbit polyclonal pATF2 Ser490/498 (1:1000 dilution) from Rockland, Inc. (Gilbertsville, PA).

Techniques: Control

Journal: Life sciences in space research

Article Title: Comparison of signaling profiles in the low dose range following low and high LET radiation.

doi: 10.1016/j.lssr.2020.02.002

Figure Lengend Snippet: Fig. 6. Average total fold intensity of pATF2 signal intensity divided by fluence and graphed versus LET. Average fold intensity levels are shown at 2 h post radiation for 0.05 Gy (A), 0.1 Gy (B) and 0.5 Gy (C). Persistent effects are shown at 24 h for 0.05 Gy (D), 0.1 Gy (E) and 0.5 Gy (F).

Article Snippet: For staining, 0.5 × 106 fixed cells were washed in phosphate buffered saline (PBS), resuspended in blocking buffer (2% FBS/PBS) and incubated for 1 h with primary antibody on ice, with resuspension of the pellet every 15 min. Primary antibodies used in flow cytometry include mouse monoclonal γH2AXSer139 (1:800 dilution) and pSMC1Ser957 (1:800 dilution) from Millipore (Temecula, CA) and rabbit polyclonal pATF2 Ser490/498 (1:1000 dilution) from Rockland, Inc. (Gilbertsville, PA).

Techniques:

Journal: International Journal of Molecular Sciences

Article Title: Strontium Attenuates Hippocampal Damage via Suppressing Neuroinflammation in High-Fat Diet-Induced NAFLD Mice

doi: 10.3390/ijms241210248

Figure Lengend Snippet: Sr restrained the HFD-induced apoptosis by altering expression levels of proteins related to the ERS pathway. ( A ) Western blot analysis of caspase-3, GRP78, IRE1α, p-IRE1α, XBP1, eIF2α, p-eIF2α, ATF4, ATF6, CHOP, and β-actin. ( B – K ) Relative protein expression of caspase-3 ( B ), GRP78 ( C ), IRE1α ( D ), p-IRE1α ( E ), XBP1 ( F ), eIF2α ( G ), p-eIF2α ( H ), ATF4 ( I ), ATF6 ( J ), and CHOP ( K ) in the hippocampi of each group of mice was examined through Western blotting ( n = 6 per group). Data were normalized with respect to the band of β-actin: the expression of target protein = the intensity of target protein band/the intensity of β-actin band. Results are shown as the ratio of the experimental group to the control group, and the values of the control group were taken as 1. All data are presented as mean ± SEM. * p < 0.05, ** p < 0.01, *** p < 0.001, and **** p < 0.0001.

Article Snippet: Subsequently, the following primary antibodies were used to incubate the membranes overnight at 4 °C: rabbit anti- NF-κB (#8242), rabbit anti- p38 (#9212), rabbit anti- ERK (#9102), rabbit anti-phospho- ERK ( p-ERK , #4370), rabbit anti-phospho- p38 ( p-p38 , #4511), and anti- caspase-3 (#9662) (purchased from Cell Signaling Technology (Danvers, MA, USA)); mouse anti- ATF6 (EM1701-94) (purchased from Hangzhou Huaan Biotechnology Co., Ltd., Hangzhou, China); rabbit anti- XBP1 (A1731), rabbit anti-phospho- NF-κB ( p- NF-κB , AP0475), rabbit anti- GRP78 (A0241), and mouse anti- β-actin (purchased from Wuhan ABclonal Technology Co., Ltd., Wuhan, China); rabbit anti- eIF2α (ab115822), rabbit anti- TLR4 (ab13556), and rabbit anti-phospho- eIF2α ( p-eIF2α , ab32157) (purchased from Abcam (Cambridge, MA, USA)); and rabbit anti- CHOP (BM4962), anti-phospho- IRE1α ( p-IRE1α , BM4444), rabbit anti- ATF4 (BM5179), and rabbit anti- IRE1α (A00683-1) (purchased from Wuhan BOSTER Biological Technology Co., Ltd., Wuhan, China).

Techniques: Expressing, Western Blot, Control

Journal: Neurochemical Research

Article Title: Morin Improves Cognitive Deficits in an in Vivo Model of Vascular Dementia by Modulating the N-methyl-D-aspartate Receptor Signaling Pathways

doi: 10.1007/s11064-026-04717-7

Figure Lengend Snippet: Morin modulated the expression of NMDA receptors in the hippocampus of VaD rats. a the expression levels of NR1 ; b the expression levels of NR2A ; c the expression levels of NR2B ; d the expression levels of NR1 protein; e the expression levels of NR2A protein; f the expression levels of NR2B protein; g protein levels of p-CREB; h protein levels of p-CAMK2A; i protein levels of p-CAMK2D. Protein levels of NR1, NR2A, and NR2B were quantified by ELISA. Data are presented as mean ± SD ( n = 8 per group). Statistical analysis was performed by one-way ANOVA with Tukey’s post-hoc test (data met assumptions of normality and homoscedasticity)/Kruskal-Wallis with Dunn’s test (data did not meet assumptions). * indicates a significant difference from the Sham group; # indicates a significant difference 2VO group; *# indicates a significant difference from both the Sham and 2VO groups, with a p-value of less than 0.05 considered statistically significant

Article Snippet: The phosphorylation status of another key NMDAR downstream effector, CREB at Ser133 (p-CREB), was quantified in hippocampal homogenates using a commercial ELISA kit (Phospho-CREB [Ser133] ELISA Kit, Boster Bio, #EKC2382).

Techniques: Expressing, Enzyme-linked Immunosorbent Assay