Journal: Neurochemical Research

Article Title: Prolactin is an Endogenous Antioxidant Factor in Astrocytes That Limits Oxidative Stress-Induced Astrocytic Cell Death via the STAT3/NRF2 Signaling Pathway

doi: 10.1007/s11064-024-04147-3

Figure Lengend Snippet: Involvement of STAT3 in the protective effect of PRL on rat astrocytes. a Rat cortical astrocytes were pre-incubated for 24 h in the absence or presence of 10 nM prolactin (PRL), then 400 μM hydrogen peroxide (H 2 O 2 ) or vehicle (Veh) was added and incubated for 3 h. Prolactin receptor ( Prlr ) mRNA levels were measured by quantitative RT-PCR. Data were normalized using the Hprt housekeeping gene as an internal control. Data are means ± SEM of four independent experiments (n = 4). b Effect of PRL on STAT3 phosphorylation in rat cortical astrocytes. Active STAT3 was detected by Western blotting in 30 μg of astrocyte lysate using an antibody against phosphorylated STAT3 (pSTAT3) and quantified by using total STAT3 and β-tubulin as internal controls. Data are means ± SEM of three independent experiments (n = 3). Rat cortical astrocytes were pre-incubated in the absence or presence of 10 nM PRL and/or 100 μM STAT3 inhibitor S3I-201 for 24 h, then 400 μM H 2 O 2 or vehicle was added and incubated for 3 h. c Cell viability was quantified by MTT assay. Data are means ± SEM of three independent experiments (n = 3) carried out in triplicate. d H 2 O 2 -induced cytotoxicity was quantified by the measurement of LDH release. Results are normalized to the control with vehicle or expressed as percent of total LDH release obtained by treating non incubated cells with lysis solution prior to the assay to induce maximum LDH release, respectively. Data are means ± SEM of four independent experiments (n = 4) carried out in triplicate. One-way ANOVA followed by Tukey’s test; *p < 0.05, **p < 0.001 versus vehicle or between group; n.s. no significant difference

Article Snippet: The blots were then incubated overnight at 4 °C with polyclonal antibodies against phospho-STAT3 (1:2000 dilution; cat. no. 9145, RRID:AB_2491009; Cell Signaling Technology, MA, USA), STAT3 (1:350 dilution; cat. no. sc-483, RRID:AB_632441; Santa Cruz Biotechnology, TX, USA), GPX1(1:100 dilution; cat. no. bs-3882R, RRID:AB_10857071; Bioss Antibodies, MA, USA) and β-tubulin (1:500 dilution; cat. no. ab6046, RRID:AB_2210370; Abcam, Cambridge, UK); or monoclonal antibodies against SOD1 (1:700 dilution; cat. no. sc-101523, RRID:AB_2191632; Santa Cruz Biotechnology, TX, USA) and SOD2 (1:100 dilution; cat. no. sc-133134, RRID:AB_2191814; Santa Cruz Biotechnology, TX, USA).

Techniques: Incubation, Quantitative RT-PCR, Control, Phospho-proteomics, Western Blot, MTT Assay, Lysis

Journal: Neurochemical Research

Article Title: Prolactin is an Endogenous Antioxidant Factor in Astrocytes That Limits Oxidative Stress-Induced Astrocytic Cell Death via the STAT3/NRF2 Signaling Pathway

doi: 10.1007/s11064-024-04147-3

Figure Lengend Snippet: Effect of PRL on H 2 O 2 -induced ROS production and oxidative damage in rat astrocytes. Rat cortical astrocytes were pre-incubated in the absence or presence of 10 nM prolactin (PRL),100 μM STAT3 inhibitor (S3I-201) or both for 24 h, then 400 μM hydrogen peroxide (H 2 O 2 ) or vehicle (Veh) was added and incubated for 3 h. a Generation of reactive oxygen species (ROS) was quantified using 2′,7′-dichlorodihydrofluorescein diacetate (DCF-DA). Values are expressed as DCF fluorescence after 30 min of incubation. Data are means ± SEM of three independent experiments (n = 3) carried out in triplicate. b Generation of superoxide anion was measured using dihydroethidium (DHE). Values are expressed as DHE fluorescence after 30 min of incubation. Data are means ± SEM of three independent experiments (n = 3) carried out in triplicate. c Protein oxidation was estimated by measuring the protein carbonyl levels with the DNPH colorimetric assay. The concentration of the protein carbonyls was adjusted to the total protein concentration. Data are means ± SEM of three independent experiments (n = 3) carried out in duplicate. d Total sulfhydryl groups were measured by the reaction of free thiols in native proteins with DTNB. The concentration of the free thiols was adjusted to the total protein concentration. Data are means ± SEM of three independent experiments (n = 3) carried out in duplicate. e Lipid peroxidation was determined as the increase in malondialdehyde (MDA), a thiobarbituric acid reactive substance (TBARS). The concentration of the MDA was adjusted to the total protein concentration. Data are means ± SEM of four independent experiments (n = 4). f Antioxidant capacity was detected by ABTS assay. Data are means ± SEM of three independent experiments (n = 3) carried out in duplicate. One-way ANOVA followed by Tukey’s test; *p < 0.05, **p < 0.001 versus indicated group; n.s. no significant difference

Article Snippet: The blots were then incubated overnight at 4 °C with polyclonal antibodies against phospho-STAT3 (1:2000 dilution; cat. no. 9145, RRID:AB_2491009; Cell Signaling Technology, MA, USA), STAT3 (1:350 dilution; cat. no. sc-483, RRID:AB_632441; Santa Cruz Biotechnology, TX, USA), GPX1(1:100 dilution; cat. no. bs-3882R, RRID:AB_10857071; Bioss Antibodies, MA, USA) and β-tubulin (1:500 dilution; cat. no. ab6046, RRID:AB_2210370; Abcam, Cambridge, UK); or monoclonal antibodies against SOD1 (1:700 dilution; cat. no. sc-101523, RRID:AB_2191632; Santa Cruz Biotechnology, TX, USA) and SOD2 (1:100 dilution; cat. no. sc-133134, RRID:AB_2191814; Santa Cruz Biotechnology, TX, USA).

Techniques: Incubation, Fluorescence, Colorimetric Assay, Concentration Assay, Protein Concentration, ABTS Assay

Journal: Neurochemical Research

Article Title: Prolactin is an Endogenous Antioxidant Factor in Astrocytes That Limits Oxidative Stress-Induced Astrocytic Cell Death via the STAT3/NRF2 Signaling Pathway

doi: 10.1007/s11064-024-04147-3

Figure Lengend Snippet: Effect of PRL on transcription of antioxidant genes in rat astrocytes. Rat cortical astrocytes were incubated in the absence or presence of 10 nM prolactin (PRL) for 4, 8, 16 or 24 h, and changes in mRNA levels of a superoxide dismutase 1 ( Sod1) , b superoxide dismutase 2 ( Sod2) , c glutathione peroxidase 1 ( Gpx1) , and d catalase were measured by quantitative RT-PCR. Data were initially normalized using the Hprt housekeeping gene as an internal control and then to the corresponding gene expression in the 4 h vehicle-treated group (Veh). Data in a , b , c and d are means ± SEM of four independent experiments (n = 4). Unpaired two-tailed Student’s t-test. Rat cortical astrocytes were incubated in the absence or presence of either 10 nM PRL, 100 μM STAT3 inhibitor S3I-201 or both for 24 h. mRNA levels of e Sod1 , f Sod2 , and g Gpx1 were measured by quantitative RT-PCR. Data were initially normalized using the Hprt housekeeping gene as an internal control and then to the corresponding gene expression in the vehicle-treated group. Data in e , f , and g are means ± SEM of four independent experiments (n = 4). Protein expression of SOD1 ( h ), SOD2 ( i ), and GPX1 ( j ) was detected by Western blotting in 5, 10 or 20 μg of astrocyte lysate, respectively; and quantified by using β-tubulin as loading control. Data are means ± SEM of ( h , j ) four (n = 4) or (j) three (n = 3) independent experiments. One-way ANOVA followed by Tukey’s test. *p < 0.05, **p < 0.001 versus vehicle or indicated group. For GPX1 protein, Kruskal–Wallis’s test followed by Dunn’s test

Article Snippet: The blots were then incubated overnight at 4 °C with polyclonal antibodies against phospho-STAT3 (1:2000 dilution; cat. no. 9145, RRID:AB_2491009; Cell Signaling Technology, MA, USA), STAT3 (1:350 dilution; cat. no. sc-483, RRID:AB_632441; Santa Cruz Biotechnology, TX, USA), GPX1(1:100 dilution; cat. no. bs-3882R, RRID:AB_10857071; Bioss Antibodies, MA, USA) and β-tubulin (1:500 dilution; cat. no. ab6046, RRID:AB_2210370; Abcam, Cambridge, UK); or monoclonal antibodies against SOD1 (1:700 dilution; cat. no. sc-101523, RRID:AB_2191632; Santa Cruz Biotechnology, TX, USA) and SOD2 (1:100 dilution; cat. no. sc-133134, RRID:AB_2191814; Santa Cruz Biotechnology, TX, USA).

Techniques: Incubation, Quantitative RT-PCR, Control, Gene Expression, Two Tailed Test, Expressing, Western Blot

Journal: Neurochemical Research

Article Title: Prolactin is an Endogenous Antioxidant Factor in Astrocytes That Limits Oxidative Stress-Induced Astrocytic Cell Death via the STAT3/NRF2 Signaling Pathway

doi: 10.1007/s11064-024-04147-3

Figure Lengend Snippet: Effect of PRL on Nrf2 activation in rat astrocytes. a Representative NFE2 like bZIP transcription factor 2 (NRF2) immunostaining images of rat cortical astrocytes treated with 10 nM prolactin (PRL) for 4 h. Right panels are merged images of NRF2 (red, left panels) and the nuclear marker Sytox (blue, center panels). Scale bar 20 μm. b Quantification of nuclear NRF2 in control versus PRL-exposed rat cortical astrocytes. Data are means ± SEM of three independent experiments (n = 3). Rat cortical astrocytes were incubated in the absence or presence of 10 nM PRL for 4, 8, 16, or 24 h, and changes in mRNA levels of c Nrf2 and d heme oxygenase 1 ( Hmox1) were measured by quantitative RT-PCR. Data were initially normalized using the Hprt housekeeping gene as an internal control and then to the corresponding gene expression in the 4 h vehicle-treated group (Veh). Data in c and d are means ± SEM of four independent experiments (n = 4). Unpaired two-tailed Student’s t-test. Rat cortical astrocytes were incubated in the absence or presence of either 10 nM PRL, 100 μM STAT3 inhibitor S3I-201 or both for 8 h. mRNA levels of e Nrf2 and f Hmox1 were measured by quantitative RT-PCR. Data were initially normalized using the Hprt housekeeping gene as an internal control and then to the corresponding gene expression in the vehicle-treated group. Data in e and f are means ± SEM of four independent experiments (n = 4). One-way ANOVA followed by Tukey’s test. *p < 0.05, **p < 0.001 versus vehicle or indicated group

Article Snippet: The blots were then incubated overnight at 4 °C with polyclonal antibodies against phospho-STAT3 (1:2000 dilution; cat. no. 9145, RRID:AB_2491009; Cell Signaling Technology, MA, USA), STAT3 (1:350 dilution; cat. no. sc-483, RRID:AB_632441; Santa Cruz Biotechnology, TX, USA), GPX1(1:100 dilution; cat. no. bs-3882R, RRID:AB_10857071; Bioss Antibodies, MA, USA) and β-tubulin (1:500 dilution; cat. no. ab6046, RRID:AB_2210370; Abcam, Cambridge, UK); or monoclonal antibodies against SOD1 (1:700 dilution; cat. no. sc-101523, RRID:AB_2191632; Santa Cruz Biotechnology, TX, USA) and SOD2 (1:100 dilution; cat. no. sc-133134, RRID:AB_2191814; Santa Cruz Biotechnology, TX, USA).

Techniques: Activation Assay, Immunostaining, Marker, Control, Incubation, Quantitative RT-PCR, Gene Expression, Two Tailed Test

Journal: Neurochemical Research

Article Title: Prolactin is an Endogenous Antioxidant Factor in Astrocytes That Limits Oxidative Stress-Induced Astrocytic Cell Death via the STAT3/NRF2 Signaling Pathway

doi: 10.1007/s11064-024-04147-3

Figure Lengend Snippet: Effect of PRL on antioxidant enzymes activity in rat astrocytes. Rat cortical astrocytes were pre-incubated in the absence or presence of either 10 nM prolactin (PRL), 100 μM STAT3 inhibitor S3I-201 or both for 24 h, then 400 μM hydrogen peroxide (H 2 O 2 ) or vehicle (Veh) was added and incubated for 3 h. a Superoxide dismutase (SOD), b glutathione peroxidase (GPX), and c catalase (CAT) activities were detected in samples of each treatment group containing equal amounts of protein using the corresponding commercial assay. Data in a , b and c are means ± SEM of three independent experiments (n = 3) carried out in duplicate. One-way ANOVA followed by Tukey’s test. *p < 0.05, **p < 0.001 versus indicated group; n.s., no significant difference

Article Snippet: The blots were then incubated overnight at 4 °C with polyclonal antibodies against phospho-STAT3 (1:2000 dilution; cat. no. 9145, RRID:AB_2491009; Cell Signaling Technology, MA, USA), STAT3 (1:350 dilution; cat. no. sc-483, RRID:AB_632441; Santa Cruz Biotechnology, TX, USA), GPX1(1:100 dilution; cat. no. bs-3882R, RRID:AB_10857071; Bioss Antibodies, MA, USA) and β-tubulin (1:500 dilution; cat. no. ab6046, RRID:AB_2210370; Abcam, Cambridge, UK); or monoclonal antibodies against SOD1 (1:700 dilution; cat. no. sc-101523, RRID:AB_2191632; Santa Cruz Biotechnology, TX, USA) and SOD2 (1:100 dilution; cat. no. sc-133134, RRID:AB_2191814; Santa Cruz Biotechnology, TX, USA).

Techniques: Activity Assay, Incubation

Journal: Neurochemical Research

Article Title: Prolactin is an Endogenous Antioxidant Factor in Astrocytes That Limits Oxidative Stress-Induced Astrocytic Cell Death via the STAT3/NRF2 Signaling Pathway

doi: 10.1007/s11064-024-04147-3

Figure Lengend Snippet: Schematic representation of the mechanism involved in the protective effect of PRL against H 2 O 2 -induced oxidative stress. PRL interaction with its receptor stimulates phosphorylation of transcription factor STAT3, which promotes the expression of enzymatic antioxidant systems (GPX, SOD) via NRF2, and thus increases the total antioxidant capacity. This cascade of events triggered by PRL reduces H 2 O 2 -induced ROS formation, protein oxidation, lipid peroxidation, and ultimately cell death. GPX, glutathione peroxidase; SOD, superoxide dismutase; ARE, antioxidant response element

Article Snippet: The blots were then incubated overnight at 4 °C with polyclonal antibodies against phospho-STAT3 (1:2000 dilution; cat. no. 9145, RRID:AB_2491009; Cell Signaling Technology, MA, USA), STAT3 (1:350 dilution; cat. no. sc-483, RRID:AB_632441; Santa Cruz Biotechnology, TX, USA), GPX1(1:100 dilution; cat. no. bs-3882R, RRID:AB_10857071; Bioss Antibodies, MA, USA) and β-tubulin (1:500 dilution; cat. no. ab6046, RRID:AB_2210370; Abcam, Cambridge, UK); or monoclonal antibodies against SOD1 (1:700 dilution; cat. no. sc-101523, RRID:AB_2191632; Santa Cruz Biotechnology, TX, USA) and SOD2 (1:100 dilution; cat. no. sc-133134, RRID:AB_2191814; Santa Cruz Biotechnology, TX, USA).

Techniques: Phospho-proteomics, Expressing

Journal: Cell Reports Medicine

Article Title: Discovery of galectin-8 as an LILRB4 ligand driving M-MDSCs defines a class of antibodies to fight solid tumors

doi: 10.1016/j.xcrm.2023.101374

Figure Lengend Snippet:

Article Snippet: Rabbit polyclonal anti-phospho-STAT3(Ser727) , CST , Cat#9134T; RRID: AB_331589.

Techniques: Recombinant, Derivative Assay, Microarray, Enzyme-linked Immunosorbent Assay, Lysis, Blocking Assay, Staining, Transfection, Marker, Flow Cytometry, shRNA, Plasmid Preparation, Negative Control, Silver Staining, Bicinchoninic Acid Protein Assay, Selection, Extraction, Labeling, RNA Expression, Cell Culture, Software