Journal: Human reproduction (Oxford, England)

Article Title: A novel selective progesterone receptor modulator asoprisnil (J867) down-regulates the expression of EGF, IGF-I, TGFbeta3 and their receptors in cultured uterine leiomyoma cells.

doi: 10.1093/humrep/del035

Figure Lengend Snippet: Figure 1. Effects of asoprisnil on the expression of immunoreactive epidermal growth factor (EGF), insulin-like growth factor-I (IGF-I) and transforming growth factor β (TGFβ3) protein in leiomyoma and normal myometrial cells cultured for 48 or 72 h, as assessed by immunocyto- chemistry. The expression of immunoreactive EGF, IGF-I and TGFβ3 protein in leiomyoma and normal myometrial cells cultured for 48 h (upper panel) and for 72 h (lower panel). EGF in leiomyoma cells cultured without any treatment (a, A), EGF in leiomyoma cells cultured in the presence of 10–7 M asoprisnil (d, D), IGF-I in leiomyoma cells cultured without any treatment (b, B), IGF-I in leiomyoma cells cultured in the presence of 10–7 M asoprisnil (e, E), TGFβ3 in leiomyoma cells cultured without any treatment (c, C) and TGFβ3 in leiomyoma cells cultured in the presence of 10–7 M asoprisnil (f, F). EGF in normal myometrial cells cultured without any treatment (g, G), EGF in normal myometrial cells cultured in the presence of 10–7 M asoprisnil (j, J), IGF-I in normal myometrial cells cultured without any treatment (h, H), IGF-I in normal myometrial cells cultured in the presence of 10–7 M asoprisnil (k, K), TGFβ3 in normal myometrial cells cultured without any treatment (i, I) and TGFβ3 in normal myometrial cells cultured in the presence of 10–7 M asoprisnil (l, L). Bars represent 50 μm. Original magnification 40.

Article Snippet: A mouse monoclonal antibody to human EGF (MAB236, R&D Systems), a goat polyclonal antibody to human IGF-I (AF-291-NA, R&D Systems) and a mouse monoclonal antibody to TGFβ3 (MAB643, R&D Systems) were used as the primary antibodies at a dilution of 1:20, 1:50 and 1:50, respectively.

Techniques: Expressing, Cell Culture

Journal: Human reproduction (Oxford, England)

Article Title: A novel selective progesterone receptor modulator asoprisnil (J867) down-regulates the expression of EGF, IGF-I, TGFbeta3 and their receptors in cultured uterine leiomyoma cells.

doi: 10.1093/humrep/del035

Figure Lengend Snippet: Figure 4. Effects of asoprisnil on the number of viable leiomyoma and normal myometrial cells cultured for 72 h in the presence of either recombinant epidermal growth factor (EGF), insulin-like growth factor-I (IGF-I) or transforming growth factor β (TGFβ3), as assessed by 3-(4,5-dimethylthi- azol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium (MTS) assay. Treatment with 100 ng/ml recombinant EGF signifi- cantly increased the number of viable cultured leiomyoma cells compared with untreated control cultures, and the concomitant treatment with 10–

Article Snippet: A mouse monoclonal antibody to human EGF (MAB236, R&D Systems), a goat polyclonal antibody to human IGF-I (AF-291-NA, R&D Systems) and a mouse monoclonal antibody to TGFβ3 (MAB643, R&D Systems) were used as the primary antibodies at a dilution of 1:20, 1:50 and 1:50, respectively.

Techniques: Cell Culture, Recombinant, MTS Assay, Control

Journal: Acta Pharmaceutica Sinica. B

Article Title: Cerebral endothelial 3-mercaptopyruvate sulfurtransferase improves ischemia-induced cognitive impairment via interacting with protein phosphatase 2A

doi: 10.1016/j.apsb.2024.11.015

Figure Lengend Snippet: Endothelial PP2A deficiency induces hippocampal dysfunction in mice. (A–C) Bioinformation analysis. Gene Ontology (GO) biological process analysis of genes related to VCD (A, B). Protein interaction network of genes enriched in “positive regulation of protein phosphorylation”, “response to hydrogen sulfide” and PP2ACA via STRING (C). (D) Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis of different genes in RNA sequencing ( n = 3). (E) Immunoblots of PP2AC α proteins level in PBMECs isolated from WT sham or WT LCA mice ( n = 4). (F, G) Representative confocal image (F) and quantification (G) of PP2AC α in brain endothelium of wild type mice after LCA, PP2Ac α (Green), lectin + endothelium (red) ( n = 4). Scale bar, 20 μm. (H) Survival curves of PP2A EC-cKO versus PP2AC α f/f mice after partial ligation on left carotid artery (LCA). (I–L) Cognition of male mice was evaluated by Y-maze test (I) and Morris water maze test 28 days after LCA. (J) The typical swim path of mice in the Morris water maze test. (K) Latency to reach the hidden platform during the acquisition trial after LCA. (L) Percentage occupancy in the goal quadrant in the probe trial ( n = 10–13). (M) Nissl staining for neurodegeneration in the hippocampus after LCA ( n = 5). (N, O) Representative confocal image (N) and quantification (O) of NeuN + neurons (red) in the CA3 and DG hippocampal subfield of PP2AC α f/f and PP2A EC-cKO mice ( n = 6). Scale bar, 20 or 50 μm. (P, Q) Reduced hippocampal fEPSP slope (P) and averages of the last 10 min of fEPSP recording(Q) in the CA1 region of PP2A EC-cKO mice compared to PP2AC α f/f mice. Representative traces before (1) and after (2) tetanic stimulation was shown above fEPSP recording. Scale bars depict 10 ms or 0.25 mV. PP2AC α f/f : n = 10 from 4 mice; PP2A EC-cKO : n = 10 from 3 mice. Data are expressed mean ± SEM, ∗ P < 0.05, ∗∗ P < 0.01.

Article Snippet: Cell lysates from PBMECs or bEnd.3 Cells infected with lenti-Ppp2ca-shRNA or lenti-vector were subjected to PP2A immunoprecipitation (IP) using anti-PP2A catalytic subunit (PP2AC) antibody (cell signaling technology, 2238S) overnight at 4 °C, followed by immobilization on protein A/G magnetic beads (MCE HYK, 0202) for 2 h. The immunoprecipitate was then incubated with phosphopeptide substrate (KRpTIRR) for 30 min at 37 °C, and the reaction was terminated using Malachite green reagent before measuring the absorbance at 620 nm.

Techniques: Phospho-proteomics, RNA Sequencing, Western Blot, Isolation, Ligation, Staining

Journal: Acta Pharmaceutica Sinica. B

Article Title: Cerebral endothelial 3-mercaptopyruvate sulfurtransferase improves ischemia-induced cognitive impairment via interacting with protein phosphatase 2A

doi: 10.1016/j.apsb.2024.11.015

Figure Lengend Snippet: PP2A deficiency in brain endothelium via AAV-Tie-Cre also induces cognitive dysfunction. (A) Experimental schematic of AAV-Tie-Cre intrahippocampal injection. (B, C) Representative confocal image (B) and transduction ratio (C) of Ppp2Cα deletion in hippocampal endothelium at 3 weeks after AAV-Tie-CRE injection. PP2Ac α f/f mice injected with AAV-Tie-CRE or AAV-zsGreen as control. PP2AC α (Green), lectin + endothelium (red) ( n = 6–7). Scale bar, 50 μm. (D–G) Cognition was evaluated by Y-maze test (D) and Morris water maze test (E–G) at 3 weeks after AAV-Tie-CRE injection. (E) The typical swim path of PP2Ac α f/f -zsGreen and PP2Ac α f/f -AAV-Tie-cre mice in the Morris water maze test. (F) Latency to reach the hidden platform during the acquisition trial. (G) Percentage occupancy in the goal quadrant in the probe trial, ( n = 10–11). (H) Nissl staining for neurodegeneration in the hippocampus of PP2Ac α f/f -zsGreen and PP2Ac α f/f -AAV-Tie-cre mice at 3 weeks after AAV-Tie-CRE injection, ( n = 5). (I, J) Representative confocal image (I) and quantification (J) of NeuN + neurons (red) in the CA3 and DG hippocampal subfield of PP2Ac α f/f -zsGreen and PP2Ac α f/f -AAV-Tie-cre mice at 3 weeks after AAV-Tie-CRE injection ( n = 6–8). Scale bar, 50 μm. Data are expressed mean ± SEM, ∗ P < 0.05, ∗∗ P < 0.01.

Article Snippet: Cell lysates from PBMECs or bEnd.3 Cells infected with lenti-Ppp2ca-shRNA or lenti-vector were subjected to PP2A immunoprecipitation (IP) using anti-PP2A catalytic subunit (PP2AC) antibody (cell signaling technology, 2238S) overnight at 4 °C, followed by immobilization on protein A/G magnetic beads (MCE HYK, 0202) for 2 h. The immunoprecipitate was then incubated with phosphopeptide substrate (KRpTIRR) for 30 min at 37 °C, and the reaction was terminated using Malachite green reagent before measuring the absorbance at 620 nm.

Techniques: Injection, Transduction, Control, Staining

Journal: Acta Pharmaceutica Sinica. B

Article Title: Cerebral endothelial 3-mercaptopyruvate sulfurtransferase improves ischemia-induced cognitive impairment via interacting with protein phosphatase 2A

doi: 10.1016/j.apsb.2024.11.015

Figure Lengend Snippet: Restoration of endothelial PP2A in the brain impedes cognitive dysfunction in PP2A EC-cKO mice. (A, B) Representative confocal image (A) and ratio of Ppp2Cα restoration in hippocampal endothelium (B) at 3 weeks after AAV virus injection. PP2Ac α (Green), lectin + (red) is the marker of endothelium ( n = 6–7). Scale bar, 50 μm. (C)–(F) Cognition was evaluated by Y-maze test (C) and Morris water maze test (D)–(F) at 3 weeks after AAV virus injection. (D) The typical swim path of PP2AC α f/f -zsGreen, PP2AC α f/f -AAV-Tie- Ppp2cα , PP2A EC-cKO -zsGreen, PP2A EC-cKO -AAV-Tie- Ppp2cα mice in the Morris water maze test. (E) Latency to reach the hidden platform during the acquisition trial. (F) Percentage occupancy in the goal quadrant in the probe trial ( n = 11–14). (G) Nissl staining for neurodegeneration in the hippocampus of PP2AC α f/f -zsGreen, PP2AC α f/f -AAV-Tie- Ppp2cα , PP2A EC-cKO -zsGreen, PP2A EC-cKO -AAV-Tie- Ppp2cα mice at 3 weeks after AAV virus injection ( n = 5). Scale bar, 100 μm. (H, I) Representative confocal image (H) and quantification (I) of NeuN + neurons (red) in the CA3 and DG hippocampal subfield of PP2AC α f/f -zsGreen, PP2AC α f/f -AAV-Tie- Ppp2cα , PP2A EC-cKO -zsGreen, PP2A EC-cKO -AAV-Tie- Ppp2cα mice at 3 weeks after AAV virus injection ( n = 6–8). Scale bar, 20 or 50 μm. Data are expressed mean ± SEM, ∗ P < 0.05, ∗∗ P < 0.01.

Article Snippet: Cell lysates from PBMECs or bEnd.3 Cells infected with lenti-Ppp2ca-shRNA or lenti-vector were subjected to PP2A immunoprecipitation (IP) using anti-PP2A catalytic subunit (PP2AC) antibody (cell signaling technology, 2238S) overnight at 4 °C, followed by immobilization on protein A/G magnetic beads (MCE HYK, 0202) for 2 h. The immunoprecipitate was then incubated with phosphopeptide substrate (KRpTIRR) for 30 min at 37 °C, and the reaction was terminated using Malachite green reagent before measuring the absorbance at 620 nm.

Techniques: Virus, Injection, Marker, Staining

Journal: Acta Pharmaceutica Sinica. B

Article Title: Cerebral endothelial 3-mercaptopyruvate sulfurtransferase improves ischemia-induced cognitive impairment via interacting with protein phosphatase 2A

doi: 10.1016/j.apsb.2024.11.015

Figure Lengend Snippet: Endothelial PP2A deficiency resulted in a reduction of H 2 S production in mice. (A, B) Bioinformation analysis for the interaction between PP2A and hydrogen sulfide. (C–E) Measurement of H 2 S concentration. (C) PP2AC α f/f and PP2A EC-cKO PBMECs, ( n = 5), (D) PP2AC α f/f PBMECs infected with AAV-Tie-cre ( n = 4), (E) bEnd.3 cells were transfected with Ppp2ca shRNA, ( n = 5). (F–I) Cognition was evaluated by Y-maze test (F) and Morris water maze test (G–I) at 2 days after termination of NaHS administration. (G) The typical swim path of PP2AC α f/f -saline, PP2AC α f/f -NaHS, PP2A EC-cKO -saline, PP2A EC-cKO -NaHS mice. (H) Latency to reach the hidden platform during the acquisition trial. (I) Percentage occupancy in the goal quadrant in the probe trial, ( n = 9–14). (J) Nissl staining for neurodegeneration in the hippocampus of PP2AC α f/f -saline, PP2AC α f/f -NaHS, PP2A EC-cKO -saline, PP2A EC-cKO -NaHS mice ( n = 5). Scale bar, 100 μm. (K, L) Representative confocal image (K) and quantification (L) of NeuN + neurons (red) in the CA3 and DG hippocampal subfield of PP2AC α f/f -saline, PP2AC α f/f -NaHS, PP2A EC-cKO -saline, PP2A EC-cKO -NaHS mice at 7 days after termination of NaHS administration ( n = 5∼7). Scale bar, 20 or 50 μm. Data are expressed mean ± SEM, ∗ P < 0.05, ∗∗ P < 0.01.

Article Snippet: Cell lysates from PBMECs or bEnd.3 Cells infected with lenti-Ppp2ca-shRNA or lenti-vector were subjected to PP2A immunoprecipitation (IP) using anti-PP2A catalytic subunit (PP2AC) antibody (cell signaling technology, 2238S) overnight at 4 °C, followed by immobilization on protein A/G magnetic beads (MCE HYK, 0202) for 2 h. The immunoprecipitate was then incubated with phosphopeptide substrate (KRpTIRR) for 30 min at 37 °C, and the reaction was terminated using Malachite green reagent before measuring the absorbance at 620 nm.

Techniques: Concentration Assay, Infection, Transfection, shRNA, Saline, Staining

Journal: Acta Pharmaceutica Sinica. B

Article Title: Cerebral endothelial 3-mercaptopyruvate sulfurtransferase improves ischemia-induced cognitive impairment via interacting with protein phosphatase 2A

doi: 10.1016/j.apsb.2024.11.015

Figure Lengend Snippet: Endothelial PP2A deficiency suppressed MPST expression in mice. (A, B) Representative confocal images (A) and quantification (B) of MPST in hippocampal endothelium of PP2AC α f/f and PP2A EC-cKO mice. MPST (green), Lectin (red) ( n = 7–8). Scale bar, 50 μm. (C, D) Representative confocal images (C) and quantification (D) of MPST in hippocampal endothelium of PP2AC α f/f -zsGreen and PP2AC α f/f -AAV-Tie-cre mice ( n = 6). MPST (green), Lectin (red). Scale bar = 50 μm. (E) Immunoblots of MPST proteins level in PBMECs and bEnd.3 cells ( n = 4 for PBMECs; n = 5 for bEnd.3 cells). (F) Immunoblots of MPST proteins level in bEnd.3 cells. bEnd.3 cells were treated with 100 nmol/L okadaic acid (OA) to inhibit PP2A activity ( n = 6). (G) Immunoblots of MPST proteins level in PBMECs lysates isolated from PP2AC α f/f mice infected with AAV-zsGreen or AAV-Tie-cre ( n = 7). (H) Immunoblots of MPST proteins level in PBMECs lysates isolated from PP2AC α f/f and PP2A EC-cKO mice infected with AAV-zsGreen or AAV-Tie- Mpst ( n = 4). (I) H 2 S concentration in PBMECs lysates isolated from PP2AC α f/f and PP2A EC-cKO mice infected with AAV-zsGreen or AAV-Tie- Mpst ( n = 5–6). (J–M) Cognition was evaluated by Y-maze test (J) and Morris water maze test (K–M) at 3 weeks after AAV-Tie- Mpst injection. (K) The typical swim path of PP2AC α f/f -zsGreen, PP2AC α f/f - Mpst , PP2A EC-cKO -zsGreen, PP2A EC-cKO - Mpst mice. (L) Latency to reach the hidden platform during the acquisition trial. (M) Percentage occupancy in the goal quadrant in the probe trial ( n = 10–13). (N, O) Representative confocal image (N) and quantification (O) of NeuN + neurons (red) in the CA3 and DG hippocampal subfield of PP2AC α f/f -zsGreen, PP2AC α f/f - Mpst , PP2A EC-cKO -zsGreen, PP2A EC-cKO - Mpst ( n = 5–7). Scale bar, 50 μm. Data are expressed as mean ± SEM, ∗ P < 0.05, ∗∗ P < 0.01.

Article Snippet: Cell lysates from PBMECs or bEnd.3 Cells infected with lenti-Ppp2ca-shRNA or lenti-vector were subjected to PP2A immunoprecipitation (IP) using anti-PP2A catalytic subunit (PP2AC) antibody (cell signaling technology, 2238S) overnight at 4 °C, followed by immobilization on protein A/G magnetic beads (MCE HYK, 0202) for 2 h. The immunoprecipitate was then incubated with phosphopeptide substrate (KRpTIRR) for 30 min at 37 °C, and the reaction was terminated using Malachite green reagent before measuring the absorbance at 620 nm.

Techniques: Expressing, Western Blot, Activity Assay, Isolation, Infection, Concentration Assay, Injection

Journal: Acta Pharmaceutica Sinica. B

Article Title: Cerebral endothelial 3-mercaptopyruvate sulfurtransferase improves ischemia-induced cognitive impairment via interacting with protein phosphatase 2A

doi: 10.1016/j.apsb.2024.11.015

Figure Lengend Snippet: Endothelial PP2A deficiency promotes calpain2-mediated degradation of MPST. (A, B) Relative mRNA levels of MPST in PBMECs isolated from PP2AC α f/f or PP2A EC-cKO mice ( n = 4) (A) and bEnd.3 cells ( n = 6) (B). (C, D) Relative mRNA levels of Capn1 (C) and Capn2 (D) in PBMECs lysates isolated from PP2AC α f/f or PP2A EC-cKO mice ( n = 4–5). (E, F) Immunoblots of calpain 2 (E) and calpain1 (F) proteins level in PBMECs lysates isolated from PP2AC α f/f or PP2A EC-cKO mice ( n = 4–5). (G) Representative line-scan images of Ca 2+ sparks and time-dependent profiles of global Ca 2+ influx induced by application of 0.3 mol/L CaCl 2 for PP2AC α f/f and PP2A EC-cKO PBMECs. (H) Maximum amplitude of CaCl 2 induced Ca 2+ transients. (I) Immunoblot analysis of calpain2 phosphorylation level in PP2AC α f/f and PP2A EC-cKO PBMECs using phospho-serine antibody. (J) Immunoblot analysis of calpain2 and MPST proteins in PBMECs lysates immunoprecipitated with control IgG or anti-calpain2 antibody. (K) Immunoblot analysis of calpain2 and MPST proteins in PBMECs lysates immunoprecipitated with control IgG or anti-MPST antibody. (L–N) Immunoblots of calpain2 and MPST in PP2AC α f/f and PP2A EC-cKO PBMECs. PBMECs were treated with vehicle or 40 μmol/L PD150606 for 12 h. Data are expressed as mean ± SEM, ∗ P < 0.05, ∗∗ P < 0.01.

Article Snippet: Cell lysates from PBMECs or bEnd.3 Cells infected with lenti-Ppp2ca-shRNA or lenti-vector were subjected to PP2A immunoprecipitation (IP) using anti-PP2A catalytic subunit (PP2AC) antibody (cell signaling technology, 2238S) overnight at 4 °C, followed by immobilization on protein A/G magnetic beads (MCE HYK, 0202) for 2 h. The immunoprecipitate was then incubated with phosphopeptide substrate (KRpTIRR) for 30 min at 37 °C, and the reaction was terminated using Malachite green reagent before measuring the absorbance at 620 nm.

Techniques: Isolation, Western Blot, Phospho-proteomics, Immunoprecipitation, Control

Journal: Acta Pharmaceutica Sinica. B

Article Title: Cerebral endothelial 3-mercaptopyruvate sulfurtransferase improves ischemia-induced cognitive impairment via interacting with protein phosphatase 2A

doi: 10.1016/j.apsb.2024.11.015

Figure Lengend Snippet: PP2A activator FTY720 restores ischemic neuron dysfunction and endothelial MPST expression. (A, B) Representative confocal image (A) and quantification (B) of MPST expression in brain endothelium. MPST (Green), lectin + (red). Scale bar, 20 μm. (C) Measurement of H 2 S concentration after FTY720 treatment. (D–G) Cognition was evaluated by Morris water maze test (D–F) and Y-maze test (G) at 2 weeks after FTY720 treatment. (D) The typical swim path of WT or LCA mice treated with either vehicle (0.4% dimethyl sulfoxide in PBS) or FTY720 (2 mg/kg) by daily gavage. (E) Latency to reach the hidden platform during the acquisition trial. (F) Percentage occupancy in the goal quadrant in the probe trial. (G) Percentage time spent in the novel arm ( n = 10–11). (H, I) Representative confocal image (H) and quantification (I) of NeuN + neurons in the brain ( n = 6). Data are expressed as mean ± SEM, ∗ P < 0.05.

Article Snippet: Cell lysates from PBMECs or bEnd.3 Cells infected with lenti-Ppp2ca-shRNA or lenti-vector were subjected to PP2A immunoprecipitation (IP) using anti-PP2A catalytic subunit (PP2AC) antibody (cell signaling technology, 2238S) overnight at 4 °C, followed by immobilization on protein A/G magnetic beads (MCE HYK, 0202) for 2 h. The immunoprecipitate was then incubated with phosphopeptide substrate (KRpTIRR) for 30 min at 37 °C, and the reaction was terminated using Malachite green reagent before measuring the absorbance at 620 nm.

Techniques: Expressing, Concentration Assay

Journal: Acta Pharmaceutica Sinica. B

Article Title: Cerebral endothelial 3-mercaptopyruvate sulfurtransferase improves ischemia-induced cognitive impairment via interacting with protein phosphatase 2A

doi: 10.1016/j.apsb.2024.11.015

Figure Lengend Snippet: 3MP sodium restores ischemic neuron dysfunction and endothelial MPST expression. (A) Measurement of H 2 S concentration after 3MP sodium treatment. (B, C) Representative confocal image (B) and quantification (C) of MPST expression in brain endothelium. MPST (Green), lectin + (red). Scale bar, 20 μm. (D, E) Representative confocal image (D) and quantification (E) of PP2AC α expression in brain endothelium. PP2AC α (Green), lectin + (red). Scale bar, 20 μm. (F–H) Cognition was evaluated by Novel object recognition (F–G) and Y-maze test (H) at 12 days after 3MP sodium treatment. (F) The typical path of WT or LCA mice treated with either vehicle (0.4% dimethyl sulfoxide in PBS) or 3MP sodium (1 mg/kg) by daily i.p. (G) Discrimination index toward a novel object. (H) Percentage time spent in the novel arm ( n = 9). (I, J) Representative confocal image (I) and quantification (J) of NeuN + neurons in the brain ( n = 6). Data are expressed mean ± SEM, ∗ P < 0.05, ∗∗ P < 0.01.

Article Snippet: Cell lysates from PBMECs or bEnd.3 Cells infected with lenti-Ppp2ca-shRNA or lenti-vector were subjected to PP2A immunoprecipitation (IP) using anti-PP2A catalytic subunit (PP2AC) antibody (cell signaling technology, 2238S) overnight at 4 °C, followed by immobilization on protein A/G magnetic beads (MCE HYK, 0202) for 2 h. The immunoprecipitate was then incubated with phosphopeptide substrate (KRpTIRR) for 30 min at 37 °C, and the reaction was terminated using Malachite green reagent before measuring the absorbance at 620 nm.

Techniques: Expressing, Concentration Assay

Journal: Scientific Reports

Article Title: Altered fibroblast-like synoviocyte epigenetics is responsible for deficient NUB1 expression in rheumatoid arthritis

doi: 10.1038/s41598-026-38420-y

Figure Lengend Snippet: Immunohistochemical staining of NUB1, NEDD8, IL-6, NF-kB in RA and OA synovial tissues. ( a ) Representative images (magnification, ×200) of immunohistochemistry for NUB1, NEDD8, and IL-6 in synovial tissues from patients with RA and OA. Expression of NEDD8 and IL-6 was higher in the intimal lining of RA synovium, and expression of NUB1 was lower compared with OA (see Fig. for quantification). Scale bar = 100 μm, ( b ) Representative images (magnification, ×400) of immunohistochemistry for NUB1 and p65 in synovial tissues from patients with RA and OA. In RA, regions with reduced NUB1 expression showed prominent nuclear localization of p65, whereas in OA, areas with higher NUB1 expression displayed weaker p65 nuclear staining. Negative control (NC) sections were stained with rabbit IgG under identical conditions. Scale bar = 50 μm.

Article Snippet: For immunohistochemistry, anti-NEDD8 (19E3) rabbit mAb (#2754), and anti-p65 rabbit mAb (#8242), were purchased from Cell Signaling Technology, and a rabbit polyclonal anti-NUB1 antibody (#14343-1-AP), and anti-rabbit polyclonal anti-IL-6 antibody (#21865-1-AP) were purchased from Proteintech, and normal Rabbit IgG control (#AB-105-C) was purchased from R༆D).

Techniques: Immunohistochemical staining, Staining, Immunohistochemistry, Expressing, Negative Control

Journal: Scientific Reports

Article Title: Altered fibroblast-like synoviocyte epigenetics is responsible for deficient NUB1 expression in rheumatoid arthritis

doi: 10.1038/s41598-026-38420-y

Figure Lengend Snippet: Immunohistochemical scoring of NUB1, NEDD8, and IL-6 in RA and OA synovial tissues. Synovial tissue sections from RA (n = 5) and OA (n = 5) patients were immunostained for NUB1, NEDD8, IL-6, and CCL5. For each patient, three randomly selected fields were evaluated in the lining and sublining zones. Staining was semiquantitatively scored as described in Methods. Graphs show the total scores as well as the average scores for the lining and sublining zones. For the total scores, NUB1 was higher in OA than in RA, whereas NEDD8 and IL-6 were significantly higher in RA; these differences were most prominent in the lining layer. Data are presented as mean ± SEM. Statistical analysis was performed using unpaired t test with Welch’s correction or Mann-Whitney U test (* p <0.05; ** p <0.01).

Article Snippet: For immunohistochemistry, anti-NEDD8 (19E3) rabbit mAb (#2754), and anti-p65 rabbit mAb (#8242), were purchased from Cell Signaling Technology, and a rabbit polyclonal anti-NUB1 antibody (#14343-1-AP), and anti-rabbit polyclonal anti-IL-6 antibody (#21865-1-AP) were purchased from Proteintech, and normal Rabbit IgG control (#AB-105-C) was purchased from R༆D).

Techniques: Immunohistochemical staining, Staining, MANN-WHITNEY

Journal: Scientific Reports

Article Title: Altered fibroblast-like synoviocyte epigenetics is responsible for deficient NUB1 expression in rheumatoid arthritis

doi: 10.1038/s41598-026-38420-y

Figure Lengend Snippet: Basal and IL-1-induced expression of NUB1 in RA and OA FLS. ( a ) Left panel: Basal NUB1 mRNA levels in RA and OA FLS were quantified by RT-qPCR and normalized to GAPDH. Right panel: Following stimulation with IL-1β (2 ng/mL) for 6 h, the fold change in NUB1 expression relative to unstimulated cells was calculated for each group (n = 6 each). Basal NUB1 mRNA expression was comparable between RA and OA FLS ( p = 0.153), whereas IL-1β–induced upregulation of NUB1 was significantly lower in RA FLS than in OA FLS. ( b ) NUB1 protein expression in RA and OA FLS following IL-1β stimulation. Left panel: RA and OA FLS were stimulated with IL-1β (2 ng/mL) for 24 h, and NUB1 protein expression was evaluated by Western blotting. Right panel: NUB1 band intensities were quantified after normalization to α-tubulin. IL-1β-induced NUB1 protein expression was also significantly reduced in RA FLS compared with OA FLS. These indicate defective IL-1–mediated NUB1 induction in RA FLS. Data are presented as mean ± SEM. Statistical significance was determined using unpaired t test with Welch’s correction. (** p < 0.01, *** p < 0.001).

Article Snippet: For immunohistochemistry, anti-NEDD8 (19E3) rabbit mAb (#2754), and anti-p65 rabbit mAb (#8242), were purchased from Cell Signaling Technology, and a rabbit polyclonal anti-NUB1 antibody (#14343-1-AP), and anti-rabbit polyclonal anti-IL-6 antibody (#21865-1-AP) were purchased from Proteintech, and normal Rabbit IgG control (#AB-105-C) was purchased from R༆D).

Techniques: Expressing, Quantitative RT-PCR, Western Blot

Journal: Scientific Reports

Article Title: Altered fibroblast-like synoviocyte epigenetics is responsible for deficient NUB1 expression in rheumatoid arthritis

doi: 10.1038/s41598-026-38420-y

Figure Lengend Snippet: Regulation of NUB1 expression by MAPKs . RA and OA FLS (n = 5 each) were stimulated with IL-1β (2 ng/mL) for 6h in the presence or absence of MAPK inhibitors: SP600125 (JNK inhibitor), SB203580 (p38 MAPK inhibitor), and U0126 (MEK1/2–ERK inhibitor). NUB1 mRNA expression is shown as fold change relative to IL-1β stimulation alone. ( a ) Quantitative analysis demonstrated that none of the MAPK inhibitors significantly reduced IL-1β–induced NUB1 mRNA expression in either RA or OA FLS (IL-1β vs. IL-1β + SB203580, adjusted p > 0.9999; IL-1β vs. IL-1β + SP600125, adjusted p > 0.9999; IL-1β vs. IL-1β + U0126, adjusted p = 0.8285). ( b ) IL-6 mRNA expression was quantified by RT-qPCR and normalized to GAPDH. IL-6 expression was significantly reduced by each MAPK inhibitor compared with IL-1β stimulation alone (**** p < 0.0001), confirming effective inhibition of MAPK-dependent signaling. Data are presented as mean ± SEM. Statistical significance was determined using one-way ANOVA with Tukey’s multiple comparisons test.

Article Snippet: For immunohistochemistry, anti-NEDD8 (19E3) rabbit mAb (#2754), and anti-p65 rabbit mAb (#8242), were purchased from Cell Signaling Technology, and a rabbit polyclonal anti-NUB1 antibody (#14343-1-AP), and anti-rabbit polyclonal anti-IL-6 antibody (#21865-1-AP) were purchased from Proteintech, and normal Rabbit IgG control (#AB-105-C) was purchased from R༆D).

Techniques: Expressing, Quantitative RT-PCR, Inhibition

Journal: Scientific Reports

Article Title: Altered fibroblast-like synoviocyte epigenetics is responsible for deficient NUB1 expression in rheumatoid arthritis

doi: 10.1038/s41598-026-38420-y

Figure Lengend Snippet: ( a ) mRNA half life of NUB1. RA and OA FLS (n = 5 each) were stimulated with IL-1β (2 ng/mL) for 6 h, followed by treatment with actinomycin D (10 μg/mL) to block transcription. NUB1 mRNA expression was then measured by qRT-PCR at 0, 4, 8, 12, and 18 h after actinomycin D treatment. Expression levels were normalized to GAPDH and presented as fold change relative to the 0 h time point (i.e., the level observed immediately after 6 h of IL-1β stimulation). Decay curves showed no significant differences between groups. mRNA decay curves were fitted using a one-phase exponential decay model by nonlinear regression to calculate NUB1 mRNA half-life, and decay constants (K) for RA and OA were compared using an extra sum-of-squares F -test. ( b ) Effect of SNHG12 knockdown on NUB1 expression in RA and OA FLS. SNHG12 (Small Nucleolar RNA Host Gene 12), a long non-coding RNA associated with NUB1, was depleted in RA and OA FLS using SNHG12 siRNA (knockdown efficiency >80%). RA and OA (n = 7 each) FLS were stimulated with IL-1β (2 ng/mL) for 6 h, and NUB1 mRNA expression was quantified by RT-qPCR and normalized to GAPDH. Left panel: Fold change in NUB1 expression following IL-1β stimulation in siCT-transfected cells (IL-1/unstimulated). Right panel: Fold change in NUB1 expression following IL-1β stimulation after SNHG12 knockdown (siSNHG12 + IL-1/siSNHG12 unstimulated). Knockdown of SNHG12 had little effect on the RA–OA difference in NUB1 induction. Data are presented as mean ± SEM. Statistical analysis was performed using unpaired t test with Welch’s correction (** p < 0.01, *** p < 0.001). ( c ) NUB1 promoter activity in RA and OA FLS. RA and OA FLS (n = 5 each) were transfected with a luciferase reporter construct containing the NUB1 promoter region and co-transfected with Renilla vector for normalization (see Material and Methods). After transfection, cells were stimulated with IL-1β (2 ng/mL). Firefly luciferase activity was normalized to Renilla and expressed as fold change calculated by dividing the IL-1β–stimulated value by the corresponding unstimulated (media) control at each time point. Limited promoter induction was noted, possibly because transcription requires epigenetic marks or the three-dimensional structure of chromatin not present in plasmids. No significant differences in IL-1β–induced NUB1 promoter activity were observed between RA and OA FLS at 1 h ( p = 0.209), 3 h ( p = 0.415), or 6 h ( p = 0.446). Data are presented as mean ± SEM. Statistical analysis was performed using an unpaired t test with Welch’s correction.

Article Snippet: For immunohistochemistry, anti-NEDD8 (19E3) rabbit mAb (#2754), and anti-p65 rabbit mAb (#8242), were purchased from Cell Signaling Technology, and a rabbit polyclonal anti-NUB1 antibody (#14343-1-AP), and anti-rabbit polyclonal anti-IL-6 antibody (#21865-1-AP) were purchased from Proteintech, and normal Rabbit IgG control (#AB-105-C) was purchased from R༆D).

Techniques: Blocking Assay, Expressing, Quantitative RT-PCR, Knockdown, Transfection, Activity Assay, Luciferase, Construct, Plasmid Preparation, Control

Journal: Scientific Reports

Article Title: Altered fibroblast-like synoviocyte epigenetics is responsible for deficient NUB1 expression in rheumatoid arthritis

doi: 10.1038/s41598-026-38420-y

Figure Lengend Snippet: Effects of epigenetic inhibitors on IL-1β–induced NUB1 expression in RA and OA fibroblast-like synoviocytes (FLS). RA and OA FLS were pretreated with epigenetic inhibitors and subsequently stimulated with IL-1β (2 ng/mL). NUB1 mRNA expression was quantified by qRT-PCR. Fold change was calculated as the ratio of NUB1 mRNA expression in IL-1β–stimulated cells to the corresponding unstimulated control; specifically, DMSO alone for the IL-1/DMSO condition and the inhibitor-alone control for inhibitor-treated conditions. ( a ) 5-aza-deoxycytidine (5-aza-dC; DNA methyltransferase inhibitor). RA and OA FLS were treated with 5-aza-dC for 14 days prior to stimulation with IL-1β (n = 7 each). 5-aza-dC partially reduced the difference in IL-1β–induced NUB1 expression between RA and OA. Under control conditions, IL-1β–induced NUB1 expression was significantly higher in OA than in RA ( p = 0.024). Following 5-aza-dC treatment, the magnitude of the OA–RA difference was no longer statistically significant ( p = 0.488), indicating partial attenuation of the baseline RA–OA difference. ( b ) EPZ6438 (EZH2 inhibitor; histone methylation inhibitor). RA and OA FLS were treated with EPZ6438 for 12 or 24 hours before IL-1β stimulation (n = 5 each). treatment with EPZ6438 partially reversed the difference in IL-1β–induced NUB1 expression between RA and OA. Under control conditions, IL-1β–induced NUB1 expression was significantly higher in OA than in RA. This difference was no longer significant after EPZ6438 treatment at 12 h ( p = 0.329) or 24 h ( p = 0.512). ( c ) Left panel: ITF2357 (pan-HDAC inhibitor); Right panel: MS275 (HDAC1/3 selective inhibitor). RA and OA FLS were treated with each HDAC inhibitor for 12 or 24 hours, followed by IL-1β stimulation (ITF2357; n = 5 each, MS275; n = 6 each). Under control conditions, IL-1β–induced NUB1 expression was significantly higher in OA than in RA in the ITF2357 panel. Following ITF2357 treatment, the OA–RA difference was no longer statistically significant at 12 h ( p = 0.596) or 24 h ( p = 0.072). Under control conditions, IL-1β–induced NUB1 expression was also significantly higher in OA than in RA in the MS275 panel. Following MS275 treatment, the OA–RA difference was no longer statistically significant at either 12 h ( p = 0.944) or 24 h ( p = 0.846). These data indicate that histone modifications are required for differential induction of NUB1 in OA compared with RA. Circles and squares represent mean fold change for OA and RA FLS, respectively. Statistical analysis was performed at each time point and condition performed by comparing the fold change values between RA and OA FLS using unpaired t test with Welch’s correction (* p < 0.05, ** p < 0.01).

Article Snippet: For immunohistochemistry, anti-NEDD8 (19E3) rabbit mAb (#2754), and anti-p65 rabbit mAb (#8242), were purchased from Cell Signaling Technology, and a rabbit polyclonal anti-NUB1 antibody (#14343-1-AP), and anti-rabbit polyclonal anti-IL-6 antibody (#21865-1-AP) were purchased from Proteintech, and normal Rabbit IgG control (#AB-105-C) was purchased from R༆D).

Techniques: Expressing, Quantitative RT-PCR, Control, Methylation

Journal: Clinical Cancer Research

Article Title: Optimized EGFR Blockade Strategies in EGFR Addicted Gastroesophageal Adenocarcinomas

doi: 10.1158/1078-0432.ccr-20-0121

Figure Lengend Snippet: Figure 1. EGFR CNG. The graphs illustrate the percentage of tumors displaying EGFR CNG in four different cohorts. Real-time PCR analysis of IRCC gastric/gastroesophageal junction (GEJ) adenocarcinomas displaying EGFR gain (4–8 copies or >8 copies)or heterogeneity (significantly different EGFR CNG in diverse analyzed samples from the same tumor, with one tumor sample displaying >8 copies and one tumor sample having≤8 copies).CGP of FMI gastricand esophageal/gastroesophageal junction cases, FISH analysis of COG esophageal/gastroesophageal junction cases, and SISH analysis of INT gastric/gastroesophageal junction adenocarcinomas.

Article Snippet: Primary antibodies, anti-EGFR (1005: sc-03) and anti-Actin, were from Santa Cruz Biotechnology, and antibodies against phosphorylated EGFR (Tyr 845), ERK (Thr202/Tyr204), phosphorylated AKT (Ser473) (Clone D9E), total AKT, and ERK were from Cell Signaling Technology.

Techniques: Real-time Polymerase Chain Reaction

Journal: Clinical Cancer Research

Article Title: Optimized EGFR Blockade Strategies in EGFR Addicted Gastroesophageal Adenocarcinomas

doi: 10.1158/1078-0432.ccr-20-0121

Figure Lengend Snippet: Figure 2. Survival analysis of patients with EGFR CNG. A, The graphs show the cumulative survival (cum survival) of patients of the COG (left) and INT (right) cohorts related to EGFR CNG. B, The graphs show the OS (left) and the disease-free survival (right) of patients of the gastroesophageal TCGA dataset, related to EGFR CNG.

Article Snippet: Primary antibodies, anti-EGFR (1005: sc-03) and anti-Actin, were from Santa Cruz Biotechnology, and antibodies against phosphorylated EGFR (Tyr 845), ERK (Thr202/Tyr204), phosphorylated AKT (Ser473) (Clone D9E), total AKT, and ERK were from Cell Signaling Technology.

Techniques:

Journal: Clinical Cancer Research

Article Title: Optimized EGFR Blockade Strategies in EGFR Addicted Gastroesophageal Adenocarcinomas

doi: 10.1158/1078-0432.ccr-20-0121

Figure Lengend Snippet: Figure 3. Clinical history of patients treated with EGFR-targeted drugs. Summarized clinical course of INT patients with EGFR CNG. Red-lined boxes indicate periods of administration of the indicated therapeutic agents. Blue vertical lines indicate timing of tumor specimen acquisition from surgical procedures or biopsies, as well as dates of tumor assessment by CT scan. PD and SD according to RECIST 1.1. 5FU, 5-fluorouracil; CCDP, cisplatin, vinorelbine, ifosfamide, and epirubicin; EOX, epirubicin, oxaliplatin, and capecitabine; FOLFIRI, folinic acid, 5-fluorouracil, and irinotecan; OGD, esophago-gastro-duodenoscopy; TCF, docetaxel, carboplatin, and 5-fluorouracil; XELOX, capecitabine and oxaliplatin.

Article Snippet: Primary antibodies, anti-EGFR (1005: sc-03) and anti-Actin, were from Santa Cruz Biotechnology, and antibodies against phosphorylated EGFR (Tyr 845), ERK (Thr202/Tyr204), phosphorylated AKT (Ser473) (Clone D9E), total AKT, and ERK were from Cell Signaling Technology.

Techniques: Computed Tomography

Journal: Clinical Cancer Research

Article Title: Optimized EGFR Blockade Strategies in EGFR Addicted Gastroesophageal Adenocarcinomas

doi: 10.1158/1078-0432.ccr-20-0121

Figure Lengend Snippet: Figure 4. RTK/KRAS pathway activation in EGFR-amplified cases. A, The scatter plot shows a significant inverse correlation between the extent of RTKs coamplification (HER2, HER3, HER4, MET, FGFR1, FGFR2, FGFR3, FGFR4, IGF1R, PDGFR2, VEGFR1, VEGR2, and VEGFR3 ≥4 gene copies) and OS in 12 EGFR FISH–positive gastroesophageal adenocar- cinomas treated with gefitinib in the COG trial. Red dots indicate cases with high polysomy and blue dots represent cases with EGFR amplification. B, The graph shows the cooccurrence of EGFR amplification and genomic events affecting the RTK/KRAS pathway in EGFR-amplified gastro- esophageal adenocarcinoma tumors in the FMI dataset.

Article Snippet: Primary antibodies, anti-EGFR (1005: sc-03) and anti-Actin, were from Santa Cruz Biotechnology, and antibodies against phosphorylated EGFR (Tyr 845), ERK (Thr202/Tyr204), phosphorylated AKT (Ser473) (Clone D9E), total AKT, and ERK were from Cell Signaling Technology.

Techniques: Activation Assay

Journal: Clinical Cancer Research

Article Title: Optimized EGFR Blockade Strategies in EGFR Addicted Gastroesophageal Adenocarcinomas

doi: 10.1158/1078-0432.ccr-20-0121

Figure Lengend Snippet: Figure 5. Dual EGFR blockade is the most effective treatment in EGFR-amplified PDXs. Tumor growth curves in mice cohorts derived from GTR0060 (A), GTR0110 (C), and GTR0511 (D) patients treated with the EGFR inhibitors, cetuximab (CETUX), erlotinib (ERL), and lapatinib (LAP), alone or in combination, as indicated. The red lines indicate the day when treatment was started. The response in mice has been evaluated using RECIST 1.1–like criteria, that is, PD: ≥35% increase from baseline; PR: ≥ 50% reduction from baseline; and SD: intermediate variations from baseline. B, Spaghetti plot illustrating drug response in the xenotrial performed on the cohort of mice derived from GTR0060 PDX. Individual lines represent, for each mouse, the percentage variation in tumor burden, from start of treatment (day 0). Blue lines, cetuximab þ lapatinib–treated mice and red lines, cetuximab þ erlotinib–treated mice. Dashed line indicates treatment stop. E, Western blot analysis of the activation state of EGFR and its downstream targets (AKT, MAPK, and S6) in GTR0060 tumor–derived cells treated with the indicated drugs/drug combinations. Actin was used as loading control. Statistical significance is indicated (, P <0.01; , P < 0.001).

Article Snippet: Primary antibodies, anti-EGFR (1005: sc-03) and anti-Actin, were from Santa Cruz Biotechnology, and antibodies against phosphorylated EGFR (Tyr 845), ERK (Thr202/Tyr204), phosphorylated AKT (Ser473) (Clone D9E), total AKT, and ERK were from Cell Signaling Technology.

Techniques: Derivative Assay, Western Blot, Activation Assay, Control

Journal: Clinical Cancer Research

Article Title: Optimized EGFR Blockade Strategies in EGFR Addicted Gastroesophageal Adenocarcinomas

doi: 10.1158/1078-0432.ccr-20-0121

Figure Lengend Snippet: Figure 6. TSC2 inactivation is a mechanism of resistance to EGFR-targeted therapies. A, Tumor growth curves in the mice cohorts derived from GTR0078 and treated with the EGFR inhibitors, cetuximab (CETUX), erlotinib (ERL), and lapatinib (LAP), alone or in combination, as indicated. The red line indicates the day when treatment was started. B, The table shows the two TSC2 mutations identified in GTR0078 PDX. C, Tumor growth curves in the mice cohorts derived from GTR0078 and treated with erlotinib or the combination erlotinib þ everolimus (ERLþ EVEROL). The red line indicates the day when treatment was started. D, Western blot analysis of the activation state of EGFR and its downstream targets (AKT, MAPK, and S6) in GTR0078 tumor–derived cells treated with the indicated drugs/drug combinations. Actin was used as loading control. Statistical significance is indicated (, P < 0.001).

Article Snippet: Primary antibodies, anti-EGFR (1005: sc-03) and anti-Actin, were from Santa Cruz Biotechnology, and antibodies against phosphorylated EGFR (Tyr 845), ERK (Thr202/Tyr204), phosphorylated AKT (Ser473) (Clone D9E), total AKT, and ERK were from Cell Signaling Technology.

Techniques: Derivative Assay, Western Blot, Activation Assay, Control