Journal: Redox Biology

Article Title: Inhibition of NLRP3-mediated crosstalk between hepatocytes and liver macrophages by geniposidic acid alleviates cholestatic liver inflammatory injury

doi: 10.1016/j.redox.2022.102404

Figure Lengend Snippet: GPA directly binds to NLRP3 and represses its expression. (A) Dual-luciferase reporter assay results. RAW264.7 cells were transiently co‐transfected with NLRP3-luc plasmids for 12 h and treated with DMSO (vehicle control), GPA-H (100 μM), GPA-M (50 μM), GPA-L (25 μM), or MCC950 (10 μM) for 24 h. Relative luciferase activity was calculated by ration of firefly luciferase/renilla luciferase activity (n = 5). * P < 0.05, ** P < 0.01, compared to the DMSO control transfected with empty vector. Data are expressed as the mean ± SEM of 5 independent experiments ( n = 5). (B) mRNA levels of NLRP3, NFκB1, and IL-1β in THP-1 monocyte-derived macrophages and BMDM as analyzed by real-time PCR after 24 h treatment with different concentrations of GPA (25–100 μM). Results are expression as fold changes compared to the DMSO group. Data are expressed as the mean ± SEM of 5 independent experiments ( n = 5). * P < 0.05, ** P < 0.01 versus DMSO control group. (C) Protein levels of NLRP3, ASC, CASP-1, and IL-1β in THP-1 monocyte-derived macrophages were determined by Western blot analysis after 24 h treatment with different concentrations of GPA. β-ACTIN was used as internal reference protein. Data are expressed as the mean ± SEM of 3 independent experiments( n = 3). (D) Cell lysates of LPS-primed THP-1 monocytes were incubated with GPA (100 μM) or Bio-GPA (100 μM) for 4 h and subjected to pull-down assays with streptavidin beads. Total (input), bound (pull-down), and remaining proteins (after pull-down) were immunoblotted as indicated. β-ACTIN was used as internal reference protein.Data are expressed as the mean ± SEM of 3–4 independent experiments ( n = 3–4). (E) Competitive experiment. Cell lysates of LPS-primed THP-1 monocytes were incubated with Bio-GPA (100 μM) and different concentrations of free GPA (50 or 100 μM) and subjected to pull-down assays with streptavidin beads. β-ACTIN was used as internal reference protein. Data are expressed as the mean ± SEM of 3–4 independent experiments ( n = 3–4). (F) Human recombinant NLRP3 proteins were incubated with the indicated doses of Bio-GPA (50 or 100 μM) and subjected to pull-down assays with streptavidin beads. NLRP3 was used as internal reference protein. Data are expressed as the mean ± SEM of 3–4 independent experiments ( n = 3–4). (G) Human recombinant NLRP3 proteins were incubated with Bio-GPA (100 μM) or/and β-Mercaptoethanol (50 μM) and subjected to pull-down assays with streptavidin beads. NLRP3 was used as internal reference protein. Data are expressed as the mean ± SEM of 3–4 independent repeats ( n = 3–4).

Article Snippet: TaqMan probes, including those for human-specific NLRP3 (Hs00918082), NF κ B1 (Hs00765730), IL-1 β (Hs00174097), IL-6 (Hs00174131 ) , ICAM-1 (Hs00164932), COX-2 (Hs00153133), TNF- α (Hs00174128), and GAPDH (Hs02786624); and mouse-specific Nlrp3 (Mm00840904), Nf κ b1 (Mm00476361), Il-1 β (Mm00434228), Il-6 (Mm00446190 ) , Icam-1 (Mm00516023), Cox-2 (Mm00478374), Tnf- α (Mm00443258), Cyp7a1 (Mm00484150), Cyp8b1 (Mm00501637), Cyp27a1 (Mm00470430), Cyp2b10 (Mm01972453), Cyp3a11 (Mm00731567), Ugt1a1 (Mm02603337), Abcb11 (Mm00445168), Abcc2 (Mm00496899), Abcc3 (Mm00551550), Abcc4 (Mm01226381), Slc01b2 (Mm00451510), Slc10a1 (Mm00441421), and Gapdh (Mm99999915), were acquired from Invitrogen (Carlsbad, CA, USA).

Techniques: Expressing, Luciferase, Reporter Assay, Transfection, Control, Activity Assay, Plasmid Preparation, Derivative Assay, Real-time Polymerase Chain Reaction, Western Blot, Incubation, Recombinant

Journal: Redox Biology

Article Title: Inhibition of NLRP3-mediated crosstalk between hepatocytes and liver macrophages by geniposidic acid alleviates cholestatic liver inflammatory injury

doi: 10.1016/j.redox.2022.102404

Figure Lengend Snippet: GPA reduces LPS-induced NLRP3 expression via inhibiting NF-κB signaling. (A – C) THP-1 monocytes were pretreated with the indicated concentrations of GPA for 24 h and stimulated with LPS (1 μg/ml) for 24 h. The levels of mRNA (A) , protein (B) , and pro-inflammatory cytokines in supernatants (C) were measured by real-time PCR, Western blot, and ELISA, respectively. mRNA results are expressed as fold changes compared to the DMSO group.β-ACTIN was used as internal reference protein. Data are expressed as the mean ± SEM of 3–5 independent experiments ( n = 5, A and C; n = 3, B). # P < 0.05, ## P < 0.01 versus DMSO-treated control group, * P < 0.05, ** P < 0.01 versus LPS-treated group. (D – E) BMDM were pretreated with indicated concentrations of GPA for 24 h and stimulated with LPS (1 μg/ml) for 24 h. The levels of mRNA (D) and pro-inflammatory cytokines in supernatants (E) were determined by real-time PCR and ELISA, respectively. mRNA results are expressed as fold changes compared to the DMSO group. Data are expressed as the mean ± SEM of 5 independent experiments ( n = 5). # P < 0.05, ## P < 0.01 versus DMSO-treated control group, * P < 0.05, ** P < 0.01 versus LPS-treated group. (F and G) Nlrp3 −/− BMDM were pretreated with indicated concentrations of GPA for 24 h and stimulated with LPS (1 μg/ml) for 24 h. The levels of mRNA (F) and pro-inflammatory cytokines in supernatants (G) were determined by real-time PCR and ELISA, respectively. mRNA results are expressed as fold changes compared to the DMSO group. Data are expressed as the mean ± SEM of 5 independent experiments ( n = 5). # P < 0.05, ## P < 0.01 versus DMSO-treated control group, * P < 0.05, ** P < 0.01 versus LPS-treated group. (H) Dual-luciferase reporter assay results. RAW274.6 cells were transiently co‐transfected with NF-κB-luc plasmids for 12 h and treated with DMSO (vehicle control), GPA-H (100 μM), GPA-M (50 μM), GPA-L (25 μM), or MCC950 (10 μM) for 12 h, followed by 24 h induction with LPS (1 μg/ml). Data are expressed as the mean ± SEM of 5 independent experiments ( n = 5). (I and J) The protein expression levels of NF-κB (I), NLRP3 and ASC (J) in THP-1 monocytes were measured by immunofluorescence assay. The nuclei were stained with DAPI in blue, and targeted protein was stained in green or red (magnification: 630 × ). (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)

Article Snippet: TaqMan probes, including those for human-specific NLRP3 (Hs00918082), NF κ B1 (Hs00765730), IL-1 β (Hs00174097), IL-6 (Hs00174131 ) , ICAM-1 (Hs00164932), COX-2 (Hs00153133), TNF- α (Hs00174128), and GAPDH (Hs02786624); and mouse-specific Nlrp3 (Mm00840904), Nf κ b1 (Mm00476361), Il-1 β (Mm00434228), Il-6 (Mm00446190 ) , Icam-1 (Mm00516023), Cox-2 (Mm00478374), Tnf- α (Mm00443258), Cyp7a1 (Mm00484150), Cyp8b1 (Mm00501637), Cyp27a1 (Mm00470430), Cyp2b10 (Mm01972453), Cyp3a11 (Mm00731567), Ugt1a1 (Mm02603337), Abcb11 (Mm00445168), Abcc2 (Mm00496899), Abcc3 (Mm00551550), Abcc4 (Mm01226381), Slc01b2 (Mm00451510), Slc10a1 (Mm00441421), and Gapdh (Mm99999915), were acquired from Invitrogen (Carlsbad, CA, USA).

Techniques: Expressing, Real-time Polymerase Chain Reaction, Western Blot, Enzyme-linked Immunosorbent Assay, Control, Luciferase, Reporter Assay, Transfection, Immunofluorescence, Staining

Journal: Redox Biology

Article Title: Inhibition of NLRP3-mediated crosstalk between hepatocytes and liver macrophages by geniposidic acid alleviates cholestatic liver inflammatory injury

doi: 10.1016/j.redox.2022.102404

Figure Lengend Snippet: GPA suppresses NLRP3 inflammasome activation. (A – C) THP-1 monocytes were pretreated with indicated concentrations of GPA for 24 h, stimulated with LPS (1 μg/ml) for 24 h and ATP (2.5 mM) for 30 min. (A) The levels of pro-inflammatory cytokines in cell-culture supernatants, including IL-1β, IL-6, and TNF-α were quantified by ELISA. Data are expressed as the mean ± SEM of 5 independent experiments ( n = 5). The levels of protein (B) and mRNA (C) were determined by Western blot analysis and real-time PCR, respectively. mRNA results are expressed as fold changes compared to the DMSO group. β-ACTIN was used as internal reference protein. Data are expressed as the mean ± SEM of 3–5 independent experiments ( n = 3–4, B; n = 5, C). # P < 0.05, ## P < 0.01 versus DMSO-treated control group, * P < 0.05, ** P < 0.01 versus LPS + ATP-treated group. (D) The NLRP3-ASC interaction in THP-1 monocytes were measured by immuno-fluorescence assay. The nuclei were stained with DAPI in blue, and targeted protein was stained in green or red (magnification: 630 × ×). (E) ROS levels were detected using an ROS fluorescent probe in THP-1 monocytes (magnification: 400 × ). (F and G) Intracellular potassium (F) and ionic calcium (G) levels in the cell lysates of THP-1 monocytes. Data are expressed as the mean ± SEM of 5 independent experiments ( n = 5). # P < 0.05, ## P < 0.01 versus DMSO-treated control group, * P < 0.05, ** P < 0.01 versus LPS + ATP-treated group. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)

Article Snippet: TaqMan probes, including those for human-specific NLRP3 (Hs00918082), NF κ B1 (Hs00765730), IL-1 β (Hs00174097), IL-6 (Hs00174131 ) , ICAM-1 (Hs00164932), COX-2 (Hs00153133), TNF- α (Hs00174128), and GAPDH (Hs02786624); and mouse-specific Nlrp3 (Mm00840904), Nf κ b1 (Mm00476361), Il-1 β (Mm00434228), Il-6 (Mm00446190 ) , Icam-1 (Mm00516023), Cox-2 (Mm00478374), Tnf- α (Mm00443258), Cyp7a1 (Mm00484150), Cyp8b1 (Mm00501637), Cyp27a1 (Mm00470430), Cyp2b10 (Mm01972453), Cyp3a11 (Mm00731567), Ugt1a1 (Mm02603337), Abcb11 (Mm00445168), Abcc2 (Mm00496899), Abcc3 (Mm00551550), Abcc4 (Mm01226381), Slc01b2 (Mm00451510), Slc10a1 (Mm00441421), and Gapdh (Mm99999915), were acquired from Invitrogen (Carlsbad, CA, USA).

Techniques: Activation Assay, Cell Culture, Enzyme-linked Immunosorbent Assay, Western Blot, Real-time Polymerase Chain Reaction, Control, Fluorescence, Staining

Journal: Redox Biology

Article Title: Inhibition of NLRP3-mediated crosstalk between hepatocytes and liver macrophages by geniposidic acid alleviates cholestatic liver inflammatory injury

doi: 10.1016/j.redox.2022.102404

Figure Lengend Snippet: GPA protects against ANIT-induced acute liver inflammation by inhibiting NLRP3 inflammasome activation. C57BL/6 mice were orally administered with the vehicle (0.5% carboxymethylcellulose) as control group, UDCA (75 mg/kg) as positive control, GPA-H (100 mg/kg), GPA-M (50 mg/kg), or GPA-L (25 mg/kg) daily for seven days. On day 5, they were orally administered with ANIT (100 mg/kg). (A) Representative FACS image of mouse liver sample. After sacrificing the animals, liver macrophage were isolated, and the inflammatory infiltration was evaluated by FACS (n = 5). Data are expressed as the mean ± SEM of 5 independent mice liver samples ( n = 5). # P < 0.05, ## P < 0.01 versus vehicle-treated control group, * P < 0.05, ** P < 0.01 versus ANIT-treated group. (B) Hepatic F4/80 and CD68 protein expression measured by immunofluorescence assay. The nuclei were stained with DAPI in blue, and targeted protein was stained in green (F4/80) or red (CD68) (magnification: 630 × ). (C) Levels of pro-inflammatory cytokines in mouse liver, including Il-1β, Il-6, and Tnf-α were detected by ELISA. Data are expressed as the mean ± SEM of 5–6 independent mouse samples ( n = 5–6). # P < 0.05, ## P < 0.01 versus vehicle-treated control group, * P < 0.05, ** P < 0.01 versus ANIT-treated group. (D) Changes in spleen weight index (ratio of spleen weight to body weight) in mice with ANIT-induced cholestasis. Data are expressed as the mean ± SEM of 5 independent mouse samples ( n = 5). (E) mRNAs levels of NLRP3 and other proinflammatory genes in mouse liver were analyzed by real-time PCR. Data are expressed as the mean ± SEM of 5 independent mouse samples ( n = 5). Results are expressed as fold changes compared to the vehicle-treated control group. # P < 0.05, ## P < 0.01 versus vehicle-treated control group, * P < 0.05, ** P < 0.01 versus ANIT-treated group. (F) Levels of NLRP3 and NF-κB signaling proteins in liver lysates were analyzed by western blotting. β-ACTIN was used as internal reference protein. Data are expressed as the mean ± SEM of 3–4 independent mouse samples ( n = 3–4). # P < 0.05, ## P < 0.01 versus vehicle-treated control group, * P < 0.05, ** P < 0.01 versus ANIT-treated group. (G) ANIT-induced hepatic ROS levels were decreased after GPA treatment ( n = 3). # P < 0.05, ## P < 0.01 versus vehicle-treated control group, * P < 0.05, ** P < 0.01 versus ANIT-treated group. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)

Article Snippet: TaqMan probes, including those for human-specific NLRP3 (Hs00918082), NF κ B1 (Hs00765730), IL-1 β (Hs00174097), IL-6 (Hs00174131 ) , ICAM-1 (Hs00164932), COX-2 (Hs00153133), TNF- α (Hs00174128), and GAPDH (Hs02786624); and mouse-specific Nlrp3 (Mm00840904), Nf κ b1 (Mm00476361), Il-1 β (Mm00434228), Il-6 (Mm00446190 ) , Icam-1 (Mm00516023), Cox-2 (Mm00478374), Tnf- α (Mm00443258), Cyp7a1 (Mm00484150), Cyp8b1 (Mm00501637), Cyp27a1 (Mm00470430), Cyp2b10 (Mm01972453), Cyp3a11 (Mm00731567), Ugt1a1 (Mm02603337), Abcb11 (Mm00445168), Abcc2 (Mm00496899), Abcc3 (Mm00551550), Abcc4 (Mm01226381), Slc01b2 (Mm00451510), Slc10a1 (Mm00441421), and Gapdh (Mm99999915), were acquired from Invitrogen (Carlsbad, CA, USA).

Techniques: Activation Assay, Control, Positive Control, Isolation, Expressing, Immunofluorescence, Staining, Enzyme-linked Immunosorbent Assay, Real-time Polymerase Chain Reaction, Western Blot

Journal: Redox Biology

Article Title: Inhibition of NLRP3-mediated crosstalk between hepatocytes and liver macrophages by geniposidic acid alleviates cholestatic liver inflammatory injury

doi: 10.1016/j.redox.2022.102404

Figure Lengend Snippet: GPA reduces BA-induced cellular inflammation by blocking the activation of NLRP3 inflammasome in hepatocytes and macrophages. (A) Wild-type primary (WT) mouse hepatocytes (PMHs) were pretreated with GPA for 12 h and treated with 200 μM TCA for 24 h. Levels of NLRP3 and proinflammatory mRNAs were determined by real-time PCR. Results are expression as fold changes compared to the DMSO group. Data are expressed as the mean ± SEM of 5 independent experiments ( n = 5). (B) Levels of Il-1β and Il-6 in cell-culture supernatants were quantified by ELISA. Data are expressed as the mean ± SEM of 5 independent experiments ( n = 5). # P < 0.05, ## P < 0.01 versus DMSO-treated control group, * P < 0.05, ** P < 0.01 versus TCA-treated group. Primary mouse hepatocytes with Nlrp3 -knockout ( Nlrp3 −/− PMHs) were pretreated with GPA for 12 h and treated with 200 μM TCA for 24 h. Levels of Nfκb1, Il-1β , and Tnf-α mRNA (C) and Il-1β and Tnf-α in cell-culture supernatants (D) were determined by real-time PCR and ELISA, respectively. mRNA results are expression as fold changes compared to the DMSO group. Data are expressed as the mean ± SEM of 5 independent experiments ( n = 5). # P < 0.05, ## P < 0.01 versus DMSO-treated control group, * P < 0.05, ** P < 0.01 versus TCA-treated group. PMHs and BMDM were isolated from WT and Nlrp3 −/− mice and cultivated on a conditioned medium (CM)-associated culture. (E – H) WT BMDM were exposed for 24 h to the CM derived from WT-PMHs(E and F) and Nlrp3 −/− PMHs(G and H) that were pretreated with or without GPA for 24 h and TCA for another 24 h. Levels of NLRP3 and proinflammatory mRNAs (E and G) and IL-1β (F and H) in cell-culture supernatants were determined by real-time PCR and ELISA, respectively. mRNA results are expressed as fold changes compared to the DMSO group. Data are expressed as the mean ± SEM of 5 independent experiments ( n = 5). # P < 0.05, ## P < 0.01 versus DMSO-treated control group, * P < 0.05, ** P < 0.01 versus TCA-treated group. (I – J) BMDM were pretreated with GPA for 12 h and treated with 200 μM TCA for 24 h. Levels of NLRP3 and proinflammatory mRNAs and pro-inflammatory cytokines in cell-culture supernatants were quantified by real-time PCR (I) and ELISA analysis (J), respectively. mRNA results are expressed as fold changes compared to the DMSO group. Data are expressed as the mean ± SEM of 5 independent experiments ( n = 5). # P < 0.05, ## P < 0.01 versus DMSO-treated control group, * P < 0.05, ** P < 0.01 versus TCA-treated group. (K–N) WT PMHs were exposed for 24 h to the CM derived from WT-BMDM (K and L) and Nlrp3 −/− (M and N) BMDM that were pretreated with or without GPA for 24 h, followed by TCA for 24 h. Levels of NLRP3 and proinflammatory mRNAs (K and M) and IL-1β (L and N) in cell-culture supernatants were determined by real-time PCR and ELISA analysis, respectively. mRNA results are expressed as fold changes compared to the DMSO group. Data are expressed as the mean ± SEM of 5 independent experiments ( n = 5). # P < 0.05, ## P < 0.01 versus DMSO-treated control group, * P < 0.05, ** P < 0.01 versus TCA-treated group.

Article Snippet: TaqMan probes, including those for human-specific NLRP3 (Hs00918082), NF κ B1 (Hs00765730), IL-1 β (Hs00174097), IL-6 (Hs00174131 ) , ICAM-1 (Hs00164932), COX-2 (Hs00153133), TNF- α (Hs00174128), and GAPDH (Hs02786624); and mouse-specific Nlrp3 (Mm00840904), Nf κ b1 (Mm00476361), Il-1 β (Mm00434228), Il-6 (Mm00446190 ) , Icam-1 (Mm00516023), Cox-2 (Mm00478374), Tnf- α (Mm00443258), Cyp7a1 (Mm00484150), Cyp8b1 (Mm00501637), Cyp27a1 (Mm00470430), Cyp2b10 (Mm01972453), Cyp3a11 (Mm00731567), Ugt1a1 (Mm02603337), Abcb11 (Mm00445168), Abcc2 (Mm00496899), Abcc3 (Mm00551550), Abcc4 (Mm01226381), Slc01b2 (Mm00451510), Slc10a1 (Mm00441421), and Gapdh (Mm99999915), were acquired from Invitrogen (Carlsbad, CA, USA).

Techniques: Blocking Assay, Activation Assay, Real-time Polymerase Chain Reaction, Expressing, Cell Culture, Enzyme-linked Immunosorbent Assay, Control, Knock-Out, Isolation, Derivative Assay

Journal: Redox Biology

Article Title: Inhibition of NLRP3-mediated crosstalk between hepatocytes and liver macrophages by geniposidic acid alleviates cholestatic liver inflammatory injury

doi: 10.1016/j.redox.2022.102404

Figure Lengend Snippet: Anti-inflammatory effect of GPA against ANIT‐induced acute liver injury is largely abolished in Nlrp3 -knockout mice. WT mice and Nlrp3 −/− mice were orally administered with the vehicle (0.5% carboxymethylcellulose), UDCA (75 mg/kg) as positive control, or GPA-H (100 mg/kg) once daily for seven days and with ANIT (100 mg/kg) on day 5. (A) Representative FACS image of mouse liver sample. After sacrificing the animals, liver macrophage were isolated, and the inflammatory infiltration was evaluated by FACS (n = 5). Data are expressed as the mean ± SEM of 5 independent mice liver samples ( n = 5). # P < 0.05, ## P < 0.01 versus vehicle-treated control group in WT and Nlrp3 −/− mice; * P < 0.05, ** P < 0.01 compared to ANIT-treated alone group. (B) Hepatic F4/80 and CD68 protein expression measured by immunofluorescence assay. The nuclei were stained with DAPI in blue, and targeted protein was stained in red (F4/80) or green (CD68) (magnification: 630 × ).Data are expressed as the mean ± SEM of 3 independent mice liver samples ( n = 3). # P < 0.05, ## P < 0.01 versus vehicle-treated control group in WT and Nlrp3 −/− mice; * P < 0.05, ** P < 0.01 compared to ANIT-treated alone group. (C) Levels of NLRP3 and proinflammatory mRNAs in mouse liver were determined by real-time PCR. Data are expressed as the mean ± SEM of 5 independent mouse samples ( n = 5). Results are expressed as fold changes compared to the vehicle-treated control group. # P < 0.05, ## P < 0.01 versus vehicle-treated control group in WT and Nlrp3 −/− mice; * P < 0.05, ** P < 0.01 compared to ANIT-treated alone group. (D) Levels of inflammatory cytokines including Il-1β, Il-6, and Tnf-α in mouse liver were detected by ELISA. Data are expressed as the mean ± SEM of 5 independent mouse samples ( n = 5). # P < 0.05, ## P < 0.01 versus vehicle-treated control group, * P < 0.05, ** P < 0.01 versus ANIT-treated group. (E) Effects of GPA treatment on spleen weight index (ratio of spleen weight to body weight) of ANIT-induced mice. Data are expressed as the mean ± SEM of 5 independent mouse samples ( n = 5). Results are expressed as fold changes compared to the vehicle-treated control group. # P < 0.05, ## P < 0.01 versus vehicle-treated control group in WT and Nlrp3 −/− mice; * P < 0.05, ** P < 0.01 compared to ANIT-treated alone group. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)

Article Snippet: TaqMan probes, including those for human-specific NLRP3 (Hs00918082), NF κ B1 (Hs00765730), IL-1 β (Hs00174097), IL-6 (Hs00174131 ) , ICAM-1 (Hs00164932), COX-2 (Hs00153133), TNF- α (Hs00174128), and GAPDH (Hs02786624); and mouse-specific Nlrp3 (Mm00840904), Nf κ b1 (Mm00476361), Il-1 β (Mm00434228), Il-6 (Mm00446190 ) , Icam-1 (Mm00516023), Cox-2 (Mm00478374), Tnf- α (Mm00443258), Cyp7a1 (Mm00484150), Cyp8b1 (Mm00501637), Cyp27a1 (Mm00470430), Cyp2b10 (Mm01972453), Cyp3a11 (Mm00731567), Ugt1a1 (Mm02603337), Abcb11 (Mm00445168), Abcc2 (Mm00496899), Abcc3 (Mm00551550), Abcc4 (Mm01226381), Slc01b2 (Mm00451510), Slc10a1 (Mm00441421), and Gapdh (Mm99999915), were acquired from Invitrogen (Carlsbad, CA, USA).

Techniques: Knock-Out, Positive Control, Isolation, Control, Expressing, Immunofluorescence, Staining, Real-time Polymerase Chain Reaction, Enzyme-linked Immunosorbent Assay

Journal: Redox Biology

Article Title: Inhibition of NLRP3-mediated crosstalk between hepatocytes and liver macrophages by geniposidic acid alleviates cholestatic liver inflammatory injury

doi: 10.1016/j.redox.2022.102404

Figure Lengend Snippet: Hepatoprotective effect of GPA against ANIT‐induced acute liver injury is partly mitigated in Nlrp3 -knockout mice. (A) Serum biochemical indexes of cholestatic liver injury including ALT, AST, TBA and TBIL levels were evaluated. Data are expressed as the mean ± SEM from 5 to 6 independent mice samples (n = 5–6). # P < 0.05, ## P < 0.01 versus vehicle-treated control group in WT and Nlrp3 −/− mice; * P < 0.05, ** P < 0.01 compared to ANIT-treated alone group. (B) Representative images of haematoxylin and eosin staining in liver sections (magnification:200 × ). The circle indicated bleeding, inflammatory infiltration and hepatic necrosis.Data are expressed as the mean ± SEM of 5 independent mouse samples ( n = 5). Results are expressed as fold changes compared to the vehicle-treated control group. # P < 0.05, ## P < 0.01 versus vehicle-treated control group in WT and Nlrp3 −/− mice; * P < 0.05, ** P < 0.01 compared to ANIT-treated alone group. (C) Relative mRNA levels of BA synthesis- and transport-related genes in mouse liver were tested by real-time PCR. Data are expressed as the mean ± SEM of 5 independent mouse samples ( n = 5). Results are expressed as fold changes compared to the vehicle-treated control group. # P < 0.05, ## P < 0.01 versus vehicle-treated control group in WT and Nlrp3 −/− mice; * P < 0.05, ** P < 0.01 compared to ANIT-treated alone group.

Article Snippet: TaqMan probes, including those for human-specific NLRP3 (Hs00918082), NF κ B1 (Hs00765730), IL-1 β (Hs00174097), IL-6 (Hs00174131 ) , ICAM-1 (Hs00164932), COX-2 (Hs00153133), TNF- α (Hs00174128), and GAPDH (Hs02786624); and mouse-specific Nlrp3 (Mm00840904), Nf κ b1 (Mm00476361), Il-1 β (Mm00434228), Il-6 (Mm00446190 ) , Icam-1 (Mm00516023), Cox-2 (Mm00478374), Tnf- α (Mm00443258), Cyp7a1 (Mm00484150), Cyp8b1 (Mm00501637), Cyp27a1 (Mm00470430), Cyp2b10 (Mm01972453), Cyp3a11 (Mm00731567), Ugt1a1 (Mm02603337), Abcb11 (Mm00445168), Abcc2 (Mm00496899), Abcc3 (Mm00551550), Abcc4 (Mm01226381), Slc01b2 (Mm00451510), Slc10a1 (Mm00441421), and Gapdh (Mm99999915), were acquired from Invitrogen (Carlsbad, CA, USA).

Techniques: Knock-Out, Control, Staining, Real-time Polymerase Chain Reaction

Journal: Redox Biology

Article Title: Inhibition of NLRP3-mediated crosstalk between hepatocytes and liver macrophages by geniposidic acid alleviates cholestatic liver inflammatory injury

doi: 10.1016/j.redox.2022.102404

Figure Lengend Snippet: Liver-specific overexpression of NLRP3 antagonizes the hepatoprotective effect of GPA against ANIT-induced acute inflammatory liver injury. WT mice were intravenously injected with Adv-Con or Adv-Nlrp3 adenovirus (1 × 10 9 pf) via the tail vein for three days and then orally administered with MCC950 (20 mg/kg) or GPA (100 mg/kg). On day 8, they were orally administered with ANIT(100 mg/kg) to induce acute liver injury. (A) Protein levels of NLRP3 in the liver tissues. Actin was used as internal reference protein. Data are expressed as the mean ± SEM of 3 independent mouse samples ( n = 3). (B) Serum Il-1β and Tnf-α levels were detected by ELISA. Data are expressed as the mean ± SEM of 5 independent mouse samples ( n = 5). # P < 0.05, ## P < 0.01 versus vehicle-treated control group in WT and Nlrp3 oe mice; * P < 0.05, ** P < 0.01 compared to ANIT-treated alone group. (C) Serum biochemical indexes of cholestatic liver injury including ALT, AST, TBA and TBIL levels were evaluated. Data are expressed as the mean ± SE from 5 to 6 independent mice samples (n = 5–6). # P < 0.05, ## P < 0.01 versus vehicle-treated control group in WT and Nlrp3 oe mice; * P < 0.05, ** P < 0.01 compared to ANIT-treated alone group. (D) Representative images of haematoxylin and eosin staining in liver sections (magnification:200 × ). The circle indicated bleeding, inflammatory infiltration and hepatic necrosis. Data are expressed as the mean ± SE from 5 independent mice samples (n = 5). # P < 0.05, ## P < 0.01 versus vehicle-treated control group in WT and Nlrp3 oe mice; * P < 0.05, ** P < 0.01 compared to ANIT-treated alone group.

Article Snippet: TaqMan probes, including those for human-specific NLRP3 (Hs00918082), NF κ B1 (Hs00765730), IL-1 β (Hs00174097), IL-6 (Hs00174131 ) , ICAM-1 (Hs00164932), COX-2 (Hs00153133), TNF- α (Hs00174128), and GAPDH (Hs02786624); and mouse-specific Nlrp3 (Mm00840904), Nf κ b1 (Mm00476361), Il-1 β (Mm00434228), Il-6 (Mm00446190 ) , Icam-1 (Mm00516023), Cox-2 (Mm00478374), Tnf- α (Mm00443258), Cyp7a1 (Mm00484150), Cyp8b1 (Mm00501637), Cyp27a1 (Mm00470430), Cyp2b10 (Mm01972453), Cyp3a11 (Mm00731567), Ugt1a1 (Mm02603337), Abcb11 (Mm00445168), Abcc2 (Mm00496899), Abcc3 (Mm00551550), Abcc4 (Mm01226381), Slc01b2 (Mm00451510), Slc10a1 (Mm00441421), and Gapdh (Mm99999915), were acquired from Invitrogen (Carlsbad, CA, USA).

Techniques: Over Expression, Injection, Enzyme-linked Immunosorbent Assay, Control, Staining

Journal: Cell reports

Article Title: Extracellular vesicles are carriers of adiponectin with insulin-sensitizing and anti-inflammatory properties.

doi: 10.1016/j.celrep.2023.112866

Figure Lengend Snippet: Figure 1. Adiponectin is the most enriched adipokine in VAT-derived EVs (A and B) Lean (Ctrl) and obese (Ob) VAT-derived lEV and sEV mode size comparison. Size-distribution curves (A) and mean mode size (B). Ctrl and Ob EV subtype size-distribution curves (A) are represented by plain or dashed lines, respectively, and are presented as the mean ± SEM (n = 4–6 independent EV preparations for each condition). (C and D) EV marker analysis from lean (Ctrl) or ob/ob (Ob) VAT explant-derived EV subpopulations. A significant enrichment in CD63 was observed for obese VAT-derived sEVs (D). (E) lEV and sEV secretion from VAT, SAT, and BAT explants. EV number secreted per gram of AT per 24 h is presented. (F) Increased lEV and sEV secretion from mouse VAT with obesity. EV secreted by total mouse VAT is presented. (G) EV secretion of human omental, mesenteric (Mesent.), and subcutaneous (Subcut.) AT collected from obese subjects. Secretion of EVs is presented as the number of EVs secreted per gram of human AT.

Article Snippet: REAGENT or RESOURCE SOURCE IDENTIFIER Antibodies Adiponectin (murine) Thermofisher Scientific #PA1-054, RRID:AB_325789 Adiponectin (human) Abeomics #10-7597, RRID:AB_2943077 AKT pan (40D4) Cell signaling Cat #2920, RRID:AB_1147620 AKT pan polyclonal (in vivo studies) Cell signaling Cat #4691, RRID:AB_915783 Phospho-AKT (pAKT, Ser 473) Cell signaling Cat #4060S, RRID:AB_2315049 Alix BD Biosciences Cat #611620, RRID:AB_399062 Murine CD9 BD Pharmingen Cat #553758, RRID:AB_395032 human CD9 Santa Cruz Cat #SC-13118, RRID:AB_627213 Murine CD63 MBL Cat #D263-3, RRID:AB_1278815 Flotillin-2 BD Pharmingen Cat #610383, RRID:AB_397766 Mac2 Cedarlane Cat #CL8942AP, RRID:AB_10060357 MIF (human) R&D systems Cat #mab289, RRID:AB_2281975 Syntenin-1 Abcam Cat #ab19903, RRID:AB_445200 Zs-Green Clontech Cat #632598, RRID:AB_2943078 Goat anti-rat IgG2a Secondary antibody HRP conjugate (used for MaC2 staining) Invitrogen Cat# PA1-84709, RRID:AB_933942 Anti-adiponectin FITC coupled antibody Assay-Pro Cat #10361-05041, RRID:AB_2943079 IRDye 800CW and 680RD secondary antibody (anti-mouse and anti-rabbit) LI-COR Biosciences Cat #926–32211, RRID:AB_621843; Cat #926–32210,RRID:AB_621842; Cat #926–68071, RRID:AB_10956166; Cat #926-68070, RRID:AB_10956588 Chemicals, peptides, and recombinant proteins Bovine serum albumin (BSA) FFA free Sigma Cat #A7030 Collagenase A Roche Cat #10103586001 DAB OB Super Sensitive detection kit ImPath Cat #46538 DAB Quanto Thermofischer Scientific Cat #TA-125-QHDX Wash buffer for IHC ImPath Cat #45002 Antigen retrieval solution pH6.0 ImPath Cat #44998 Ultravision Hydrogen Peroxyde block Thermofisher Scientific Cat #TS-12-H2O2Q Goat serum Immunoreagents Cat #SP-004-VX10 IHC mounting medium CellPath Cat #SEA-1604-00A Qiazol Lysis reagent Qiagen Cat #79306 RNeasy mini kit Qiagen Cat #74104 SuperScriptII reverse Transcriptase Invitrogen Cat #18064-014 dNTP set 100mM Invitrogen Cat #10297-018 Random hexamers Invitrogen Cat #48190-011 QIAquick PCR purification kit Qiagen Cat #28106 RNaseOut Ribonuclease inhibitor Invitrogen Cat #10777-019 Maxima SYBR Green qPCR master mix 2X Thermofisher Scientific Cat #K0253 96-Well PCR plates Thermofisher Scientific Cat #AB0700 DMEM 4.5 g/L glucose Gibco Cat #41966-029 DMEM 1 g/L glucose Gibco Cat #31885-023 Elisa mouse Adiponectin/Acrp30 R&DSystems Cat #DY1119 and #DY008 (Continued on next page) 16 Cell Reports 42, 112866, August 29, 2023

Techniques: Derivative Assay, Comparison, Marker

Journal: Cell reports

Article Title: Extracellular vesicles are carriers of adiponectin with insulin-sensitizing and anti-inflammatory properties.

doi: 10.1016/j.celrep.2023.112866

Figure Lengend Snippet: Figure 3. Plasma EVs represent stable carriers of adiponectin (A) VAT-derived EVs are retrieved in the blood circulation, as illustrated by flow-cytometry detection of ZsGreen+ EVs in platelet-free plasma (PFP). Ctrl, control; AdipoZS1, Cre AdipoZs1 mice; AdipoZS1, Cre+ AdipoZs1 mice. (B) Adiponectinemia significantly decreases upon plasma EV removal in lean mice. (C) The presence of adiponectin was confirmed in mouse plasma circulating EVs. Plasma EV subtypes were isolated from lean and obese mice. Ten micrograms of each EV subtype was resolved by SDS-PAGE under reducing conditions (R) or nonreducing unheated conditions (NR). One representative blot (out of two experiments performed) is presented. (D) Adiponectin clearance measurement following the injection of serum or EV-depleted serum in adiponectin KO mice. n = 6 mice injected per group. (E) Western blot of human plasma sEVs confirms the decreased amount of adiponectin in sEVs isolated from obese patients compared to control patients. A representative blot (out of three independent experiments performed) is presented with quantification of the Adpn blot signal intensity.

Article Snippet: REAGENT or RESOURCE SOURCE IDENTIFIER Antibodies Adiponectin (murine) Thermofisher Scientific #PA1-054, RRID:AB_325789 Adiponectin (human) Abeomics #10-7597, RRID:AB_2943077 AKT pan (40D4) Cell signaling Cat #2920, RRID:AB_1147620 AKT pan polyclonal (in vivo studies) Cell signaling Cat #4691, RRID:AB_915783 Phospho-AKT (pAKT, Ser 473) Cell signaling Cat #4060S, RRID:AB_2315049 Alix BD Biosciences Cat #611620, RRID:AB_399062 Murine CD9 BD Pharmingen Cat #553758, RRID:AB_395032 human CD9 Santa Cruz Cat #SC-13118, RRID:AB_627213 Murine CD63 MBL Cat #D263-3, RRID:AB_1278815 Flotillin-2 BD Pharmingen Cat #610383, RRID:AB_397766 Mac2 Cedarlane Cat #CL8942AP, RRID:AB_10060357 MIF (human) R&D systems Cat #mab289, RRID:AB_2281975 Syntenin-1 Abcam Cat #ab19903, RRID:AB_445200 Zs-Green Clontech Cat #632598, RRID:AB_2943078 Goat anti-rat IgG2a Secondary antibody HRP conjugate (used for MaC2 staining) Invitrogen Cat# PA1-84709, RRID:AB_933942 Anti-adiponectin FITC coupled antibody Assay-Pro Cat #10361-05041, RRID:AB_2943079 IRDye 800CW and 680RD secondary antibody (anti-mouse and anti-rabbit) LI-COR Biosciences Cat #926–32211, RRID:AB_621843; Cat #926–32210,RRID:AB_621842; Cat #926–68071, RRID:AB_10956166; Cat #926-68070, RRID:AB_10956588 Chemicals, peptides, and recombinant proteins Bovine serum albumin (BSA) FFA free Sigma Cat #A7030 Collagenase A Roche Cat #10103586001 DAB OB Super Sensitive detection kit ImPath Cat #46538 DAB Quanto Thermofischer Scientific Cat #TA-125-QHDX Wash buffer for IHC ImPath Cat #45002 Antigen retrieval solution pH6.0 ImPath Cat #44998 Ultravision Hydrogen Peroxyde block Thermofisher Scientific Cat #TS-12-H2O2Q Goat serum Immunoreagents Cat #SP-004-VX10 IHC mounting medium CellPath Cat #SEA-1604-00A Qiazol Lysis reagent Qiagen Cat #79306 RNeasy mini kit Qiagen Cat #74104 SuperScriptII reverse Transcriptase Invitrogen Cat #18064-014 dNTP set 100mM Invitrogen Cat #10297-018 Random hexamers Invitrogen Cat #48190-011 QIAquick PCR purification kit Qiagen Cat #28106 RNaseOut Ribonuclease inhibitor Invitrogen Cat #10777-019 Maxima SYBR Green qPCR master mix 2X Thermofisher Scientific Cat #K0253 96-Well PCR plates Thermofisher Scientific Cat #AB0700 DMEM 4.5 g/L glucose Gibco Cat #41966-029 DMEM 1 g/L glucose Gibco Cat #31885-023 Elisa mouse Adiponectin/Acrp30 R&DSystems Cat #DY1119 and #DY008 (Continued on next page) 16 Cell Reports 42, 112866, August 29, 2023

Techniques: Clinical Proteomics, Derivative Assay, Cytometry, Control, Isolation, SDS Page, Injection, Western Blot

Journal: Cell reports

Article Title: Extracellular vesicles are carriers of adiponectin with insulin-sensitizing and anti-inflammatory properties.

doi: 10.1016/j.celrep.2023.112866

Figure Lengend Snippet: Figure 4. EV-associated adiponectin maintains insulin sensitivity in target cells (A and B) Adiponectin-enriched sEVs reverse insulin resistance in hepatocytes. Ctrl sEVs and Ob sEVs correspond to VAT-derived sEVs isolated from either lean or obese VAT, respectively. Adpn-Ob sEVs refer to the enrichment of Ob sEVs with adiponectin (following their preincubation with EV-free conditioned media). Ins, insulin; Palm, palmitate. (C) Silencing of both AdipoR1 and AdipoR2 significantly reduces the VAT-derived sEV insulin-sensitizing effects. Scr, Scramble; R1, AdipoR1; R2, AdipoR2. (D) Rapid and time-dependent internalization of fluorescent adiponectin-Venus (Adpn-Venus) internalization in hepatocytes. Scale bars, 50 mm. (E) Adiponectin enrichment in sEVs is responsible for their insulin-sensitizing effects, independent of their sEV cellular origin. Dot plots represent independent experiments. Data are presented as the mean ± SEM. Statistical differences were assessed using one-way ANOVA. Statistical significancy is indicated for each panel as follows : *p % 0.05, **p % 0.01, ***p % 0.01,****p % 0.0001.

Article Snippet: REAGENT or RESOURCE SOURCE IDENTIFIER Antibodies Adiponectin (murine) Thermofisher Scientific #PA1-054, RRID:AB_325789 Adiponectin (human) Abeomics #10-7597, RRID:AB_2943077 AKT pan (40D4) Cell signaling Cat #2920, RRID:AB_1147620 AKT pan polyclonal (in vivo studies) Cell signaling Cat #4691, RRID:AB_915783 Phospho-AKT (pAKT, Ser 473) Cell signaling Cat #4060S, RRID:AB_2315049 Alix BD Biosciences Cat #611620, RRID:AB_399062 Murine CD9 BD Pharmingen Cat #553758, RRID:AB_395032 human CD9 Santa Cruz Cat #SC-13118, RRID:AB_627213 Murine CD63 MBL Cat #D263-3, RRID:AB_1278815 Flotillin-2 BD Pharmingen Cat #610383, RRID:AB_397766 Mac2 Cedarlane Cat #CL8942AP, RRID:AB_10060357 MIF (human) R&D systems Cat #mab289, RRID:AB_2281975 Syntenin-1 Abcam Cat #ab19903, RRID:AB_445200 Zs-Green Clontech Cat #632598, RRID:AB_2943078 Goat anti-rat IgG2a Secondary antibody HRP conjugate (used for MaC2 staining) Invitrogen Cat# PA1-84709, RRID:AB_933942 Anti-adiponectin FITC coupled antibody Assay-Pro Cat #10361-05041, RRID:AB_2943079 IRDye 800CW and 680RD secondary antibody (anti-mouse and anti-rabbit) LI-COR Biosciences Cat #926–32211, RRID:AB_621843; Cat #926–32210,RRID:AB_621842; Cat #926–68071, RRID:AB_10956166; Cat #926-68070, RRID:AB_10956588 Chemicals, peptides, and recombinant proteins Bovine serum albumin (BSA) FFA free Sigma Cat #A7030 Collagenase A Roche Cat #10103586001 DAB OB Super Sensitive detection kit ImPath Cat #46538 DAB Quanto Thermofischer Scientific Cat #TA-125-QHDX Wash buffer for IHC ImPath Cat #45002 Antigen retrieval solution pH6.0 ImPath Cat #44998 Ultravision Hydrogen Peroxyde block Thermofisher Scientific Cat #TS-12-H2O2Q Goat serum Immunoreagents Cat #SP-004-VX10 IHC mounting medium CellPath Cat #SEA-1604-00A Qiazol Lysis reagent Qiagen Cat #79306 RNeasy mini kit Qiagen Cat #74104 SuperScriptII reverse Transcriptase Invitrogen Cat #18064-014 dNTP set 100mM Invitrogen Cat #10297-018 Random hexamers Invitrogen Cat #48190-011 QIAquick PCR purification kit Qiagen Cat #28106 RNaseOut Ribonuclease inhibitor Invitrogen Cat #10777-019 Maxima SYBR Green qPCR master mix 2X Thermofisher Scientific Cat #K0253 96-Well PCR plates Thermofisher Scientific Cat #AB0700 DMEM 4.5 g/L glucose Gibco Cat #41966-029 DMEM 1 g/L glucose Gibco Cat #31885-023 Elisa mouse Adiponectin/Acrp30 R&DSystems Cat #DY1119 and #DY008 (Continued on next page) 16 Cell Reports 42, 112866, August 29, 2023

Techniques: Derivative Assay, Isolation

Journal: Cell reports

Article Title: Extracellular vesicles are carriers of adiponectin with insulin-sensitizing and anti-inflammatory properties.

doi: 10.1016/j.celrep.2023.112866

Figure Lengend Snippet: Figure 5. EV-associated adiponectin reverses HFD-induced insulin resistance in mice (A) NHS ester-labeled VAT-derived sEV organ biodistribution. Licor fluorescent tissue imaging is presented in Figure S4A. (B) Adoptive transfer of sEV-associated adiponectin limits weight gain induced by a high-fat diet (HFD). A standard diet (SD) is presented as a control to demonstrate HFD-induced weight gain compared to body weight on a regular chow diet. Number of animals per group: SD, n = 3; NaCl, n = 15; Ctrl sEVs, n = 13; AdpnKO sEVs, n = 11. (C) Tissue weights at sacrifice of mice that received i.p. injections of Ctrl sEVs, AdpnKO sEVs, or NaCl (vehicle) over the course of 5 weeks of HFD compared to mice maintained on an SD.

Article Snippet: REAGENT or RESOURCE SOURCE IDENTIFIER Antibodies Adiponectin (murine) Thermofisher Scientific #PA1-054, RRID:AB_325789 Adiponectin (human) Abeomics #10-7597, RRID:AB_2943077 AKT pan (40D4) Cell signaling Cat #2920, RRID:AB_1147620 AKT pan polyclonal (in vivo studies) Cell signaling Cat #4691, RRID:AB_915783 Phospho-AKT (pAKT, Ser 473) Cell signaling Cat #4060S, RRID:AB_2315049 Alix BD Biosciences Cat #611620, RRID:AB_399062 Murine CD9 BD Pharmingen Cat #553758, RRID:AB_395032 human CD9 Santa Cruz Cat #SC-13118, RRID:AB_627213 Murine CD63 MBL Cat #D263-3, RRID:AB_1278815 Flotillin-2 BD Pharmingen Cat #610383, RRID:AB_397766 Mac2 Cedarlane Cat #CL8942AP, RRID:AB_10060357 MIF (human) R&D systems Cat #mab289, RRID:AB_2281975 Syntenin-1 Abcam Cat #ab19903, RRID:AB_445200 Zs-Green Clontech Cat #632598, RRID:AB_2943078 Goat anti-rat IgG2a Secondary antibody HRP conjugate (used for MaC2 staining) Invitrogen Cat# PA1-84709, RRID:AB_933942 Anti-adiponectin FITC coupled antibody Assay-Pro Cat #10361-05041, RRID:AB_2943079 IRDye 800CW and 680RD secondary antibody (anti-mouse and anti-rabbit) LI-COR Biosciences Cat #926–32211, RRID:AB_621843; Cat #926–32210,RRID:AB_621842; Cat #926–68071, RRID:AB_10956166; Cat #926-68070, RRID:AB_10956588 Chemicals, peptides, and recombinant proteins Bovine serum albumin (BSA) FFA free Sigma Cat #A7030 Collagenase A Roche Cat #10103586001 DAB OB Super Sensitive detection kit ImPath Cat #46538 DAB Quanto Thermofischer Scientific Cat #TA-125-QHDX Wash buffer for IHC ImPath Cat #45002 Antigen retrieval solution pH6.0 ImPath Cat #44998 Ultravision Hydrogen Peroxyde block Thermofisher Scientific Cat #TS-12-H2O2Q Goat serum Immunoreagents Cat #SP-004-VX10 IHC mounting medium CellPath Cat #SEA-1604-00A Qiazol Lysis reagent Qiagen Cat #79306 RNeasy mini kit Qiagen Cat #74104 SuperScriptII reverse Transcriptase Invitrogen Cat #18064-014 dNTP set 100mM Invitrogen Cat #10297-018 Random hexamers Invitrogen Cat #48190-011 QIAquick PCR purification kit Qiagen Cat #28106 RNaseOut Ribonuclease inhibitor Invitrogen Cat #10777-019 Maxima SYBR Green qPCR master mix 2X Thermofisher Scientific Cat #K0253 96-Well PCR plates Thermofisher Scientific Cat #AB0700 DMEM 4.5 g/L glucose Gibco Cat #41966-029 DMEM 1 g/L glucose Gibco Cat #31885-023 Elisa mouse Adiponectin/Acrp30 R&DSystems Cat #DY1119 and #DY008 (Continued on next page) 16 Cell Reports 42, 112866, August 29, 2023

Techniques: Labeling, Derivative Assay, Imaging, Adoptive Transfer Assay, Control

Journal: Cell reports

Article Title: Extracellular vesicles are carriers of adiponectin with insulin-sensitizing and anti-inflammatory properties.

doi: 10.1016/j.celrep.2023.112866

Figure Lengend Snippet: Figure 6. EV-associated adiponectin dis- plays anti-inflammatory properties (A) Macrophage (Mac2) staining in SAT (upper panels) and VAT (lower panels) depots from sEV-injected mice reveal the anti-inflammatory properties of Ctrl sEVs treatment. HFD-induced adipocyte hypertrophy and HFD-related macro- phage infiltration are observed in both SAT and VAT compared to SD-fed conditions. Scale bars, 50 mm. (B and C) qPCR mRNA expression of inflammatory markers in VAT (B) and liver (C) tissues. Dot plots represent the number of independent animals analyzed. Data are presented as the mean ± SEM. Statistical differences were calculated compared to the HFD + NaCl group using one-way ANOVA followed by the Kruskal-Wallis test. Statistical significancy is indicated for each panel as follows : *p % 0.05, **p % 0.01, ***p % 0.01,****p % 0.0001.

Article Snippet: REAGENT or RESOURCE SOURCE IDENTIFIER Antibodies Adiponectin (murine) Thermofisher Scientific #PA1-054, RRID:AB_325789 Adiponectin (human) Abeomics #10-7597, RRID:AB_2943077 AKT pan (40D4) Cell signaling Cat #2920, RRID:AB_1147620 AKT pan polyclonal (in vivo studies) Cell signaling Cat #4691, RRID:AB_915783 Phospho-AKT (pAKT, Ser 473) Cell signaling Cat #4060S, RRID:AB_2315049 Alix BD Biosciences Cat #611620, RRID:AB_399062 Murine CD9 BD Pharmingen Cat #553758, RRID:AB_395032 human CD9 Santa Cruz Cat #SC-13118, RRID:AB_627213 Murine CD63 MBL Cat #D263-3, RRID:AB_1278815 Flotillin-2 BD Pharmingen Cat #610383, RRID:AB_397766 Mac2 Cedarlane Cat #CL8942AP, RRID:AB_10060357 MIF (human) R&D systems Cat #mab289, RRID:AB_2281975 Syntenin-1 Abcam Cat #ab19903, RRID:AB_445200 Zs-Green Clontech Cat #632598, RRID:AB_2943078 Goat anti-rat IgG2a Secondary antibody HRP conjugate (used for MaC2 staining) Invitrogen Cat# PA1-84709, RRID:AB_933942 Anti-adiponectin FITC coupled antibody Assay-Pro Cat #10361-05041, RRID:AB_2943079 IRDye 800CW and 680RD secondary antibody (anti-mouse and anti-rabbit) LI-COR Biosciences Cat #926–32211, RRID:AB_621843; Cat #926–32210,RRID:AB_621842; Cat #926–68071, RRID:AB_10956166; Cat #926-68070, RRID:AB_10956588 Chemicals, peptides, and recombinant proteins Bovine serum albumin (BSA) FFA free Sigma Cat #A7030 Collagenase A Roche Cat #10103586001 DAB OB Super Sensitive detection kit ImPath Cat #46538 DAB Quanto Thermofischer Scientific Cat #TA-125-QHDX Wash buffer for IHC ImPath Cat #45002 Antigen retrieval solution pH6.0 ImPath Cat #44998 Ultravision Hydrogen Peroxyde block Thermofisher Scientific Cat #TS-12-H2O2Q Goat serum Immunoreagents Cat #SP-004-VX10 IHC mounting medium CellPath Cat #SEA-1604-00A Qiazol Lysis reagent Qiagen Cat #79306 RNeasy mini kit Qiagen Cat #74104 SuperScriptII reverse Transcriptase Invitrogen Cat #18064-014 dNTP set 100mM Invitrogen Cat #10297-018 Random hexamers Invitrogen Cat #48190-011 QIAquick PCR purification kit Qiagen Cat #28106 RNaseOut Ribonuclease inhibitor Invitrogen Cat #10777-019 Maxima SYBR Green qPCR master mix 2X Thermofisher Scientific Cat #K0253 96-Well PCR plates Thermofisher Scientific Cat #AB0700 DMEM 4.5 g/L glucose Gibco Cat #41966-029 DMEM 1 g/L glucose Gibco Cat #31885-023 Elisa mouse Adiponectin/Acrp30 R&DSystems Cat #DY1119 and #DY008 (Continued on next page) 16 Cell Reports 42, 112866, August 29, 2023

Techniques: Staining, Injection, Expressing

Journal: EMBO Reports

Article Title: Functional BRI2-TREM2 interactions in microglia: implications for Alzheimer’s and related dementias

doi: 10.1038/s44319-024-00077-x

Figure Lengend Snippet: ( A ) Quantification of Adam10 and Adam17 mRNA expression in WT and Itm2b-KO primary microglia using quantitative RT-PCR. ( B ) The experiment is a replicate of the one described in ( A ), using separate biological replicates. ( C ) Quantification of Adam17 in cell lysates from WT and Itm2b-KO primary microglia taken 2 h post E. coli stimulation (including controls treated solely with vehicle, Veh). The Western blot corresponding to the quantification is shown to the right and was performed using the membrane probed with α-pSyk, α-pPlcγ1, and α-p-p38 antibodies in Fig. . ( D ) The experiment is a replicate of the one described in ( C ), using separate biological replicates. The Western blot corresponding to the quantification is shown to the right and was performed using the membrane probed with α-pSyk, α-pPlcγ1, and α-p-p38 antibodies in Fig. . ( E ) ELISA-based quantification of TNFα secretion in the culture supernatants of WT and Itm2b-KO primary microglia treated with E. coli for 1 h, including controls treated solely with vehicle (Veh). ( F ) The experiment is a replicate of the one described in ( E ), using separate biological replicates. Data information: This figure encompasses the comprehensive dataset employed for these specific experiments. We have included the images of the complete membranes used for Western blot analyses, without any cropping of information above or below the targeted signals. Statistical comparisons among the groups were conducted using either a two-tailed unpaired t -test ( A , B ) or a two-way ANOVA followed by post-hoc Sidak’s multiple comparisons test when ANOVA indicated significant differences ( C – F ). * P < 0.05, *** P < 0.001, **** P < 0.0001. The data presented are derived from WT primary microglia cultures ( n = 3) and Itm2b-KO primary microglia ( n = 3); the letter “n” indicates biological replicates. Each biological replicate was composed of primary microglia generated from 2 P2 pups. All data are expressed as means +/− SEM. .

Article Snippet: The probes Mm01310552_mH (exon junction 1–2) and Mm04209424_g1 (exon junction 3–4), Mm00545742_m1 (exon junction 14–15) and Mm00456428_m1 (exon junction 3-4), were used to detect mouse Itm2b , Trem2, Adam10 and Adam17 .

Techniques: Expressing, Quantitative RT-PCR, Western Blot, Membrane, Enzyme-linked Immunosorbent Assay, Two Tailed Test, Derivative Assay, Generated

Journal: EMBO Reports

Article Title: Functional BRI2-TREM2 interactions in microglia: implications for Alzheimer’s and related dementias

doi: 10.1038/s44319-024-00077-x

Figure Lengend Snippet: Reagents and tools.

Article Snippet: The probes Mm01310552_mH (exon junction 1–2) and Mm04209424_g1 (exon junction 3–4), Mm00545742_m1 (exon junction 14–15) and Mm00456428_m1 (exon junction 3-4), were used to detect mouse Itm2b , Trem2, Adam10 and Adam17 .

Techniques: Recombinant, Enzyme-linked Immunosorbent Assay, Sequencing, Gene Expression, Blocking Assay, Western Blot, Isolation, Biomarker Discovery, Software, Imaging, Real-time Polymerase Chain Reaction

Journal: Stem cells (Dayton, Ohio)

Article Title: Mesenchymal stem cells shape microglia effector functions through the release of CX3CL1.

doi: 10.1002/stem.1174

Figure Lengend Snippet: Figure 4. Silencing of CX3CL1 in MSC modulates the expression of effector genes in microglia upon activation. MSC were transfected with small interfering RNA for CX3CL1 for 24 hours and utilized in the culture with N9 in the presence of LPS as described in Materials and Meth- ods. The gene expression of TNF, CX3CR1, NURR1, IL1b, EP2, and IGF1 was measured by real time polymerase chain reaction on LPS-acti- vated N9 in the presence of MSC (light gray bars) or in the presence of MSC silenced for CX3CL1 (dark gray bars). White bars represent microglia in resting condition and black bars microglia activated with LPS. Results are shown as mean 6 SD of three independent experiments. *, p < .05; **, p < .01 by t test. Abbreviations: CX3CR1, fractalkine receptor; EP2, prostaglandin E2 receptor; IL1b, interleukin1b; IGF1, insulin growth factor 1; LPS, lipopolysaccharide; MSC, mesenchymal stem cell; NURR1, nuclear receptor 4 family; TNF, tumor necrosis factor.

Article Snippet: Quantitative analysis of CX3CL1 was performed by ELISA utilizing the Quantikine Mouse CX3CL1/fractalkine Immunoassay (R&D Systems) on supernatants derived from MSC cultured for 24 hours in the presence of either 5 ng/ml IFNc or 1 lg/ml LPS and in the presence of both stimuli according to the manufacturer’s instructions.

Techniques: Expressing, Activation Assay, Transfection, Small Interfering RNA, Gene Expression, Real-time Polymerase Chain Reaction

Journal: Stem cells (Dayton, Ohio)

Article Title: Mesenchymal stem cells shape microglia effector functions through the release of CX3CL1.

doi: 10.1002/stem.1174

Figure Lengend Snippet: Figure 5. CX3CL1 secreted by MSC can induce functional changes on activated microglia. MSC were cultured with the same concentration of N9 cells (8 105 cells) in the presence of 1 lg/ml of LPS and with (dark gray bars) or without (gray bars) 10 lg/ml of a blocking monoclonal anti-CX3CL1 antibody for 24 hours. Ca2þ i, phagocytic activity and TREM2 mRNA expression were measured as described in Materials and Methods. Blocking of release of CX3CL1 by MSC (dark gray bar) significantly inhibited the increase of intracellular Ca2þ concentration observed when microglia was activated with LPS in the presence of MSC (light gray bar) as determined by fluorimetric quantification (left upper panel). Similarly, CX3CL1 blockade on MSC in coculture with N9 (dark gray bar) reverted the enhanced phagocytic activity (middle panel and lower panels) and the upregulation in the expression of TREM2, quantified by real time polymerase chain reaction (Right upper panel), of LPS- activated microglia observed in the presence of MSC (light gray bar). Control LPS-activated N9 cells are depicted as black bars while resting microglia is shown as white bar. Results are shown as mean 6 SD of at least three independent experiments. *, p < .01; **, p < .05 by t test. The images were representative of results obtained in three independent experiments. Abbreviations: aCX3CL1, anti-CX3CL1 antibody; LPS, li- popolysaccharide; MSC, mesenchymal stem cell; TREM2, triggering receptor expressed on myeloid cells-2.

Article Snippet: Quantitative analysis of CX3CL1 was performed by ELISA utilizing the Quantikine Mouse CX3CL1/fractalkine Immunoassay (R&D Systems) on supernatants derived from MSC cultured for 24 hours in the presence of either 5 ng/ml IFNc or 1 lg/ml LPS and in the presence of both stimuli according to the manufacturer’s instructions.

Techniques: Functional Assay, Cell Culture, Concentration Assay, Blocking Assay, Activity Assay, Expressing, Real-time Polymerase Chain Reaction, Control

Journal: Stem cells (Dayton, Ohio)

Article Title: Mesenchymal stem cells shape microglia effector functions through the release of CX3CL1.

doi: 10.1002/stem.1174

Figure Lengend Snippet: Figure 6. Exogenous CX3CL1 mimics the effect of mesenchymal stem cell on activated microglia. 5 ng/ml of recombinant CX3CL1 was added to LPS-activated N9 cells (gray bars), and the expression of TNF, IL1b, CX3CR1, NURR1, EP2, and TREM2 in microglia was analyzed by real time polymerase chain reaction while Ca2þ i and phagocytic activity were measured as described in Materials and Methods and compared to LPS-activated N9 (black bars) and resting microglia (white bars). Results are shown as mean 6 SD of at least three independent experiments. *, p < .05; **, p < .05 by t test. Abbreviations: CX3CR1, fractalkine receptor; EP2, prostaglandin E2 receptor; IL1b, interleukin1b; LPS, lipo- polysaccharide; NURR1, nuclear receptor 4 family; TNF, tumor necrosis factor; TREM2, triggering receptor expressed on myeloid cells-2.

Article Snippet: Quantitative analysis of CX3CL1 was performed by ELISA utilizing the Quantikine Mouse CX3CL1/fractalkine Immunoassay (R&D Systems) on supernatants derived from MSC cultured for 24 hours in the presence of either 5 ng/ml IFNc or 1 lg/ml LPS and in the presence of both stimuli according to the manufacturer’s instructions.

Techniques: Recombinant, Expressing, Real-time Polymerase Chain Reaction, Activity Assay

Journal: Nucleic Acids Research

Article Title: MAZ induces MYB expression during the exit from quiescence via the E2F site in the MYB promoter

doi: 10.1093/nar/gkx641

Figure Lengend Snippet: Myb-sp contains the transcription factor MAZ. ( A ) Sequence of WT and mutant MYB-E2F probes. For each mutant, alterations relative to the WT sequence are indicated in bold. Lines indicate the binding sites for E2F and Myb-sp. ( B ) Complexes formed between DG-75 nuclear factors and the radiolabeled MYB-E2F probe were analyzed by EMSA. Reaction mixtures were incubated in the absence (indicated as None) or in the presence of a 100-fold excess of the indicated unlabeled competitor oligonucleotides. The position of complexes I–V is denoted. The free probe is not shown. ( C ) A nuclear extract derived from DG-75 cells was subjected to sequence-specific DNA affinity chromatography employing concatemerized MYB-Sp or MYB-Null oligonucleotides bound to sepharose beads. Samples were eluted with an excess of MYB-Sp oligonucleotides and analyzed by 10% SDS-PAGE and silver staining. A 30-kDa band (B30) was excised and subjected to mass spectrometry analysis. ( D ) Sequence of the seven tryptic peptides derived from B30. ( E ) Schematic representation of major MAZ isoforms. The Uniprot code of these isoforms and the approximate position of the detected peptides are indicated. ( F ) EMSA analysis of complex formation employing the radiolabeled MYB-E2F probe and a nuclear extract from DG-75 cells (NE), a control reticulocyte lysate (Mock) or in vitro translated MAZ-2s or MAZ-1. Reaction mixtures were incubated in the absence or in the presence of a 100-fold excess of the indicated unlabeled MYB-E2F competitor oligonucleotides.

Article Snippet: The genomic facility at Instituto de Investigaciones Biomedicas synthesized complementary DNA using M-MLV retro transcriptase (ThermoFischer Scientific) and analyzed gene expression by real-time quantitative (RT-qPCR) as described ( ) using TaqMan Gene Expression Assays (ThermoFischer Scientific) specific for human MAZ (Hs00911157_g1), MYB (Hs00193527_m1), CYCNG2 (Hs00171119_m1), E2F1 (Hs00153451_m1) and GUSB (Hs99999908_m1).

Techniques: Sequencing, Mutagenesis, Binding Assay, Incubation, Derivative Assay, Affinity Chromatography, SDS Page, Silver Staining, Mass Spectrometry, In Vitro

Journal: Nucleic Acids Research

Article Title: MAZ induces MYB expression during the exit from quiescence via the E2F site in the MYB promoter

doi: 10.1093/nar/gkx641

Figure Lengend Snippet: Production of antibodies against various MAZ isoforms. ( A ) Schematic representation of major MAZ isoforms indicating the approximate position of the peptides employed as immunogens in the production of the indicated anti-MAZ antibodies. ( B ) Immunoblot analysis employing the indicated anti-MAZ antibodies and protein extracts derived from HEK-293T overexpressing MAZ1, MAZ-2, MAZ-2s or from Mock-transfected cells. Arrowheads indicate the position of the major proteins identified by these antibodies.

Article Snippet: The genomic facility at Instituto de Investigaciones Biomedicas synthesized complementary DNA using M-MLV retro transcriptase (ThermoFischer Scientific) and analyzed gene expression by real-time quantitative (RT-qPCR) as described ( ) using TaqMan Gene Expression Assays (ThermoFischer Scientific) specific for human MAZ (Hs00911157_g1), MYB (Hs00193527_m1), CYCNG2 (Hs00171119_m1), E2F1 (Hs00153451_m1) and GUSB (Hs99999908_m1).

Techniques: Western Blot, Derivative Assay, Transfection

Journal: Nucleic Acids Research

Article Title: MAZ induces MYB expression during the exit from quiescence via the E2F site in the MYB promoter

doi: 10.1093/nar/gkx641

Figure Lengend Snippet: Myb-sp contains various MAZ isoforms. ( A ) EMSA analysis of complex formation employing the radiolabeled MYB-E2F probe and a control reticulocyte lysate (Mock), or in vitro translated MAZ-2s or MAZ-1. Reaction mixtures were incubated in the absence (none) or in the presence of the indicated anti-MAZ antibodies, the M-123 pre-immune serum (Pre-123) and an anti-HA or an anti-Smad2/3 antibody. The free probe is not shown. ( B ) EMSA analysis of complex formation employing the radiolabeled MYB-E2F probe and nuclear extracts from DG-75 cells. Reaction mixtures were incubated in the absence (none) or in the presence of the indicated anti-MAZ antibodies; the M-13, M-123 and M-12 pre-immune sera (Pre-13, Pre-123 and Pre-12, respectively); and an anti-HA or an anti-DP1 antibody. The free probe is not shown.

Article Snippet: The genomic facility at Instituto de Investigaciones Biomedicas synthesized complementary DNA using M-MLV retro transcriptase (ThermoFischer Scientific) and analyzed gene expression by real-time quantitative (RT-qPCR) as described ( ) using TaqMan Gene Expression Assays (ThermoFischer Scientific) specific for human MAZ (Hs00911157_g1), MYB (Hs00193527_m1), CYCNG2 (Hs00171119_m1), E2F1 (Hs00153451_m1) and GUSB (Hs99999908_m1).

Techniques: In Vitro, Incubation

Journal: Nucleic Acids Research

Article Title: MAZ induces MYB expression during the exit from quiescence via the E2F site in the MYB promoter

doi: 10.1093/nar/gkx641

Figure Lengend Snippet: MAZ proteins bind the MYB -E2F element in vivo and overcome RB-mediated transcriptional repression through this element. ( A ) Schematic representation of the luciferase reporter plasmids used, 2xE2F/MAZ-Luc . Two copies of the WT E2F site from the MYB promoter or its mutant derivatives that interact only with E2F ( E2F ), only with MAZ ( Sp ) or with none of them ( Null ) were cloned immediately upstream from the ß-globin TATA box in pBG-Luc . ( B ) The indicated 2xE2F/MAZ-Luc plasmids (1 μg) were cotransfected with pRL-null (1 μg) in asynchronously growing Saos-2 cells in the presence of plasmids encoding MAZ-1 (0.1 μg), MAZ-2 (0.1 μg), MAZ-2s (0.1 μg), DP1 (50 ng) plus E2F1 (50 ng) or empty vector (0.1 μg). Forty hours later, cell extracts were prepared and firefly and renilla luciferase assays were performed. Firefly luciferase values were normalized for renilla activity. Luciferase activity is shown relative to that in the presence of the empty vector (mean ± SEM; n = 4). ** P < 0.01, *** P < 0.001 and **** P < 0.0001 versus Null ; one-way ANOVA with Bonferroni post-test. ( C – E ) The indicated 2xE2F/MAZ-Luc (1 μg) plasmids were cotransfected with pRL-null (1 μg) in asynchronously growing Saos-2 cells in the presence of 0.1 μg of empty vector (−), or plasmids encoding either (C and D) pRB (0.1 μg) or (E) p130, together with (C) MAZ-2s (0.1 or 0.5 μg), (D) MAZ-1 (0.1 or 0.5 μg) or MAZ-1 (0.5 μg), as indicated. Forty hours later, cell extracts were prepared and firefly and renilla luciferase assays were performed. Firefly luciferase values were normalized for renilla activity. Luciferase activity is shown relative to that in the presence of the empty vector (mean ± SEM; n = 5). (C and D) * P < 0.05 and **** P < 0.0001 versus empty; #### P < 0.0001 versus RB; one-way ANOVA with Bonferroni post-test.

Article Snippet: The genomic facility at Instituto de Investigaciones Biomedicas synthesized complementary DNA using M-MLV retro transcriptase (ThermoFischer Scientific) and analyzed gene expression by real-time quantitative (RT-qPCR) as described ( ) using TaqMan Gene Expression Assays (ThermoFischer Scientific) specific for human MAZ (Hs00911157_g1), MYB (Hs00193527_m1), CYCNG2 (Hs00171119_m1), E2F1 (Hs00153451_m1) and GUSB (Hs99999908_m1).

Techniques: In Vivo, Luciferase, Mutagenesis, Clone Assay, Plasmid Preparation, Activity Assay

Journal: Nucleic Acids Research

Article Title: MAZ induces MYB expression during the exit from quiescence via the E2F site in the MYB promoter

doi: 10.1093/nar/gkx641

Figure Lengend Snippet: MAZ proteins activate the MYB promoter through the E2F/MAZ-binding element. ( A ) Schematic representation of the luciferase reporter vector driven by the MYB promoter ( MYBpr-Luc ). The position of the E2F/MAZ combined element (black box) and that of the transcription initiation site (arrow) is indicated. Reporter vectors with a WT E2F/MAZ element ( WT ) as well as vectors with a mutated site that interacted only with E2F ( E2F ), only with MAZ ( Sp ) or with none of them ( Null ) were also generated. ( B and C ) The indicated MYBpr-Luc reporter plasmids (10 μg) were cotransfected with pRL-null (1 μg) into Saos-2 cells in the presence of empty vector (0) or increasing amount of plasmids (indicated in μg) encoding (B) MAZ-1 ( n = 4) or (C) MAZ-2s ( n = 3). Forty hours later, cell extracts were prepared and firefly and renilla luciferase assays were performed. Firefly luciferase values were normalized for renilla activity. Luciferase activity is shown relative to that in the presence of the empty vector (mean ± SEM). * P < 0.05, ** P < 0.01, *** P < 0.001 and **** P < 0.0001 versus Null ; one-way ANOVA with Bonferroni post-test.

Article Snippet: The genomic facility at Instituto de Investigaciones Biomedicas synthesized complementary DNA using M-MLV retro transcriptase (ThermoFischer Scientific) and analyzed gene expression by real-time quantitative (RT-qPCR) as described ( ) using TaqMan Gene Expression Assays (ThermoFischer Scientific) specific for human MAZ (Hs00911157_g1), MYB (Hs00193527_m1), CYCNG2 (Hs00171119_m1), E2F1 (Hs00153451_m1) and GUSB (Hs99999908_m1).

Techniques: Binding Assay, Luciferase, Plasmid Preparation, Generated, Activity Assay

Journal: Nucleic Acids Research

Article Title: MAZ induces MYB expression during the exit from quiescence via the E2F site in the MYB promoter

doi: 10.1093/nar/gkx641

Figure Lengend Snippet: Activation of the MYB promoter during the exit from quiescence requires its MAZ-binding site. ( A ) Schematic representation of the luciferase reporter vector used, MYBpr(+205)-Luc . A DNA insert encompassing the MYB promoter plus its 5′UTR (a 205-bp DNA fragment) were cloned into pGL2-Basic . Reporter vectors with a WT E2F/MAZ element ( WT ) as well as vectors with a mutated site that interacted only with E2F ( E2F ), only with MAZ ( Sp ) or with none of them ( Null ) were also generated. The relative positions of the combined E2F/MAZ element (black box), the transcription initiation site (arrow) and the GC box (gray block) are shown. ( B ) The indicated MYBpr(+205)-Luc reporter plasmids (40 μg) were cotransfected with pRL-SV40 (5 μg) into primary peripheral blood lymphocytes (PBLs). Following transfection, cells were split and either activated for 24 h with leucoagglutinin plus interleukin-2 to exit from quiescence (Activated cells) or left untreated (Resting cells). Firefly and renilla luciferase activities were measured in cell extracts from both activated and untreated cells. Normalized luciferase values for each indicated reporter plasmid are shown relative to untreated cells (mean ± SEM; n = 8). ** P < 0.01, **** P < 0.0001 versus Null Activated; two-way Anova with Bonferroni post-test. ( C ) Anti-Pol II, anti-MAZ M-123 or pre-immune M-123 (Ctl) ChIP analyses of resting lymphocytes (Res) and lymphocytes activated for 2 h (Activ). Different dilutions of input chromatin DNA (1, 0.25 and 0. 1%) or DNA extracted from the indicated immunoprecipitates were analyzed by real-time PCR using oligonucleotides flanking a MYB genomic region encompassing the E2F/MAZ site and the transcription initiation site ( MYB ), the transcription initiation site of POLR2A or the 3′-UTR of BMP7 . Data are shown as enrichment in the amount of chromatin precipitated with each antibody relative to 0.1% input chromatin. Histograms show means ± SEM ( n = 3). ** P < 0.01, *** P < 0.001 versus Ctl in quiescent cells; two-way ANOVA with Bonferroni post-test.

Article Snippet: The genomic facility at Instituto de Investigaciones Biomedicas synthesized complementary DNA using M-MLV retro transcriptase (ThermoFischer Scientific) and analyzed gene expression by real-time quantitative (RT-qPCR) as described ( ) using TaqMan Gene Expression Assays (ThermoFischer Scientific) specific for human MAZ (Hs00911157_g1), MYB (Hs00193527_m1), CYCNG2 (Hs00171119_m1), E2F1 (Hs00153451_m1) and GUSB (Hs99999908_m1).

Techniques: Activation Assay, Binding Assay, Luciferase, Plasmid Preparation, Clone Assay, Generated, Blocking Assay, Transfection, Real-time Polymerase Chain Reaction

Journal: Nucleic Acids Research

Article Title: MAZ induces MYB expression during the exit from quiescence via the E2F site in the MYB promoter

doi: 10.1093/nar/gkx641

Figure Lengend Snippet: MAZ is required for MYB induction during the exit from quiescence. ( A ) Experimental timeline. The proliferation of isolated primary PBLs was induced with a combination of leucoagglutinin and IL2 and maintained by the presence of IL2 during 12 days. Cells were then made quiescent by IL2 starvation and induced to re-enter the cell cycle 48 h later by adding leucoagglutinin and IL2. ( B ) Immunoblot analysis of p130 phospho-S672; p130 total; a combination of pRB phospho-S780, phospho-S795 and phospho-S807/811; pRB total; and TFIIH (loading control) in nuclear extracts from these cells. ( C ) RT-qPCR analysis of MYB, MAZ and E2F1 mRNA expression in lymphocytes stimulated for the indicated periods of time to re-enter the cell cycle. mRNA amounts were normalized to GUSB expression. Data are shown as means ± SEM ( n = 3) relative to 0 h. * P < 0.05, ** P < 0.01, *** P < 0.001 versus 0 h; two-way ANOVA with Bonferroni post-test. ( D ) Immunoblot analysis of phospho-MAZ (S460) and MAZ total levels (employing the MAZ-123 antibody) in nuclear extracts from these cells. ( E ) Experimental timeline as in (A). Proliferating lymphocytes were transduced with lentivirus expressing GFP and either a control shRNA or MAZ -specific shRNAs sh59 or sh60 2 days before IL2 starvation. ( F and G ) Lymphocytes transduced with lentivirus expressing shRNAs as indicated were forced to become quiescent by IL2 starvation for 48 h. (E) MAZ knockdown was confirmed by RT-qPCR analysis at this time. mRNA amounts were normalized to GUSB expression (means ± SEM, n = 3). *** P < 0.001, **** P < 0.0001 versus Control ; one-way ANOVA with Bonferroni post-test. (F) Transduced, quiescent cells were treated with leucoagglutinin plus IL2 for 1 h to re-enter the cell cycle (Activated) or left untreated (Resting). MYB mRNA levels were determined by RT-qPCR analysis and normalized to GUSB expression (means ± SEM, n = 3). *** P < 0.001 versus Control Activated; #### P < 0.0001 versus Control Resting; two-way ANOVA with Bonferroni post-test.

Article Snippet: The genomic facility at Instituto de Investigaciones Biomedicas synthesized complementary DNA using M-MLV retro transcriptase (ThermoFischer Scientific) and analyzed gene expression by real-time quantitative (RT-qPCR) as described ( ) using TaqMan Gene Expression Assays (ThermoFischer Scientific) specific for human MAZ (Hs00911157_g1), MYB (Hs00193527_m1), CYCNG2 (Hs00171119_m1), E2F1 (Hs00153451_m1) and GUSB (Hs99999908_m1).

Techniques: Isolation, Western Blot, Quantitative RT-PCR, Expressing, Transduction, shRNA

Journal: PLoS Medicine

Article Title: An Immune Basis for Lung Parenchymal Destruction in Chronic Obstructive Pulmonary Disease and Emphysema

doi: 10.1371/journal.pmed.0010008

Figure Lengend Snippet: (A) CD14 + , lymphocyte-depleted lung leukocytes were cultured with and without the indicated amounts of recombinant human IP-10 and IFN-γ, and supernatants were assessed for the presence of MMP12 by Western blotting. (B) Fold increase relative to unstimulated of MMP12 mRNA from lung macrophages stimulated without ( – ) and with ( + ) 500 ng/ml of IP-10 in the presence or absence of a function-blocking antibody to CXCR3 as determined by real-time PCR. (C and D) Lung tissue from a participant with emphysema (C) shows strong immune staining for MMP12 localized to macrophages (arrows), and (D) shows lung tissue from a control participant without emphysema and with undetectable MMP12. The insets show a high-power view of lung macrophages staining positive (C) and negative (D) for MMP12 (×60) *, p = 0.04.

Article Snippet: Paraffin-embedded, and fresh-frozen lung sections (5 μm) were immunostained using monoclonal antibodies against human MMP12 (R&D Systems) or non-immune antisera by an immunoperoxidase protocol (Vectastain Elite, Vector Labs, Burlingame, California, United States) and counterstained with hematoxylin as recommended by the manufacturer.

Techniques: Cell Culture, Recombinant, Western Blot, Blocking Assay, Real-time Polymerase Chain Reaction, Staining, Control

Journal: eLife

Article Title: FRMD8 promotes inflammatory and growth factor signalling by stabilising the iRhom/ADAM17 sheddase complex

doi: 10.7554/eLife.35012

Figure Lengend Snippet: ( A ) HEK293T cells transiently transfected with human iRhom2-3xHA or UNC93B1-3xHA were stained with DAPI (blue) to label nuclei, anti-HA to label iRhom2-HA (red), and anti-calnexin to label the ER (green). Scale bar = 10 μm. ( B ) Lysates and anti-HA immunoprecipitation (HA-IP) from wild-type (WT) and FRMD8 knockout (KO) HEK293T cells stably expressing iRhom2-3xHA (where indicated) were immunoblotted for HA and FRMD8. Nonspecific bands are marked with an asterisk.

Article Snippet: Resulting cDNA was used for quantitative PCR (qPCR) using the TaqMan Gene Expression Master Mix (Applied Biosystems) and the following TaqMan probes (all Thermo Fisher Scientific): human ACTB (Hs99999903_m1), human FRMD8 (Hs00607699_mH), human RHBDF2 (Hs00226277_m1), and human TNFα (Hs00174128_m1). qPCR was performed on a StepOnePlus system (Applied Biosystems).

Techniques: Transfection, Staining, Immunoprecipitation, Knock-Out, Stable Transfection, Expressing

Journal: eLife

Article Title: FRMD8 promotes inflammatory and growth factor signalling by stabilising the iRhom/ADAM17 sheddase complex

doi: 10.7554/eLife.35012

Figure Lengend Snippet: ( A ) Volcano plot representing results from three iRhom2 co-immunoprecipitations. The fold change of label-free quantification values (in log2 ratio) was plotted against the p value (-log10 transformed). The grey dotted line indicates p-values <0.05 (analysed with a two-sample t-test). Benjamini-Hochberg correction was applied to adjust the p-value for multiple hypothesis testing (dark grey dotted line). ( B ) Lysates of HEK293T cells stably expressing human iRhom1-3xHA or iRhom2-3xHA transfected with human FRMD8-V5 (where indicated) were subjected to anti-HA and anti-V5 immunoprecipitation (HA-IP, V5–IP) and a western blot using anti-HA and anti-V5 antibodies was performed. Black arrowheads indicated the co-immunoprecipitated FRMD8-V5; white arrowheads indicated the co-immunoprecipitated iRhoms.

Article Snippet: Resulting cDNA was used for quantitative PCR (qPCR) using the TaqMan Gene Expression Master Mix (Applied Biosystems) and the following TaqMan probes (all Thermo Fisher Scientific): human ACTB (Hs99999903_m1), human FRMD8 (Hs00607699_mH), human RHBDF2 (Hs00226277_m1), and human TNFα (Hs00174128_m1). qPCR was performed on a StepOnePlus system (Applied Biosystems).

Techniques: Quantitative Proteomics, Transformation Assay, Stable Transfection, Expressing, Transfection, Immunoprecipitation, Western Blot

Journal: eLife

Article Title: FRMD8 promotes inflammatory and growth factor signalling by stabilising the iRhom/ADAM17 sheddase complex

doi: 10.7554/eLife.35012

Figure Lengend Snippet: List of iRhom2 interaction partners identified in the mass spectrometry screen that have either shown a significant adjusted p-value or been reported previously ( Adrain et al., 2012 ; McIlwain et al., 2012 ; Grieve et al., 2017 ). P-values from a two-sample t-test in Perseus are listed below. P-values were adjusted for multiple hypothesis testing with the Benjamini-Hochberg correction and are listed under ‘adjusted p-values’.

Article Snippet: Resulting cDNA was used for quantitative PCR (qPCR) using the TaqMan Gene Expression Master Mix (Applied Biosystems) and the following TaqMan probes (all Thermo Fisher Scientific): human ACTB (Hs99999903_m1), human FRMD8 (Hs00607699_mH), human RHBDF2 (Hs00226277_m1), and human TNFα (Hs00174128_m1). qPCR was performed on a StepOnePlus system (Applied Biosystems).

Techniques: Mass Spectrometry, Membrane

Journal: eLife

Article Title: FRMD8 promotes inflammatory and growth factor signalling by stabilising the iRhom/ADAM17 sheddase complex

doi: 10.7554/eLife.35012

Figure Lengend Snippet: ( A ) ADAM17 levels were analysed in HEK293T cells transfected with non-targeting siRNA control pool (ctrl) or FRMD8 SMARTpool siRNA after western blotting with anti-ADAM17 and anti-actin staining. In this and subsequent figures, pro- and mature form of ADAM17 are indicated with black and white arrowheads, respectively. Lower panel: Knockdown efficiency of FRMD8 was analysed by TaqMan PCR. ( B, C ) Lysates from wild-type (WT) and FRMD8 knockout (KO) HEK293T cells, transiently transfected with FRMD8-V5 for 72 hr (where indicated) and immunoblotted for endogenous ADAM17, ADAM10, FRMD8 and actin using western blotting. Nonspecific bands are marked with an asterisk. ( D ) Cell surface levels of endogenous ADAM10 and ADAM17 were analysed in WT and FRMD8 KO HEK293T cells after stimulation with 200 nM PMA for 5 min. Unpermeabilised cells were stained on ice with ADAM10 and ADAM17 antibodies, or only with the secondary antibody as a control (grey). The immunostaining was analysed by flow cytometry. The graph shown is one representative experiment out of four biological replicates. The geometric mean fluorescence was calculated for each experiment using FlowJo software. Statistical analysis was performed using an unpaired t-test. ( E, F ) WT and FRMD8 KO HEK293T cells were transiently transfected with alkaline phosphatase (AP)-tagged AREG, HB-EGF or TGFα, and then either incubated with 200 nM PMA, with 200 nM PMA and 1 µM GW (ADAM10/ADAM17 inhibitor), or with DMSO for 30 min. In addition, cells transfected with AP-TGFα were either left unstimulated for 20 hr or incubated with GW for 20 hr. AP activity was measured in supernatants and cell lysates. Each experiment was performed in biological triplicates. The results of three independent shedding experiments are shown. Statistical analysis was performed of using a Mann-Whitney test. ns = p value>0.05; *=p value<0.05; ***=p value<0.001; ****=p value<0.0001.

Article Snippet: Resulting cDNA was used for quantitative PCR (qPCR) using the TaqMan Gene Expression Master Mix (Applied Biosystems) and the following TaqMan probes (all Thermo Fisher Scientific): human ACTB (Hs99999903_m1), human FRMD8 (Hs00607699_mH), human RHBDF2 (Hs00226277_m1), and human TNFα (Hs00174128_m1). qPCR was performed on a StepOnePlus system (Applied Biosystems).

Techniques: Transfection, Control, Western Blot, Staining, Knockdown, Knock-Out, Immunostaining, Flow Cytometry, Fluorescence, Software, Incubation, Activity Assay, MANN-WHITNEY

Journal: eLife

Article Title: FRMD8 promotes inflammatory and growth factor signalling by stabilising the iRhom/ADAM17 sheddase complex

doi: 10.7554/eLife.35012

Figure Lengend Snippet: ( A ) Schematic representation of truncated human iRhom2 constructs used in ( B–E ). ( B, C ) Lysates and anti-HA immunoprecipitation (HA-IP) from HEK293T cells transiently co-transfected with FRMD8-V5 and either empty vector (vect) or truncated human iRhom2-3xHA constructs were immunoblotted for V5 and HA. ( D ) iRhom1/2 double knockout HEK293T cells stably expressing empty vector (vect) or human iRhom2-3xHA constructs were transiently transfected with alkaline phosphatase (AP)-tagged AREG and then incubated with 200 nM PMA or with DMSO for 30 min. AP activity was measured in supernatants and cell lysates. Each experiment was performed in biological triplicates. The results of three independent shedding experiments are shown. Statistical analysis was performed using a Mann-Whitney test. ****=p value<0.0001. ( E ) Lysates from iRhom1/2 double knockout HEK293T cells transiently transfected with empty vector (vect) or human iRhom2-3xHA constructs were immunoblotted for ADAM17 and HA.

Article Snippet: Resulting cDNA was used for quantitative PCR (qPCR) using the TaqMan Gene Expression Master Mix (Applied Biosystems) and the following TaqMan probes (all Thermo Fisher Scientific): human ACTB (Hs99999903_m1), human FRMD8 (Hs00607699_mH), human RHBDF2 (Hs00226277_m1), and human TNFα (Hs00174128_m1). qPCR was performed on a StepOnePlus system (Applied Biosystems).

Techniques: Construct, Immunoprecipitation, Transfection, Plasmid Preparation, Double Knockout, Stable Transfection, Expressing, Incubation, Activity Assay, MANN-WHITNEY

Journal: eLife

Article Title: FRMD8 promotes inflammatory and growth factor signalling by stabilising the iRhom/ADAM17 sheddase complex

doi: 10.7554/eLife.35012

Figure Lengend Snippet: The region required for FRMD8 binding is highlighted in red. Conserved phosphorylation sites that have been mutated to alanine in the iRhom2 pDEAD are marked in yellow. Grey residues indicate additional phosphorylation sites that have been reported on PhosphoSitePlus ( www.phosphosite.org ). An asterisk (*) indicates positions which have a fully conserved residue, a colon (:) indicates strongly similar properties of the amino acids, and a period (.) indicates weakly similar properties according to the Clustal Omega tool. ( B ) Lysates and anti-HA immunoprecipitation (HA-IP) from HEK293T cells transiently transfected with FRMD8-V5 and either empty vector (vect), mouse iRhom2 WT (WT) or Rhom2 cub (Δ268) were immunoblotted for V5 and HA.

Article Snippet: Resulting cDNA was used for quantitative PCR (qPCR) using the TaqMan Gene Expression Master Mix (Applied Biosystems) and the following TaqMan probes (all Thermo Fisher Scientific): human ACTB (Hs99999903_m1), human FRMD8 (Hs00607699_mH), human RHBDF2 (Hs00226277_m1), and human TNFα (Hs00174128_m1). qPCR was performed on a StepOnePlus system (Applied Biosystems).

Techniques: Binding Assay, Phospho-proteomics, Residue, Immunoprecipitation, Transfection, Plasmid Preparation

Journal: eLife

Article Title: FRMD8 promotes inflammatory and growth factor signalling by stabilising the iRhom/ADAM17 sheddase complex

doi: 10.7554/eLife.35012

Figure Lengend Snippet: ( A ) Lysates, anti-HA and anti-V5 immunoprecipitations (HA-IP, V5–IP) of HEK293T cells co-expressing human iRhom2-3xHA and human FRMD8-V5 were immunoblotted for ADAM17, HA and V5. ( B ) Lysates of wild-type (WT) and ADAM17 knockout (KO) HEK293T cells were transiently transfected with human iRhom2-3xHA and FRMD8-V5 (where indicated), anti-HA and anti-V5 immunoprecipitated (HA-IP; V5–IP) and immunoblotted for ADAM17, HA, and V5. ( C ) Lysates of WT and FRMD8 KO HEK293T cells stably expressing human iRhom2-3xHA were anti-HA immunoprecipitated (HA-IP) and stained for ADAM17 and HA. Nonspecific bands are indicated by an asterisk. ( D ) Lysates of WT and iRhom1/2 double knockout (DKO) HEK293T cells stably expressing human iRhom2 WT -3xHA or iRhom2 Δ201-300 -3xHA were anti-V5 immunoprecipitated (V5–IP) and immunoblotted for ADAM17, HA and V5.

Article Snippet: Resulting cDNA was used for quantitative PCR (qPCR) using the TaqMan Gene Expression Master Mix (Applied Biosystems) and the following TaqMan probes (all Thermo Fisher Scientific): human ACTB (Hs99999903_m1), human FRMD8 (Hs00607699_mH), human RHBDF2 (Hs00226277_m1), and human TNFα (Hs00174128_m1). qPCR was performed on a StepOnePlus system (Applied Biosystems).

Techniques: Expressing, Knock-Out, Transfection, Immunoprecipitation, Stable Transfection, Staining, Double Knockout

Journal: eLife

Article Title: FRMD8 promotes inflammatory and growth factor signalling by stabilising the iRhom/ADAM17 sheddase complex

doi: 10.7554/eLife.35012

Figure Lengend Snippet: ( A, B ) Immunofluorescence of iRhom1/2 double knockout HEK293T cells stably expressing iRhom2-3xHA or iRhom2 Δ300 -3xHA and transiently transfected with FRMD8-V5 for 72 hr. Cells were stained for HA (red), V5 (green) and DAPI for DNA (blue). Single confocal sections are shown, taken through the centre of the nucleus. ( C ) Schematic model of the FRMD8-iRhom2 Δ300 construct used in ( E ). ( D, E ) Immunofluorescence of iRhom1/2 double knockout HEK293T cells stably expressing iRhom2 Δ300 -3xHA or FRMD8-iRhom2 Δ300 -3xHA and transiently transfected with ADAM17-V5 for 72 hr. Cells were stained for HA (green), V5 (red) and DAPI for DNA (blue). Single confocal sections are shown, taken either through the centre of the nucleus (MEDIAL), or at basal regions close to the coverslip (BASAL). In all images the scale bar = 10 µm.

Article Snippet: Resulting cDNA was used for quantitative PCR (qPCR) using the TaqMan Gene Expression Master Mix (Applied Biosystems) and the following TaqMan probes (all Thermo Fisher Scientific): human ACTB (Hs99999903_m1), human FRMD8 (Hs00607699_mH), human RHBDF2 (Hs00226277_m1), and human TNFα (Hs00174128_m1). qPCR was performed on a StepOnePlus system (Applied Biosystems).

Techniques: Immunofluorescence, Double Knockout, Stable Transfection, Expressing, Transfection, Staining, Construct

Journal: eLife

Article Title: FRMD8 promotes inflammatory and growth factor signalling by stabilising the iRhom/ADAM17 sheddase complex

doi: 10.7554/eLife.35012

Figure Lengend Snippet: ( A–D ) Immunofluorescence of iRhom1/2 double knockout HEK293T cells stably expressing iRhom2-3xHA or iRhom2 Δ300 -3xHA treated with DMSO (CON) or 100 nM bafilomycin A1 (BAF) for 16 hr prior to fixation. Cells were stained for HA (green), the lysosomal marker LAMP1 (red) and DAPI for DNA (blue). LAMP1-labelled regions (within white boxes) have been magnified. Scale bar = 10 µm. ( E, F ) iRhom2 Δ300 -3xHA cells were treated as in ( A–D ), but with 72 hr expression of ADAM17-V5 and labelling of HA (green), V5 (red) and DAPI for DNA (blue). Arrows indicate colocalising puncta. Single confocal sections are shown, taken through the centre of the nucleus. HA- and V5-labelled regions (within white boxes) have been magnified. Scale bar = 10 µm. ( G ) Cell lysates of wild-type (WT) and FRMD8 knockout (KO) HEK293T cells treated with the solvent DMSO (–), 10 µM MG-132 (MG) or 200 nM bafilomycin A1 (Baf) for 16 hr were enriched for glycosylated proteins using concanavalin A (conA) beads and immunoblotted for ADAM17 and transferrin receptor 1 (TfR). TfR was used as a loading control although it is also susceptible to bafilomycin treatment. Mature ADAM17 levels from three experiments were quantified relative to TfR levels using ImageJ.

Article Snippet: Resulting cDNA was used for quantitative PCR (qPCR) using the TaqMan Gene Expression Master Mix (Applied Biosystems) and the following TaqMan probes (all Thermo Fisher Scientific): human ACTB (Hs99999903_m1), human FRMD8 (Hs00607699_mH), human RHBDF2 (Hs00226277_m1), and human TNFα (Hs00174128_m1). qPCR was performed on a StepOnePlus system (Applied Biosystems).

Techniques: Immunofluorescence, Double Knockout, Stable Transfection, Expressing, Staining, Marker, Knock-Out, Solvent, Control

Journal: eLife

Article Title: FRMD8 promotes inflammatory and growth factor signalling by stabilising the iRhom/ADAM17 sheddase complex

doi: 10.7554/eLife.35012

Figure Lengend Snippet: ( A ) iRhom1/2 double knockout HEK293T cells stably expressing iRhom2 WT -3xHA, iRhom2 Δ300 -3xHA or FRMD8-iRhom2 Δ300 -3xHA were treated with 100 µg/ml cycloheximide (CHX) for the indicated time (0–8 hr) to block protein synthesis. Cell lysates were immunoblotted for HA and actin. ( B ) Cell lysates of wild-type (WT) and FRMD8 knockout (KO) HEK293T cells treated with 10 µM MG-132 (MG), 200 nM bafilomycin A1 (Baf) or 50 mM ammonium chloride (NH 4 Cl) for 16 hr were immunoblotted for ADAM17, FRMD8, and actin. An asterisk marks a nonspecific band. ( C ) N-glycosylation of iRhom2 was analysed using EndoH and PNGase to distinguish ER/ cis- Golgi (EndoH sensitive) and late Golgi localisation (EndoH resistant). Lysates of WT and FRMD8 KO HEK293T cells transiently transfected with mouse iRhom2-3xHA were deglycosylated with EndoH or PNGase and then immunoblotted for mouse iRhom2, human FRMD8 and actin. An asterisk marks a nonspecific band. ( D ) Lysates of HEK293T cells stably expressing human iRhom1-3xHA and transfected with FRMD8-V5 (where indicated) were immunoblotted for HA, V5, and actin. ( E ) Levels of ADAM17 were analysed in HEK293T-iRhom2-3xHA and HEK293T WT cells transfected with siRNAs targeting iRhom2 where indicated. Cell lysates were immunoblotted using an anti-ADAM17 or anti-actin antibody. An asterisk marks a nonspecific band.

Article Snippet: Resulting cDNA was used for quantitative PCR (qPCR) using the TaqMan Gene Expression Master Mix (Applied Biosystems) and the following TaqMan probes (all Thermo Fisher Scientific): human ACTB (Hs99999903_m1), human FRMD8 (Hs00607699_mH), human RHBDF2 (Hs00226277_m1), and human TNFα (Hs00174128_m1). qPCR was performed on a StepOnePlus system (Applied Biosystems).

Techniques: Double Knockout, Stable Transfection, Expressing, Blocking Assay, Knock-Out, Glycoproteomics, Transfection

Journal: eLife

Article Title: FRMD8 promotes inflammatory and growth factor signalling by stabilising the iRhom/ADAM17 sheddase complex

doi: 10.7554/eLife.35012

Figure Lengend Snippet: ( A ) Unpermeabilised WT (black) and FRMD8 KO HEK293T (cyan) cells stably expressing human iRhom2-3xHA were immunostained on ice for HA. Wild-type HEK293T cells immunostained for HA served as a negative control (grey). ( B ) Cells were permeabilised and stained at room temperature with an anti-HA antibody. Immunostaining with the Alexa Fluor 488-coupled secondary antibody served as a control (grey). The flow cytometry graphs shown are one representative experiment out of three experiments. The geometric mean fluorescence was calculated for each experiment using FlowJo software. Statistical analysis was performed using an unpaired t-test; ns = p value>0.05; *=p value<0.05. ( C ) Lysates of HEK293T cells stably expressing human iRhom2-3xHA and transiently transfected with FRMD8-V5 (where indicated) were analysed by western blot for iRhom2 levels using anti-HA, anti-V5 and anti-actin immunostaining. Nonspecific bands are marked with an asterisk. ( D ) Lysates of WT and FRMD8 KO HEK293T cells stably expressing human iRhom2-3xHA (where indicated) were immunoblotted for HA, FRMD8 and actin. An asterisk marks nonspecific bands. ( E ) FRMD8 mRNA levels relative to actin mRNA levels were determined by TaqMan PCR in cells used in ( D ).

Article Snippet: Resulting cDNA was used for quantitative PCR (qPCR) using the TaqMan Gene Expression Master Mix (Applied Biosystems) and the following TaqMan probes (all Thermo Fisher Scientific): human ACTB (Hs99999903_m1), human FRMD8 (Hs00607699_mH), human RHBDF2 (Hs00226277_m1), and human TNFα (Hs00174128_m1). qPCR was performed on a StepOnePlus system (Applied Biosystems).

Techniques: Stable Transfection, Expressing, Negative Control, Staining, Immunostaining, Control, Flow Cytometry, Fluorescence, Software, Transfection, Western Blot

Journal: eLife

Article Title: FRMD8 promotes inflammatory and growth factor signalling by stabilising the iRhom/ADAM17 sheddase complex

doi: 10.7554/eLife.35012

Figure Lengend Snippet: ( A, B ) Levels of endogenously 3xHA tagged iRhom2 were analysed in HEK293T-iRhom2-3xHA cells transfected with FRMD8-V5 plasmid, siRNAs targeting iRhom2, non-targeting siRNA control pool (ctrl) or FRMD8 SMARTpool siRNA. Cell lysates were anti-HA immunoprecipitated (HA-IP) to detect endogenous iRhom2-3xHA levels and immunoblotted using anti-HA antibody. Cell lysates were immunoblotted for ADAM17, V5, and actin. ( C ) FRMD8 and iRhom2 mRNA levels relative to actin mRNA levels were determined by TaqMan PCR in cells used for the experiment shown in ( B ) to demonstrate that the destabilisation of endogenous iRhom2 was not induced by a change in iRhom2 mRNA levels.

Article Snippet: Resulting cDNA was used for quantitative PCR (qPCR) using the TaqMan Gene Expression Master Mix (Applied Biosystems) and the following TaqMan probes (all Thermo Fisher Scientific): human ACTB (Hs99999903_m1), human FRMD8 (Hs00607699_mH), human RHBDF2 (Hs00226277_m1), and human TNFα (Hs00174128_m1). qPCR was performed on a StepOnePlus system (Applied Biosystems).

Techniques: Transfection, Plasmid Preparation, Control, Immunoprecipitation

Journal: eLife

Article Title: FRMD8 promotes inflammatory and growth factor signalling by stabilising the iRhom/ADAM17 sheddase complex

doi: 10.7554/eLife.35012

Figure Lengend Snippet: ( A ) Sequencing of the genomic DNA isolated from clonal FRMD8 KO iPSCs shows a 1-nt insertion (clone 1) and a 7-nt and 10-nt deletion (clone 2). The targeting sequence of the sgRNA is shown in bold; small letters indicate the sequence within the intronic region; the protospacer adjacent motif (PAM) sequence underlined. ( B ) Parental wild-type and FRMD8 KO iPSC lines were karyotyped by SNP array. Detected copy number variations are indicated in red (DNA copy number loss in the indicated region) and green (DNA copy number increase). The AH017-13 iPSC line used was derived from a female donor, therefore the Y chromosome is marked in red (loss of Y chromosome DNA). ( C ) 25,000 iPSC-derived macrophages were either left unstimulated, stimulated with 50 ng/ml LPS, or with 50 ng/ml LPS and simultaneously with 2 μM GI or 2 μM GW for 4 hr. TNFα concentration in the cell supernatants was measured by ELISA and then normalised to the protein concentration in macrophage cell lysates to adjust the cytokine release for potential differences in cell numbers. Each experiment was performed in biological triplicates. Data from three independent experiments were statistically analysed using a Mann-Whitney test; ns = p value>0.05; ****=p value<0.0001. ( D ) TNFα mRNA levels relative to actin mRNA levels were measured by TaqMan PCR in WT and FRMD8 KO iPSC-derived macrophages without stimulation and after stimulation with 200 ng/ml LPS for 0.5 hr.

Article Snippet: Resulting cDNA was used for quantitative PCR (qPCR) using the TaqMan Gene Expression Master Mix (Applied Biosystems) and the following TaqMan probes (all Thermo Fisher Scientific): human ACTB (Hs99999903_m1), human FRMD8 (Hs00607699_mH), human RHBDF2 (Hs00226277_m1), and human TNFα (Hs00174128_m1). qPCR was performed on a StepOnePlus system (Applied Biosystems).

Techniques: Sequencing, Isolation, Derivative Assay, Concentration Assay, Enzyme-linked Immunosorbent Assay, Protein Concentration, MANN-WHITNEY

Journal: eLife

Article Title: FRMD8 promotes inflammatory and growth factor signalling by stabilising the iRhom/ADAM17 sheddase complex

doi: 10.7554/eLife.35012

Figure Lengend Snippet: ( A ) Schematic representation of the differentiation protocol of iPSCs into macrophages based on . Scale bars = 10 μm. ( B ) Lysates of iPSC-derived macrophages (on day seven after harvest from EBs) were immunoblotted for ADAM17, FRMD8, and actin. Western blots from three experiments were quantified using ImageJ with actin serving as the loading control. ( C ) 25,000 iPSC-derived macrophages were either left unstimulated or stimulated with 50 ng/ml LPS for 4 hr. TNFα concentration in the cell supernatants was measured by ELISA and then normalised to the protein concentration in macrophage cell lysates to adjust the cytokine release for potential differences in cell numbers. Each experiment was performed in biological triplicates. Data from three independent experiments were statistically analysed using a Mann-Whitney test; ***=p value<0.001; ****=p value<0.0001. ( D, E ) Lysates from tissues derived from Frmd8 -/- or Rhbdf2 -/- and their wild-type littermates were immunoblotted for ADAM17, FRMD8, iRhom2 and actin. Blots from three experiments using three different littermates of Frmd8 -/- and Frmd8 +/+ mice were quantified using ImageJ with actin serving as the loading control.

Article Snippet: Resulting cDNA was used for quantitative PCR (qPCR) using the TaqMan Gene Expression Master Mix (Applied Biosystems) and the following TaqMan probes (all Thermo Fisher Scientific): human ACTB (Hs99999903_m1), human FRMD8 (Hs00607699_mH), human RHBDF2 (Hs00226277_m1), and human TNFα (Hs00174128_m1). qPCR was performed on a StepOnePlus system (Applied Biosystems).

Techniques: Derivative Assay, Western Blot, Control, Concentration Assay, Enzyme-linked Immunosorbent Assay, Protein Concentration, MANN-WHITNEY

Journal: eLife

Article Title: FRMD8 promotes inflammatory and growth factor signalling by stabilising the iRhom/ADAM17 sheddase complex

doi: 10.7554/eLife.35012

Figure Lengend Snippet: ( A ) Schematic representation of the insertion of a lacZ/neomycin cassette into the Frdm8 locus in the ES cells used to generate Frmd8 -/- mice. ( B ) Offspring of Frmd8 +/- × Frmd8 +/- (HET x HET) crosses listed by genotype: Frmd8 +/+ (WT), Frmd8 +/- (HET), and Frmd8 -/- (KO). Two Frmd8 mouse strains were bred (both in BL6 background): one with the entire lacZ/neomycin cassette inserted and one strain in which the neomycin resistance gene has been removed from the cassette. ( C ) Lysates from skin derived from Frmd8 -/- , Rhbdf2 -/- mice and their wild-type littermate were immunoblotted for iRhom2 and actin.

Article Snippet: Resulting cDNA was used for quantitative PCR (qPCR) using the TaqMan Gene Expression Master Mix (Applied Biosystems) and the following TaqMan probes (all Thermo Fisher Scientific): human ACTB (Hs99999903_m1), human FRMD8 (Hs00607699_mH), human RHBDF2 (Hs00226277_m1), and human TNFα (Hs00174128_m1). qPCR was performed on a StepOnePlus system (Applied Biosystems).

Techniques: Derivative Assay

Journal: eLife

Article Title: FRMD8 promotes inflammatory and growth factor signalling by stabilising the iRhom/ADAM17 sheddase complex

doi: 10.7554/eLife.35012

Figure Lengend Snippet: Schematic representation of the role of FRMD8 in the iRhom2/ADAM17 pathway: under wild-type conditions ADAM17 and iRhom2 are stabilised by FRMD8 and thereby protected from degradation through the endolysosmal pathway.

Article Snippet: Resulting cDNA was used for quantitative PCR (qPCR) using the TaqMan Gene Expression Master Mix (Applied Biosystems) and the following TaqMan probes (all Thermo Fisher Scientific): human ACTB (Hs99999903_m1), human FRMD8 (Hs00607699_mH), human RHBDF2 (Hs00226277_m1), and human TNFα (Hs00174128_m1). qPCR was performed on a StepOnePlus system (Applied Biosystems).

Techniques:

Journal: eLife

Article Title: FRMD8 promotes inflammatory and growth factor signalling by stabilising the iRhom/ADAM17 sheddase complex

doi: 10.7554/eLife.35012

Figure Lengend Snippet:

Article Snippet: Resulting cDNA was used for quantitative PCR (qPCR) using the TaqMan Gene Expression Master Mix (Applied Biosystems) and the following TaqMan probes (all Thermo Fisher Scientific): human ACTB (Hs99999903_m1), human FRMD8 (Hs00607699_mH), human RHBDF2 (Hs00226277_m1), and human TNFα (Hs00174128_m1). qPCR was performed on a StepOnePlus system (Applied Biosystems).

Techniques: Generated, Control, Transduction, CRISPR, Knock-In, Double Knockout, Clone Assay, Recombinant, Plasmid Preparation, Transfection, Construct, Sequencing, Bicinchoninic Acid Protein Assay, Enzyme-linked Immunosorbent Assay, cDNA Synthesis, Protease Inhibitor, Magnetic Beads, Knock-Out, Electron Microscopy, Software

Journal: eLife

Article Title: ARID1A loss in adult hepatocytes activates β-catenin-mediated erythropoietin transcription

doi: 10.7554/eLife.53550

Figure Lengend Snippet: ( a ) Cre-loxP-generated hepatocyte-specific and inducible inactivation of Apc and/or Arid1a in 20% of hepatocytes after retro-orbital injection of infectious viral particles (ivp) of adenovirus encoding Cre recombinase (AdCre). The resulting mice are referred to as [ Apc-Arid1a ] ko-focal , [ Apc ] ko-focal , and [ Arid1a ] ko-focal . ( b ) Gross examination of mouse livers, 7 months after AdCre injection. Livers from [ Apc-Arid1a ] ko-focal mice had an irregular shape and a rough surface, with multiple dark red zones (indicated by arrows). ( c ) Incidence of hepatic lesions detected in WT (n = 10) and [ Apc-Arid1a ] ko-focal (n = 24) mice by ultrasonography. ( d ) Kaplan-Meier estimated survival curves of WT and [ Apc-Arid1a ] ko-focal mice over 15 months. n = 6 for each group. Inset: Liver of one mouse at necropsy (13 months after AdCre injection, representative of the three analyzed mice). ( e ) Hematoxylin Eosin (HE)-stained sections of mouse livers at 7 months post-injection. Large vascular spaces filled with blood cells were observed only in [ Apc-Arid1a ] ko-focal livers. Related data are found in – , and source data in ‘ ; ; ’. Figure 1—source data 1. Emergence of peliosis and survival curve .

Article Snippet: Sequence-based reagent , Arid1a (total) , Thermo Fisher Scientific , Taqman Assay Mm00473838_m1 , qPCR primers Mus musculus.

Techniques: Generated, Injection, Staining

Journal: eLife

Article Title: ARID1A loss in adult hepatocytes activates β-catenin-mediated erythropoietin transcription

doi: 10.7554/eLife.53550

Figure Lengend Snippet: ( a ) Liver to Body weight (%) in mice; WT (n = 8), [ Apc ] ko-focal (n = 10), [ Arid1a ] ko-focal (n = 18), and [ Apc-Arid1a ] ko-focal (n = 19) mice. ( b ) RT-qPCR analysis of β-catenin-positive target genes from seven-month-old-mouse livers. WT (n = 5), [ Apc ] ko-TOTAL (n = 7), [ Arid1a ] ko-TOTAL (n = 12), and [ Apc-Arid1a ] ko-TOTAL (n = 10) mice. The data in ( a,b ) are expressed as the mean ± SEM and analyzed with one-way ANOVA. ( c,d ) Immunostainings against glutamine synthetase (Glul) and Arid1a after focal Apc and/or Arid1a loss. ( c ) Note the physiological staining of Glul in hepatocytes surrounding the centrolobular vein (cv). Focal activation of β-catenin signaling in single hepatocytes leads to an immunostaining of Glul, its hepatospecific target (red asterisk). Scale bars = 200 μm. ( d ) Immunofluorescence for Glul in hepatocytes without Arid1a nuclear fluorescence (white asterisk), shows the efficiency and specificity of the double Apc / Arid1a inactivation in [ Apc-Arid1a ] ko-focal livers (white asterisk). Scale bars = 100 μm. Figure 1—figure supplement 1—source data 1. Liver to body weight ratios and expression of Glul and Axin2 mRNAs .

Article Snippet: Sequence-based reagent , Arid1a (total) , Thermo Fisher Scientific , Taqman Assay Mm00473838_m1 , qPCR primers Mus musculus.

Techniques: Quantitative RT-PCR, Staining, Activation Assay, Immunostaining, Immunofluorescence, Fluorescence, Expressing

Journal: eLife

Article Title: ARID1A loss in adult hepatocytes activates β-catenin-mediated erythropoietin transcription

doi: 10.7554/eLife.53550

Figure Lengend Snippet: ( a ) Echogenicity of peliotic areas within the [ Apc-Arid1a ] ko-focal liver (arrow), showing striking tissue modification. Scale bars = 2 cm. ( b ) Dynamic contrast-enhanced ultrasound using microbubble administration. Contrast-enhanced ultrasound imaging involves the injection of gas-filled micron-sized bubbles (microbubbles) that do not extravasate. This property makes them ideal contrast agents for imaging vascularity and blood perfusion. The protocol has been described in . It revealed a decrease of hepatic vascular perfusion within echogenic areas compared to neighboring control tissue.

Article Snippet: Sequence-based reagent , Arid1a (total) , Thermo Fisher Scientific , Taqman Assay Mm00473838_m1 , qPCR primers Mus musculus.

Techniques: Modification, Imaging, Injection, Control

Journal: eLife

Article Title: ARID1A loss in adult hepatocytes activates β-catenin-mediated erythropoietin transcription

doi: 10.7554/eLife.53550

Figure Lengend Snippet: ( a ) Peliotic areas appeared as abnormal tangles of irregularly shaped, leaky, small and large blood vessels filled with red blood cells, with multiple, mottled cyst-like spaces associated with sinusoidal dilatation and liver cell dropout in the livers of [ Apc-Arid1a ] ko-focal mice (1), relative to neighboring tissue (2). Scale bars = 200 μm (left panel) and 100 μm (right panel). ( b ) Immunofluorescence, at different magnifications, against β-catenin and Pecam1 of [ Apc-Arid1a ] ko-focal liver. Peliosis-like area, showing strong enrichment of blood vessels (1) compared to neighboring tissue (2). ( c ) RT-qPCR analysis of angiogenic factors from 7-month-old-mouse livers. WT (n = 5), [ Apc ] ko-TOTAL (n = 7), [ Arid1a ] ko-TOTAL (n = 12), and [ Apc-Arid1a ] ko-TOTAL (n = 10) mice. Data are presented as the mean + SEM and analyzed by one-way ANOVA. Figure 1—figure supplement 3—source data 1. qPCR expression of angiogenic mRNAs .

Article Snippet: Sequence-based reagent , Arid1a (total) , Thermo Fisher Scientific , Taqman Assay Mm00473838_m1 , qPCR primers Mus musculus.

Techniques: Immunofluorescence, Quantitative RT-PCR, Expressing

Journal: eLife

Article Title: ARID1A loss in adult hepatocytes activates β-catenin-mediated erythropoietin transcription

doi: 10.7554/eLife.53550

Figure Lengend Snippet: ( a–c ) HCC incidence decrease in [ Apc-Arid1a ] ko-focal compared to [ Apc ] ko-focal mice. ( a ) Incidence of HCC was detected by ultrasound in [ Apc ] ko-focal (n = 13) and [ Apc-Arid1a ] ko-focal (n = 24) mice. ( b ) Two representative livers of ten-month-old [ Apc ] ko-focal and [ Apc-Arid1a ] ko-focal mouse livers presenting tumor. ( c ) Immunostaining of glutamine synthetase (Glul) and Arid1a in [ Apc ] ko-focal and [ Apc-Arid1a ] ko-focal liver sections. NT: non tumoral tissue; Tum: tumor; Scale bars = 200 μm. ( d ) EPO expression in human HCC depending on their CTNNB1 and ARID1A mutational status. Datasets in hepatocellular carcinoma were downloaded from the Cancer Genome Atlas (TCGA) data portal ( http://tcga-data.nci.nih.gov ). We extracted two types of molecular data including gene expression and somatic mutation using TCGA2STAT R package. Fours groups of tumors were clusterized based on mutation status: CTNNB1 -mutated (n = 46), ARID1A -mutated (n = 11), CTNNB1/ARID1A- mutated (n = 5) and no CTNNB1 nor ARID1A mutations (n = 133). Analysis of variance and post-hoc tests were performed to test the association between EPO expression and mutation status.

Article Snippet: Sequence-based reagent , Arid1a (total) , Thermo Fisher Scientific , Taqman Assay Mm00473838_m1 , qPCR primers Mus musculus.

Techniques: Immunostaining, Expressing, Gene Expression, Mutagenesis

Journal: eLife

Article Title: ARID1A loss in adult hepatocytes activates β-catenin-mediated erythropoietin transcription

doi: 10.7554/eLife.53550

Figure Lengend Snippet: ( a ) Experimental strategy; ( b ) Transcriptomic gene-set enrichment analysis (GSEA) of hepatic peliosis (n = 4) relative to adjacent regions (n = 4) of [ Apc-Arid1a ] ko-focal mice. ( c ) Quantitative RT-PCR showing relative expression of mRNAs for positive targets of hepatic Wnt/β-catenin pathway and angiogenic factors in hepatic peliosis (n = 10) compared to adjacent regions (n = 10) of [ Apc-Arid1a ] ko-focal mice (unpaired t test analysis); ( d ) Hematological parameters from WT (n = 7), [ Apc ] ko-focal (n = 12), [ Arid1a ] ko-focal (n = 19), and [ Apc-Arid1a ] ko-focal (n = 20) mice (One-way ANOVA analysis). ( e ) Evaluation of erythropoietin ( Epo ) mRNAs by quantitative RT-PCR in the livers analyzed by the ΔCt technique and expressed relative to those for 18S RNA for the liver, and as relative levels in the kidney (One-way ANOVA analysis). ( f ) Plasma EPO concentrations at sacrifice (WT (n = 6), [ Apc ] ko-focal (n = 5), [ Arid1a ] ko-focal (n = 2), and [ Apc-Arid1a ] ko-focal (n = 10)). Exact p-values are mentioned, ****p<0.0001. Related data are found in and source data in ‘ '. Figure 2—source data 1. Gene expression and hematological parameters .

Article Snippet: Sequence-based reagent , Arid1a (total) , Thermo Fisher Scientific , Taqman Assay Mm00473838_m1 , qPCR primers Mus musculus.

Techniques: Quantitative RT-PCR, Expressing, Clinical Proteomics, Gene Expression

Journal: eLife

Article Title: ARID1A loss in adult hepatocytes activates β-catenin-mediated erythropoietin transcription

doi: 10.7554/eLife.53550

Figure Lengend Snippet: Gene-set enrichment analysis (GSEA) was performed with the Java tool application available at the Broad Institute (Cambridge, MA, USA) in which FFPE micro-dissected RBC regions were compared with neighboring tissue. ES: enrichment score, NES: normalized enrichment score, NOM p-val: nominal p-value, FDR: false discovery rate, FWER p-val: familywise-error rate p-value. Transcriptomic gene-set enrichment analysis (GSEA) of hepatic peliosis (n = 4) relative to adjacent regions (n = 4) of [ Apc-Arid1a ] ko-focal mice showing endothelium and erythrocyte signatures. The table below is a GSEA using the hallmark database.

Article Snippet: Sequence-based reagent , Arid1a (total) , Thermo Fisher Scientific , Taqman Assay Mm00473838_m1 , qPCR primers Mus musculus.

Techniques:

Journal: eLife

Article Title: ARID1A loss in adult hepatocytes activates β-catenin-mediated erythropoietin transcription

doi: 10.7554/eLife.53550

Figure Lengend Snippet: ( a ) Gross morphology of spleens from representative control (WT) and [ Apc-Arid1a ] ko-focal mice; ( b ) Spleen/body weight ratio of WT (n = 7), [ Apc ] ko-focal (n = 11), [ Arid1a ] ko-focal (n = 11), and [ Apc-Arid1a ] ko-focal (n = 17) mice (one-way ANOVA). ( c ) Hematoxylin and Eosin staining of splenic sections. Scale bar is 200 µm. ( d,e ) FACS analysis of liver NPC, bone marrow, and spleens from control (WT) or [ Apc-Arid1a ] ko-focal mice using the erythroid markers CD71 and Ter119. ( e ) FACS quantification from WT (n = 4) and [ Apc-Arid1a ] ko-focal (n = 4) mice (multiple t-test). ( f ) Quantification of erythroid progenitors as erythroid colony-forming units (CFU-E) in the presence of EPO, using 2 × 10 5 cells from bone marrow or 2 × 10 6 cells from the liver and spleen of WT or [ Apc-Arid1a ] ko-focal mice (2-way ANOVA). ( g ) Q-PCR showing relative expression of several factors, known to be involved in stress-induced erythropoiesis, in the spleens of WT (n = 9), [ Apc ] ko-focal (n = 5), [ Arid1a ] ko-focal (n = 8), and [ Apc-Arid1a ] ko-focal (n = 8) mice (one-way ANOVA). ****p<0.0001. Related data are found in and source data in ‘ '. Figure 3—source data 1. Spleen to body weight , FACS analyses , CFU-E counts and gene expression .

Article Snippet: Sequence-based reagent , Arid1a (total) , Thermo Fisher Scientific , Taqman Assay Mm00473838_m1 , qPCR primers Mus musculus.

Techniques: Control, Staining, Expressing, Gene Expression

Journal: eLife

Article Title: ARID1A loss in adult hepatocytes activates β-catenin-mediated erythropoietin transcription

doi: 10.7554/eLife.53550

Figure Lengend Snippet: Western blot ( a ) and immunostaining ( b, c ) of hemoglobin subunit beta (Hbb) showing that Hbb-positive erythroid cells accumulated in [ Apc-Arid1a ] ko-focal livers are not nucleated, so do not correspond to immature and proliferative erythroblasts. Scale bars = 52 μm (b, c: bottom panel) or 200 μm (c: top panel).

Article Snippet: Sequence-based reagent , Arid1a (total) , Thermo Fisher Scientific , Taqman Assay Mm00473838_m1 , qPCR primers Mus musculus.

Techniques: Western Blot, Immunostaining

Journal: eLife

Article Title: ARID1A loss in adult hepatocytes activates β-catenin-mediated erythropoietin transcription

doi: 10.7554/eLife.53550

Figure Lengend Snippet: ( a ) Hematocrit before (n = 4) and after (n = 4) anti-EPO treatment (t-test). ( b,c ) FACS analysis ( b ) and quantification ( c ) of spleens with/without anti-EPO (n = 4 for each group) (t-test). ( d ) RT-qPCR showing relative expression of erythropoiesis factors in the spleens of WT (n = 9), treated [ Apc-Arid1a ] ko-focal (n = 4), untreated [ Apc-Arid1a ] ko-focal (n = 8) mice (one-way ANOVA). ( e ) Hematoxylin Eosin (HE)-stained sections of livers from representative 7-month-old mice. ( f,g ) FACS analysis ( f ) and quantification ( g ) of liver NPC with/without anti-EPO. ( h ) RT-qPCR showing relative expression of angiogenic factors in the livers with (n = 4) and without (n = 10) anti-EPO (t-test). ****p<0.0001. Related data are found in and source data in ‘ '. Figure 4—source data 1. Hematocrit , FACS quantifications and gene expression after anti-EPO treatment.

Article Snippet: Sequence-based reagent , Arid1a (total) , Thermo Fisher Scientific , Taqman Assay Mm00473838_m1 , qPCR primers Mus musculus.

Techniques: Quantitative RT-PCR, Expressing, Staining, Gene Expression

Journal: eLife

Article Title: ARID1A loss in adult hepatocytes activates β-catenin-mediated erythropoietin transcription

doi: 10.7554/eLife.53550

Figure Lengend Snippet: Hematoxylin Eosin (HE)-stained sections of livers from untreated and treated 7-month-old [ Apc-Arid1a ] ko-focal mice with anti-EPO blocking serum. Scale bars = 200 μm. The dotted outlines correspond to increasing magnification showed in .

Article Snippet: Sequence-based reagent , Arid1a (total) , Thermo Fisher Scientific , Taqman Assay Mm00473838_m1 , qPCR primers Mus musculus.

Techniques: Staining, Blocking Assay

Journal: eLife

Article Title: ARID1A loss in adult hepatocytes activates β-catenin-mediated erythropoietin transcription

doi: 10.7554/eLife.53550

Figure Lengend Snippet: ( a ) In vivo and ex vivo strategy. WT (n = 8), [ Apc ] ko-TOTAL (n = 7), [ Arid1a ] ko-TOTAL (n = 8), and [ Apc-Arid1a ] ko-TOTAL (n = 10) mice. ( b ) Inactivation efficiency of Apc and Arid1a genes in isolated hepatocytes. ( c,d ) RT-qPCR assessment of erythropoietin ( Epo ) transcription ( c ) in the hepatocyte and NPC compartments of the livers, ( d ) in the kidney (1-way ANOVA). ( e ) In vitro analysis of Axin2 , Arid1a ( Arid1a floxed-exon detection), and Epo expression by RT-qPCR of mouse hepatocytes after Wnt3a and R-Spondin3 stimulation, and si-Arid1a/si-Control treatments, showing Arid1a knockdown efficiency and Wnt/β-catenin pathway activation, as the mRNA levels of Axin2 , a canonical target gene of Wnt signaling, significantly increased (2-way ANOVA). ( f ) In vitro analysis of Apc , Arid1a , and Epo by RT-qPCR of cryopreserved human hepatocytes after siRNA transfection (one-way ANOVA analysis). Data are presented as the mean ± SEM. ****p<0.0001. Cell culture data are representative of three independent experiments. Related data are found in – , and source data in ‘ ; ; ’. Figure 5—source data 1. Efficiency of gene invalidation , and gene expression in vivo and ex vivo in mice and humans.

Article Snippet: Sequence-based reagent , Arid1a (total) , Thermo Fisher Scientific , Taqman Assay Mm00473838_m1 , qPCR primers Mus musculus.

Techniques: In Vivo, Ex Vivo, Isolation, Quantitative RT-PCR, In Vitro, Expressing, Control, Knockdown, Activation Assay, Transfection, Cell Culture, Gene Expression

Journal: eLife

Article Title: ARID1A loss in adult hepatocytes activates β-catenin-mediated erythropoietin transcription

doi: 10.7554/eLife.53550

Figure Lengend Snippet: ( a ) Hepatomegaly in mice after panlobular inactivations. WT (n = 30), [ Apc ] ko-TOTAL (n = 9), [ Arid1a ] ko-TOTAL (n = 9), and [ Apc-Arid1a ] ko-TOTAL (n = 14) mice. Data are presented as the mean + SEM and analyzed using one-way ANOVA. ( b ) Immunostaining of glutamine synthetase (Glul) and Arid1a after Apc and/or Arid1a loss in all hepatocytes in mouse liver. Note the physiological staining of Glul surrounding the centrilobular vein (cv) in WT and [ Arid1a ] ko-TOTAL livers and the remaining nonparenchymal staining of Arid1a after hepato-specific Arid1a inactivation ([ Arid1a ] KO-TOTAL and [ Apc-Arid1a ] KO-TOTAL ). Apc loss leads to overactivation of the Wnt/β-catenin pathway and, consequently, increased Glul staining of hepatocytes to the whole lobule. Scale bars = 200 μm. Figure 5—figure supplement 1—source data 1. Liver to body weight ratio .

Article Snippet: Sequence-based reagent , Arid1a (total) , Thermo Fisher Scientific , Taqman Assay Mm00473838_m1 , qPCR primers Mus musculus.

Techniques: Immunostaining, Staining

Journal: eLife

Article Title: ARID1A loss in adult hepatocytes activates β-catenin-mediated erythropoietin transcription

doi: 10.7554/eLife.53550

Figure Lengend Snippet: ( a ) No invalidation of Apc and Arid1a genes was found in NPC from [ Apc-Arid1a ] ko-TOTAL mice (Student t-test). ( b ) In vitro analysis of Axin2 , Arid1a, and Epo transcription by RT-qPCR in primary culture hepatocytes after siRNA-mediated knockdown of Arid1a (siArid1a, 20 nM) and Wnt3a and R-Spondin3 stimulation (Wnt/RSpo) relative to that of control hepatocytes. ( c ) In vitro expression of Axin2 , Arid1a, and Epo in isolated [ Apc ] ko-TOTAL hepatocytes after siArid1a. ( d ) In vitro expression of Apc , Arid1a , and Epo in the HEPA1.6 β-catenin-mutated hepatoma murine cell line after siRNA-mediated knockdown of Arid1a and/or β-catenin. ( e,f ) Western blot of Arid1a and beta-catenin demonstrating effective siRNA-mediated knockdown of Arid1a (20 nM) and Wnt/Spondin stimulation in primary culture hepatocytes ( e ) and effective inactivation of Apc and/or Arid1a in vivo ( f ). Data are presented as the mean ± SEM and analyzed with one-way ANOVA. ****p<0.0001. Cell culture data are representative of three independent experiments carried out in triplicate. Figure 5—figure supplement 2—source data 1. Efficiency of gene invalidation , mRNA expression , western blots .

Article Snippet: Sequence-based reagent , Arid1a (total) , Thermo Fisher Scientific , Taqman Assay Mm00473838_m1 , qPCR primers Mus musculus.

Techniques: In Vitro, Quantitative RT-PCR, Knockdown, Control, Expressing, Isolation, Western Blot, In Vivo, Cell Culture

Journal: eLife

Article Title: ARID1A loss in adult hepatocytes activates β-catenin-mediated erythropoietin transcription

doi: 10.7554/eLife.53550

Figure Lengend Snippet: ( a ) Seven months after Apc/Arid1a gene invalidation in single hepatocytes from two livers (#1 and #2); ( b ) 7 days after gene invalidation in more than 90% hepatocytes (two livers: #a and #b). Axin2 RNAScope probe stains β-catenin-activated hepatocytes (blue dots), and Epo RNAScope probe stains single Epo mRNAs as red dots. Related data are found in .

Article Snippet: Sequence-based reagent , Arid1a (total) , Thermo Fisher Scientific , Taqman Assay Mm00473838_m1 , qPCR primers Mus musculus.

Techniques: RNAscope

Journal: eLife

Article Title: ARID1A loss in adult hepatocytes activates β-catenin-mediated erythropoietin transcription

doi: 10.7554/eLife.53550

Figure Lengend Snippet: ( a ) Genomic environment of the Epo gene (UCSC Genome Browser, mm9 database) and ChIP-seq peaks at the 3’ Epo enhancer. In blue/red: the crude reads of ChIP-Seq data performed in adult livers against HNF-4a (54). In black: ChIP-Seq under Apc ko or βcat ko conditions with an antibody against TCF4 (16). In yellow: ENCODE data of H3K27Ac marks in eight-week-old and E14.5 embryonic livers (Histone Mods by ChIP-Seq from ENCODE/LICR). ( b ) Schematic representation of the EpoE-Luc erythropoietin luciferase reporter, driven by the 3’ enhancer. ( c–e ) Luciferase reporter assays in mouse primary hepatocytes: ( c ) after in vitro overactivation of Wnt/β-catenin signaling and Arid1a knockdown ( d ) after in vivo Cre-loxP-mediated gene inactivation; ( e ) Effect of hypoxic-mimic conditions using desferrioxamine (DFO), and effect of knockdown of HIF factors (two separate experiments carried out in triplicate). Results are in relative light units, and analyzed using 1-way ( d ) or 2-way ANOVA ( c,e ). ****p<0.0001. Related data are found in – , and source data in ‘ ; ; ’. Figure 7—source data 1. EpoE-luc luciferase relative activity .

Article Snippet: Sequence-based reagent , Arid1a (total) , Thermo Fisher Scientific , Taqman Assay Mm00473838_m1 , qPCR primers Mus musculus.

Techniques: ChIP-sequencing, Luciferase, In Vitro, Knockdown, In Vivo, Activity Assay

Journal: eLife

Article Title: ARID1A loss in adult hepatocytes activates β-catenin-mediated erythropoietin transcription

doi: 10.7554/eLife.53550

Figure Lengend Snippet: ( a ) Immunodetection of hepatic hypoxia seven days after Apc and/or Arid1a loss in all hepatocytes in mouse liver. For hypoxia detection in tissues, mice were injected with Hypoxyprobe (NPI Inc) solution via intraperitoneal injection (60 mg/100 g body weight), and livers harvested 1 hr after. Paraffin sections were processed according to the manufacturer’s instructions (Hypoxyprobe-1 Kit). Note the physiological staining surrounding the centrilobular vein (cv) in all conditions. No pathological and extended hypoxia was detected. Scale bars = 200 μm. ( b,c ) Western blots of Arid1a, Glul, Hif2α, and actin ( b ), and Hif2α detection quantification (n = 2 for each genotype) ( c ) showing no Hif2α stabilization in either condition. ( d ) RT-qPCR analysis of Hif1α and Hif2α target gene expression in livers of 2-month-old mice, 1 week after panlobular Apc and Arid1a invalidation. WT (n = 8), [ Apc ] ko-TOTAL (n = 8), [ Arid1a ] ko-TOTAL (n = 7), and [ Apc-Arid1a ] ko-TOTAL (n = 8) mice. One-way ANOVA tests were done. ns = non significant. Figure 7—figure supplement 1—source data 1. Quantification of western blots and mRNA expression .

Article Snippet: Sequence-based reagent , Arid1a (total) , Thermo Fisher Scientific , Taqman Assay Mm00473838_m1 , qPCR primers Mus musculus.

Techniques: Immunodetection, Injection, Staining, Western Blot, Quantitative RT-PCR, Targeted Gene Expression, Expressing

Journal: eLife

Article Title: ARID1A loss in adult hepatocytes activates β-catenin-mediated erythropoietin transcription

doi: 10.7554/eLife.53550

Figure Lengend Snippet: ( a ) RT-qPCR analysis of Hif1α and Hif2α expression in primary culture hepatocytes treated or not with desferrioxamine (DFO) and after siRNA mediated knockdown of Hif1α (20 nM) and/or Hif2α (20 nM). ( b ) Western blots of Hif1α, Hif2α, and actin showing that Hif1α and Hif2α were stabilized in presence of DFO and that siRNA-mediated knockdown against Hif1α, Hif2α were effective. ( c,d ) RT-qPCR analysis of Hif1α ( c ) and Hif2α ( d ) in primary culture hepatocytes from livers of 2-month-old mice. Experiments were performed 1 week after panlobular inactivation of Apc and/or Arid1a and 48 hr after siRNA mediated knockdown of Hif1α (20 nM) and/or Hif2α (20 nM). ( e ) Western blots of Arid1a, Hif1α, Hif2α, Glul and Actin showing that no increase of Hif1α and Hif2α was detected in primary culture hepatocytes from [ Apc-Arid1a ] ko-TOTAL mice compared to control ones. 1,2: WT; 3,4: [ Arid1a ] ko-TOTAL ; 5,6: [ Apc ] ko-TOTAL ; 7,8: [ Apc-Arid1a ] ko-TOTAL . Data are presented as the mean ± SEM and analyzed by one-way ANOVA ****p<0.0001. Cell culture data are representative of two independent experiments, each performed in technical triplicate. Figure 7—figure supplement 2—source data 1. mRNA expressions and western blots .

Article Snippet: Sequence-based reagent , Arid1a (total) , Thermo Fisher Scientific , Taqman Assay Mm00473838_m1 , qPCR primers Mus musculus.

Techniques: Quantitative RT-PCR, Expressing, Knockdown, Western Blot, Control, Cell Culture

Journal: eLife

Article Title: ARID1A loss in adult hepatocytes activates β-catenin-mediated erythropoietin transcription

doi: 10.7554/eLife.53550

Figure Lengend Snippet: ( a ) EMSA using nuclear proteic extracts from WT or [ Apc ] ko-TOTAL livers and 32 P-labeled probes containing Epo-HRE (DR2). ( b, c ) Competitive EMSA using 32 P-labeled DR2 ( b ) and 32 P-labeled WRE ( c ) probes and increasing concentrations of cold probes containing HNF4, WRE or control-responsive element. WRE cold probes compete with radiolabeled DR2 motif for the Tcf4/β-catenin binding and vice versa. ( d, e ) Chromatin ImmunoPrecipitation (ChIP) assays of hepatocytes from WT, [ Apc ] ko-TOTAL , [ Arid1a ] ko-TOTAL , and [ Apc-Arid1a ] ko-TOTAL livers. ChIP-qPCR against IgG, Tcf4, Acetylation of Histone3 in Lysine27 (H3K27Ac), and Tri-methylation of Histone3 in Lysine27 (H3K27me3) for Axin2 ( d ) and Epo ( e ) enhancer regions. WT (n = 3), [ Apc ] ko-TOTAL (n = 2), [ Arid1a ] ko-TOTAL (n = 2), and [ Apc-Arid1a ] ko-TOTAL (n = 3) mice. Enrichment by ChIP was assessed relative to the input DNA and normalized to the level of negative controls. ( f ) ATAC-qPCR using frozen livers from WT (n = 7), [ Apc ] ko-TOTAL (n = 7), [ Arid1a ] ko-TOTAL (n = 6), and [ Apc-Arid1a ] ko-TOTAL (n = 7) mice. Data are analyzed with one-way ANOVA. ****p<0.0001. Related data are found in – , and source data in ‘ ; ; ’. Figure 8—source data 1. EMSA , ChIP-qPCR and ATAC-qPCR data.

Article Snippet: Sequence-based reagent , Arid1a (total) , Thermo Fisher Scientific , Taqman Assay Mm00473838_m1 , qPCR primers Mus musculus.

Techniques: Labeling, Control, Binding Assay, Chromatin Immunoprecipitation, ChIP-qPCR, Methylation

Journal: eLife

Article Title: ARID1A loss in adult hepatocytes activates β-catenin-mediated erythropoietin transcription

doi: 10.7554/eLife.53550

Figure Lengend Snippet: RT-qPCR analysis of GS and Axin2 expression in hepatocytes from the livers of two-month-old mice, one week after panlobular Apc and Arid1a invalidation. WT (n = 3), [ Apc ] ko-TOTAL (n = 2), [ Arid1a ] ko-TOTAL (n = 2), and [ Apc- Arid1a ]ko -TOTAL (n = 3) mice. Data are presented as the mean + SEM and analyzed with one-way ANOVA. Figure 8—figure supplement 1—source data 1. mRNA expression .

Article Snippet: Sequence-based reagent , Arid1a (total) , Thermo Fisher Scientific , Taqman Assay Mm00473838_m1 , qPCR primers Mus musculus.

Techniques: Quantitative RT-PCR, Expressing

Journal: eLife

Article Title: ARID1A loss in adult hepatocytes activates β-catenin-mediated erythropoietin transcription

doi: 10.7554/eLife.53550

Figure Lengend Snippet: Under physiological conditions, the presence of Arid1a is associated with histone repressive marks at the Epo enhancer and β-catenin is constantly degraded; thus, Epo is not produced. In the absence of Apc, β-catenin/Tcf4 complex binds the Epo enhancer, and enhances chromatin accessibility, but the histone marks remain repressive. The loss of Arid1a increases active histone marks, which is insufficient to induce Epo transcription. After both Wnt/β-catenin activation and Arid1a inactivation, active histone marks and binding of β-catenin/Tcf4 to the Epo enhancer drive Epo liver transcription, and subsequent secretion of Epo into the bloodstream, resulting in splenic erythropoiesis and in substantial blood and liver erythrocytosis.

Article Snippet: Sequence-based reagent , Arid1a (total) , Thermo Fisher Scientific , Taqman Assay Mm00473838_m1 , qPCR primers Mus musculus.

Techniques: Produced, Activation Assay, Binding Assay

Journal: eLife

Article Title: ARID1A loss in adult hepatocytes activates β-catenin-mediated erythropoietin transcription

doi: 10.7554/eLife.53550

Figure Lengend Snippet:

Article Snippet: Sequence-based reagent , Arid1a (total) , Thermo Fisher Scientific , Taqman Assay Mm00473838_m1 , qPCR primers Mus musculus.

Techniques: Transfection, Plasmid Preparation, Sequencing, TaqMan Assay, Binding Assay, Derivative Assay, Control, Negative Control

Journal: Redox Biology

Article Title: Inhibition of NLRP3-mediated crosstalk between hepatocytes and liver macrophages by geniposidic acid alleviates cholestatic liver inflammatory injury

doi: 10.1016/j.redox.2022.102404

Figure Lengend Snippet: GPA directly binds to NLRP3 and represses its expression. (A) Dual-luciferase reporter assay results. RAW264.7 cells were transiently co‐transfected with NLRP3-luc plasmids for 12 h and treated with DMSO (vehicle control), GPA-H (100 μM), GPA-M (50 μM), GPA-L (25 μM), or MCC950 (10 μM) for 24 h. Relative luciferase activity was calculated by ration of firefly luciferase/renilla luciferase activity (n = 5). * P < 0.05, ** P < 0.01, compared to the DMSO control transfected with empty vector. Data are expressed as the mean ± SEM of 5 independent experiments ( n = 5). (B) mRNA levels of NLRP3, NFκB1, and IL-1β in THP-1 monocyte-derived macrophages and BMDM as analyzed by real-time PCR after 24 h treatment with different concentrations of GPA (25–100 μM). Results are expression as fold changes compared to the DMSO group. Data are expressed as the mean ± SEM of 5 independent experiments ( n = 5). * P < 0.05, ** P < 0.01 versus DMSO control group. (C) Protein levels of NLRP3, ASC, CASP-1, and IL-1β in THP-1 monocyte-derived macrophages were determined by Western blot analysis after 24 h treatment with different concentrations of GPA. β-ACTIN was used as internal reference protein. Data are expressed as the mean ± SEM of 3 independent experiments( n = 3). (D) Cell lysates of LPS-primed THP-1 monocytes were incubated with GPA (100 μM) or Bio-GPA (100 μM) for 4 h and subjected to pull-down assays with streptavidin beads. Total (input), bound (pull-down), and remaining proteins (after pull-down) were immunoblotted as indicated. β-ACTIN was used as internal reference protein.Data are expressed as the mean ± SEM of 3–4 independent experiments ( n = 3–4). (E) Competitive experiment. Cell lysates of LPS-primed THP-1 monocytes were incubated with Bio-GPA (100 μM) and different concentrations of free GPA (50 or 100 μM) and subjected to pull-down assays with streptavidin beads. β-ACTIN was used as internal reference protein. Data are expressed as the mean ± SEM of 3–4 independent experiments ( n = 3–4). (F) Human recombinant NLRP3 proteins were incubated with the indicated doses of Bio-GPA (50 or 100 μM) and subjected to pull-down assays with streptavidin beads. NLRP3 was used as internal reference protein. Data are expressed as the mean ± SEM of 3–4 independent experiments ( n = 3–4). (G) Human recombinant NLRP3 proteins were incubated with Bio-GPA (100 μM) or/and β-Mercaptoethanol (50 μM) and subjected to pull-down assays with streptavidin beads. NLRP3 was used as internal reference protein. Data are expressed as the mean ± SEM of 3–4 independent repeats ( n = 3–4).

Article Snippet: TaqMan probes, including those for human-specific NLRP3 (Hs00918082), NF κ B1 (Hs00765730), IL-1 β (Hs00174097), IL-6 (Hs00174131 ) , ICAM-1 (Hs00164932), COX-2 (Hs00153133), TNF- α (Hs00174128), and GAPDH (Hs02786624); and mouse-specific Nlrp3 (Mm00840904), Nf κ b1 (Mm00476361), Il-1 β (Mm00434228), Il-6 (Mm00446190 ) , Icam-1 (Mm00516023), Cox-2 (Mm00478374), Tnf- α (Mm00443258), Cyp7a1 (Mm00484150), Cyp8b1 (Mm00501637), Cyp27a1 (Mm00470430), Cyp2b10 (Mm01972453), Cyp3a11 (Mm00731567), Ugt1a1 (Mm02603337), Abcb11 (Mm00445168), Abcc2 (Mm00496899), Abcc3 (Mm00551550), Abcc4 (Mm01226381), Slc01b2 (Mm00451510), Slc10a1 (Mm00441421), and Gapdh (Mm99999915), were acquired from Invitrogen (Carlsbad, CA, USA).

Techniques: Expressing, Luciferase, Reporter Assay, Transfection, Control, Activity Assay, Plasmid Preparation, Derivative Assay, Real-time Polymerase Chain Reaction, Western Blot, Incubation, Recombinant

Journal: Redox Biology

Article Title: Inhibition of NLRP3-mediated crosstalk between hepatocytes and liver macrophages by geniposidic acid alleviates cholestatic liver inflammatory injury

doi: 10.1016/j.redox.2022.102404

Figure Lengend Snippet: GPA reduces LPS-induced NLRP3 expression via inhibiting NF-κB signaling. (A – C) THP-1 monocytes were pretreated with the indicated concentrations of GPA for 24 h and stimulated with LPS (1 μg/ml) for 24 h. The levels of mRNA (A) , protein (B) , and pro-inflammatory cytokines in supernatants (C) were measured by real-time PCR, Western blot, and ELISA, respectively. mRNA results are expressed as fold changes compared to the DMSO group.β-ACTIN was used as internal reference protein. Data are expressed as the mean ± SEM of 3–5 independent experiments ( n = 5, A and C; n = 3, B). # P < 0.05, ## P < 0.01 versus DMSO-treated control group, * P < 0.05, ** P < 0.01 versus LPS-treated group. (D – E) BMDM were pretreated with indicated concentrations of GPA for 24 h and stimulated with LPS (1 μg/ml) for 24 h. The levels of mRNA (D) and pro-inflammatory cytokines in supernatants (E) were determined by real-time PCR and ELISA, respectively. mRNA results are expressed as fold changes compared to the DMSO group. Data are expressed as the mean ± SEM of 5 independent experiments ( n = 5). # P < 0.05, ## P < 0.01 versus DMSO-treated control group, * P < 0.05, ** P < 0.01 versus LPS-treated group. (F and G) Nlrp3 −/− BMDM were pretreated with indicated concentrations of GPA for 24 h and stimulated with LPS (1 μg/ml) for 24 h. The levels of mRNA (F) and pro-inflammatory cytokines in supernatants (G) were determined by real-time PCR and ELISA, respectively. mRNA results are expressed as fold changes compared to the DMSO group. Data are expressed as the mean ± SEM of 5 independent experiments ( n = 5). # P < 0.05, ## P < 0.01 versus DMSO-treated control group, * P < 0.05, ** P < 0.01 versus LPS-treated group. (H) Dual-luciferase reporter assay results. RAW274.6 cells were transiently co‐transfected with NF-κB-luc plasmids for 12 h and treated with DMSO (vehicle control), GPA-H (100 μM), GPA-M (50 μM), GPA-L (25 μM), or MCC950 (10 μM) for 12 h, followed by 24 h induction with LPS (1 μg/ml). Data are expressed as the mean ± SEM of 5 independent experiments ( n = 5). (I and J) The protein expression levels of NF-κB (I), NLRP3 and ASC (J) in THP-1 monocytes were measured by immunofluorescence assay. The nuclei were stained with DAPI in blue, and targeted protein was stained in green or red (magnification: 630 × ). (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)

Article Snippet: TaqMan probes, including those for human-specific NLRP3 (Hs00918082), NF κ B1 (Hs00765730), IL-1 β (Hs00174097), IL-6 (Hs00174131 ) , ICAM-1 (Hs00164932), COX-2 (Hs00153133), TNF- α (Hs00174128), and GAPDH (Hs02786624); and mouse-specific Nlrp3 (Mm00840904), Nf κ b1 (Mm00476361), Il-1 β (Mm00434228), Il-6 (Mm00446190 ) , Icam-1 (Mm00516023), Cox-2 (Mm00478374), Tnf- α (Mm00443258), Cyp7a1 (Mm00484150), Cyp8b1 (Mm00501637), Cyp27a1 (Mm00470430), Cyp2b10 (Mm01972453), Cyp3a11 (Mm00731567), Ugt1a1 (Mm02603337), Abcb11 (Mm00445168), Abcc2 (Mm00496899), Abcc3 (Mm00551550), Abcc4 (Mm01226381), Slc01b2 (Mm00451510), Slc10a1 (Mm00441421), and Gapdh (Mm99999915), were acquired from Invitrogen (Carlsbad, CA, USA).

Techniques: Expressing, Real-time Polymerase Chain Reaction, Western Blot, Enzyme-linked Immunosorbent Assay, Control, Luciferase, Reporter Assay, Transfection, Immunofluorescence, Staining

Journal: Redox Biology

Article Title: Inhibition of NLRP3-mediated crosstalk between hepatocytes and liver macrophages by geniposidic acid alleviates cholestatic liver inflammatory injury

doi: 10.1016/j.redox.2022.102404

Figure Lengend Snippet: GPA suppresses NLRP3 inflammasome activation. (A – C) THP-1 monocytes were pretreated with indicated concentrations of GPA for 24 h, stimulated with LPS (1 μg/ml) for 24 h and ATP (2.5 mM) for 30 min. (A) The levels of pro-inflammatory cytokines in cell-culture supernatants, including IL-1β, IL-6, and TNF-α were quantified by ELISA. Data are expressed as the mean ± SEM of 5 independent experiments ( n = 5). The levels of protein (B) and mRNA (C) were determined by Western blot analysis and real-time PCR, respectively. mRNA results are expressed as fold changes compared to the DMSO group. β-ACTIN was used as internal reference protein. Data are expressed as the mean ± SEM of 3–5 independent experiments ( n = 3–4, B; n = 5, C). # P < 0.05, ## P < 0.01 versus DMSO-treated control group, * P < 0.05, ** P < 0.01 versus LPS + ATP-treated group. (D) The NLRP3-ASC interaction in THP-1 monocytes were measured by immuno-fluorescence assay. The nuclei were stained with DAPI in blue, and targeted protein was stained in green or red (magnification: 630 × ×). (E) ROS levels were detected using an ROS fluorescent probe in THP-1 monocytes (magnification: 400 × ). (F and G) Intracellular potassium (F) and ionic calcium (G) levels in the cell lysates of THP-1 monocytes. Data are expressed as the mean ± SEM of 5 independent experiments ( n = 5). # P < 0.05, ## P < 0.01 versus DMSO-treated control group, * P < 0.05, ** P < 0.01 versus LPS + ATP-treated group. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)

Article Snippet: TaqMan probes, including those for human-specific NLRP3 (Hs00918082), NF κ B1 (Hs00765730), IL-1 β (Hs00174097), IL-6 (Hs00174131 ) , ICAM-1 (Hs00164932), COX-2 (Hs00153133), TNF- α (Hs00174128), and GAPDH (Hs02786624); and mouse-specific Nlrp3 (Mm00840904), Nf κ b1 (Mm00476361), Il-1 β (Mm00434228), Il-6 (Mm00446190 ) , Icam-1 (Mm00516023), Cox-2 (Mm00478374), Tnf- α (Mm00443258), Cyp7a1 (Mm00484150), Cyp8b1 (Mm00501637), Cyp27a1 (Mm00470430), Cyp2b10 (Mm01972453), Cyp3a11 (Mm00731567), Ugt1a1 (Mm02603337), Abcb11 (Mm00445168), Abcc2 (Mm00496899), Abcc3 (Mm00551550), Abcc4 (Mm01226381), Slc01b2 (Mm00451510), Slc10a1 (Mm00441421), and Gapdh (Mm99999915), were acquired from Invitrogen (Carlsbad, CA, USA).

Techniques: Activation Assay, Cell Culture, Enzyme-linked Immunosorbent Assay, Western Blot, Real-time Polymerase Chain Reaction, Control, Fluorescence, Staining

Journal: Redox Biology

Article Title: Inhibition of NLRP3-mediated crosstalk between hepatocytes and liver macrophages by geniposidic acid alleviates cholestatic liver inflammatory injury

doi: 10.1016/j.redox.2022.102404

Figure Lengend Snippet: GPA protects against ANIT-induced acute liver inflammation by inhibiting NLRP3 inflammasome activation. C57BL/6 mice were orally administered with the vehicle (0.5% carboxymethylcellulose) as control group, UDCA (75 mg/kg) as positive control, GPA-H (100 mg/kg), GPA-M (50 mg/kg), or GPA-L (25 mg/kg) daily for seven days. On day 5, they were orally administered with ANIT (100 mg/kg). (A) Representative FACS image of mouse liver sample. After sacrificing the animals, liver macrophage were isolated, and the inflammatory infiltration was evaluated by FACS (n = 5). Data are expressed as the mean ± SEM of 5 independent mice liver samples ( n = 5). # P < 0.05, ## P < 0.01 versus vehicle-treated control group, * P < 0.05, ** P < 0.01 versus ANIT-treated group. (B) Hepatic F4/80 and CD68 protein expression measured by immunofluorescence assay. The nuclei were stained with DAPI in blue, and targeted protein was stained in green (F4/80) or red (CD68) (magnification: 630 × ). (C) Levels of pro-inflammatory cytokines in mouse liver, including Il-1β, Il-6, and Tnf-α were detected by ELISA. Data are expressed as the mean ± SEM of 5–6 independent mouse samples ( n = 5–6). # P < 0.05, ## P < 0.01 versus vehicle-treated control group, * P < 0.05, ** P < 0.01 versus ANIT-treated group. (D) Changes in spleen weight index (ratio of spleen weight to body weight) in mice with ANIT-induced cholestasis. Data are expressed as the mean ± SEM of 5 independent mouse samples ( n = 5). (E) mRNAs levels of NLRP3 and other proinflammatory genes in mouse liver were analyzed by real-time PCR. Data are expressed as the mean ± SEM of 5 independent mouse samples ( n = 5). Results are expressed as fold changes compared to the vehicle-treated control group. # P < 0.05, ## P < 0.01 versus vehicle-treated control group, * P < 0.05, ** P < 0.01 versus ANIT-treated group. (F) Levels of NLRP3 and NF-κB signaling proteins in liver lysates were analyzed by western blotting. β-ACTIN was used as internal reference protein. Data are expressed as the mean ± SEM of 3–4 independent mouse samples ( n = 3–4). # P < 0.05, ## P < 0.01 versus vehicle-treated control group, * P < 0.05, ** P < 0.01 versus ANIT-treated group. (G) ANIT-induced hepatic ROS levels were decreased after GPA treatment ( n = 3). # P < 0.05, ## P < 0.01 versus vehicle-treated control group, * P < 0.05, ** P < 0.01 versus ANIT-treated group. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)

Article Snippet: TaqMan probes, including those for human-specific NLRP3 (Hs00918082), NF κ B1 (Hs00765730), IL-1 β (Hs00174097), IL-6 (Hs00174131 ) , ICAM-1 (Hs00164932), COX-2 (Hs00153133), TNF- α (Hs00174128), and GAPDH (Hs02786624); and mouse-specific Nlrp3 (Mm00840904), Nf κ b1 (Mm00476361), Il-1 β (Mm00434228), Il-6 (Mm00446190 ) , Icam-1 (Mm00516023), Cox-2 (Mm00478374), Tnf- α (Mm00443258), Cyp7a1 (Mm00484150), Cyp8b1 (Mm00501637), Cyp27a1 (Mm00470430), Cyp2b10 (Mm01972453), Cyp3a11 (Mm00731567), Ugt1a1 (Mm02603337), Abcb11 (Mm00445168), Abcc2 (Mm00496899), Abcc3 (Mm00551550), Abcc4 (Mm01226381), Slc01b2 (Mm00451510), Slc10a1 (Mm00441421), and Gapdh (Mm99999915), were acquired from Invitrogen (Carlsbad, CA, USA).

Techniques: Activation Assay, Control, Positive Control, Isolation, Expressing, Immunofluorescence, Staining, Enzyme-linked Immunosorbent Assay, Real-time Polymerase Chain Reaction, Western Blot

Journal: Redox Biology

Article Title: Inhibition of NLRP3-mediated crosstalk between hepatocytes and liver macrophages by geniposidic acid alleviates cholestatic liver inflammatory injury

doi: 10.1016/j.redox.2022.102404

Figure Lengend Snippet: GPA reduces BA-induced cellular inflammation by blocking the activation of NLRP3 inflammasome in hepatocytes and macrophages. (A) Wild-type primary (WT) mouse hepatocytes (PMHs) were pretreated with GPA for 12 h and treated with 200 μM TCA for 24 h. Levels of NLRP3 and proinflammatory mRNAs were determined by real-time PCR. Results are expression as fold changes compared to the DMSO group. Data are expressed as the mean ± SEM of 5 independent experiments ( n = 5). (B) Levels of Il-1β and Il-6 in cell-culture supernatants were quantified by ELISA. Data are expressed as the mean ± SEM of 5 independent experiments ( n = 5). # P < 0.05, ## P < 0.01 versus DMSO-treated control group, * P < 0.05, ** P < 0.01 versus TCA-treated group. Primary mouse hepatocytes with Nlrp3 -knockout ( Nlrp3 −/− PMHs) were pretreated with GPA for 12 h and treated with 200 μM TCA for 24 h. Levels of Nfκb1, Il-1β , and Tnf-α mRNA (C) and Il-1β and Tnf-α in cell-culture supernatants (D) were determined by real-time PCR and ELISA, respectively. mRNA results are expression as fold changes compared to the DMSO group. Data are expressed as the mean ± SEM of 5 independent experiments ( n = 5). # P < 0.05, ## P < 0.01 versus DMSO-treated control group, * P < 0.05, ** P < 0.01 versus TCA-treated group. PMHs and BMDM were isolated from WT and Nlrp3 −/− mice and cultivated on a conditioned medium (CM)-associated culture. (E – H) WT BMDM were exposed for 24 h to the CM derived from WT-PMHs(E and F) and Nlrp3 −/− PMHs(G and H) that were pretreated with or without GPA for 24 h and TCA for another 24 h. Levels of NLRP3 and proinflammatory mRNAs (E and G) and IL-1β (F and H) in cell-culture supernatants were determined by real-time PCR and ELISA, respectively. mRNA results are expressed as fold changes compared to the DMSO group. Data are expressed as the mean ± SEM of 5 independent experiments ( n = 5). # P < 0.05, ## P < 0.01 versus DMSO-treated control group, * P < 0.05, ** P < 0.01 versus TCA-treated group. (I – J) BMDM were pretreated with GPA for 12 h and treated with 200 μM TCA for 24 h. Levels of NLRP3 and proinflammatory mRNAs and pro-inflammatory cytokines in cell-culture supernatants were quantified by real-time PCR (I) and ELISA analysis (J), respectively. mRNA results are expressed as fold changes compared to the DMSO group. Data are expressed as the mean ± SEM of 5 independent experiments ( n = 5). # P < 0.05, ## P < 0.01 versus DMSO-treated control group, * P < 0.05, ** P < 0.01 versus TCA-treated group. (K–N) WT PMHs were exposed for 24 h to the CM derived from WT-BMDM (K and L) and Nlrp3 −/− (M and N) BMDM that were pretreated with or without GPA for 24 h, followed by TCA for 24 h. Levels of NLRP3 and proinflammatory mRNAs (K and M) and IL-1β (L and N) in cell-culture supernatants were determined by real-time PCR and ELISA analysis, respectively. mRNA results are expressed as fold changes compared to the DMSO group. Data are expressed as the mean ± SEM of 5 independent experiments ( n = 5). # P < 0.05, ## P < 0.01 versus DMSO-treated control group, * P < 0.05, ** P < 0.01 versus TCA-treated group.

Article Snippet: TaqMan probes, including those for human-specific NLRP3 (Hs00918082), NF κ B1 (Hs00765730), IL-1 β (Hs00174097), IL-6 (Hs00174131 ) , ICAM-1 (Hs00164932), COX-2 (Hs00153133), TNF- α (Hs00174128), and GAPDH (Hs02786624); and mouse-specific Nlrp3 (Mm00840904), Nf κ b1 (Mm00476361), Il-1 β (Mm00434228), Il-6 (Mm00446190 ) , Icam-1 (Mm00516023), Cox-2 (Mm00478374), Tnf- α (Mm00443258), Cyp7a1 (Mm00484150), Cyp8b1 (Mm00501637), Cyp27a1 (Mm00470430), Cyp2b10 (Mm01972453), Cyp3a11 (Mm00731567), Ugt1a1 (Mm02603337), Abcb11 (Mm00445168), Abcc2 (Mm00496899), Abcc3 (Mm00551550), Abcc4 (Mm01226381), Slc01b2 (Mm00451510), Slc10a1 (Mm00441421), and Gapdh (Mm99999915), were acquired from Invitrogen (Carlsbad, CA, USA).

Techniques: Blocking Assay, Activation Assay, Real-time Polymerase Chain Reaction, Expressing, Cell Culture, Enzyme-linked Immunosorbent Assay, Control, Knock-Out, Isolation, Derivative Assay

Journal: Redox Biology

Article Title: Inhibition of NLRP3-mediated crosstalk between hepatocytes and liver macrophages by geniposidic acid alleviates cholestatic liver inflammatory injury

doi: 10.1016/j.redox.2022.102404

Figure Lengend Snippet: Anti-inflammatory effect of GPA against ANIT‐induced acute liver injury is largely abolished in Nlrp3 -knockout mice. WT mice and Nlrp3 −/− mice were orally administered with the vehicle (0.5% carboxymethylcellulose), UDCA (75 mg/kg) as positive control, or GPA-H (100 mg/kg) once daily for seven days and with ANIT (100 mg/kg) on day 5. (A) Representative FACS image of mouse liver sample. After sacrificing the animals, liver macrophage were isolated, and the inflammatory infiltration was evaluated by FACS (n = 5). Data are expressed as the mean ± SEM of 5 independent mice liver samples ( n = 5). # P < 0.05, ## P < 0.01 versus vehicle-treated control group in WT and Nlrp3 −/− mice; * P < 0.05, ** P < 0.01 compared to ANIT-treated alone group. (B) Hepatic F4/80 and CD68 protein expression measured by immunofluorescence assay. The nuclei were stained with DAPI in blue, and targeted protein was stained in red (F4/80) or green (CD68) (magnification: 630 × ).Data are expressed as the mean ± SEM of 3 independent mice liver samples ( n = 3). # P < 0.05, ## P < 0.01 versus vehicle-treated control group in WT and Nlrp3 −/− mice; * P < 0.05, ** P < 0.01 compared to ANIT-treated alone group. (C) Levels of NLRP3 and proinflammatory mRNAs in mouse liver were determined by real-time PCR. Data are expressed as the mean ± SEM of 5 independent mouse samples ( n = 5). Results are expressed as fold changes compared to the vehicle-treated control group. # P < 0.05, ## P < 0.01 versus vehicle-treated control group in WT and Nlrp3 −/− mice; * P < 0.05, ** P < 0.01 compared to ANIT-treated alone group. (D) Levels of inflammatory cytokines including Il-1β, Il-6, and Tnf-α in mouse liver were detected by ELISA. Data are expressed as the mean ± SEM of 5 independent mouse samples ( n = 5). # P < 0.05, ## P < 0.01 versus vehicle-treated control group, * P < 0.05, ** P < 0.01 versus ANIT-treated group. (E) Effects of GPA treatment on spleen weight index (ratio of spleen weight to body weight) of ANIT-induced mice. Data are expressed as the mean ± SEM of 5 independent mouse samples ( n = 5). Results are expressed as fold changes compared to the vehicle-treated control group. # P < 0.05, ## P < 0.01 versus vehicle-treated control group in WT and Nlrp3 −/− mice; * P < 0.05, ** P < 0.01 compared to ANIT-treated alone group. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)

Article Snippet: TaqMan probes, including those for human-specific NLRP3 (Hs00918082), NF κ B1 (Hs00765730), IL-1 β (Hs00174097), IL-6 (Hs00174131 ) , ICAM-1 (Hs00164932), COX-2 (Hs00153133), TNF- α (Hs00174128), and GAPDH (Hs02786624); and mouse-specific Nlrp3 (Mm00840904), Nf κ b1 (Mm00476361), Il-1 β (Mm00434228), Il-6 (Mm00446190 ) , Icam-1 (Mm00516023), Cox-2 (Mm00478374), Tnf- α (Mm00443258), Cyp7a1 (Mm00484150), Cyp8b1 (Mm00501637), Cyp27a1 (Mm00470430), Cyp2b10 (Mm01972453), Cyp3a11 (Mm00731567), Ugt1a1 (Mm02603337), Abcb11 (Mm00445168), Abcc2 (Mm00496899), Abcc3 (Mm00551550), Abcc4 (Mm01226381), Slc01b2 (Mm00451510), Slc10a1 (Mm00441421), and Gapdh (Mm99999915), were acquired from Invitrogen (Carlsbad, CA, USA).

Techniques: Knock-Out, Positive Control, Isolation, Control, Expressing, Immunofluorescence, Staining, Real-time Polymerase Chain Reaction, Enzyme-linked Immunosorbent Assay

Journal: Redox Biology

Article Title: Inhibition of NLRP3-mediated crosstalk between hepatocytes and liver macrophages by geniposidic acid alleviates cholestatic liver inflammatory injury

doi: 10.1016/j.redox.2022.102404

Figure Lengend Snippet: Hepatoprotective effect of GPA against ANIT‐induced acute liver injury is partly mitigated in Nlrp3 -knockout mice. (A) Serum biochemical indexes of cholestatic liver injury including ALT, AST, TBA and TBIL levels were evaluated. Data are expressed as the mean ± SEM from 5 to 6 independent mice samples (n = 5–6). # P < 0.05, ## P < 0.01 versus vehicle-treated control group in WT and Nlrp3 −/− mice; * P < 0.05, ** P < 0.01 compared to ANIT-treated alone group. (B) Representative images of haematoxylin and eosin staining in liver sections (magnification:200 × ). The circle indicated bleeding, inflammatory infiltration and hepatic necrosis.Data are expressed as the mean ± SEM of 5 independent mouse samples ( n = 5). Results are expressed as fold changes compared to the vehicle-treated control group. # P < 0.05, ## P < 0.01 versus vehicle-treated control group in WT and Nlrp3 −/− mice; * P < 0.05, ** P < 0.01 compared to ANIT-treated alone group. (C) Relative mRNA levels of BA synthesis- and transport-related genes in mouse liver were tested by real-time PCR. Data are expressed as the mean ± SEM of 5 independent mouse samples ( n = 5). Results are expressed as fold changes compared to the vehicle-treated control group. # P < 0.05, ## P < 0.01 versus vehicle-treated control group in WT and Nlrp3 −/− mice; * P < 0.05, ** P < 0.01 compared to ANIT-treated alone group.

Article Snippet: TaqMan probes, including those for human-specific NLRP3 (Hs00918082), NF κ B1 (Hs00765730), IL-1 β (Hs00174097), IL-6 (Hs00174131 ) , ICAM-1 (Hs00164932), COX-2 (Hs00153133), TNF- α (Hs00174128), and GAPDH (Hs02786624); and mouse-specific Nlrp3 (Mm00840904), Nf κ b1 (Mm00476361), Il-1 β (Mm00434228), Il-6 (Mm00446190 ) , Icam-1 (Mm00516023), Cox-2 (Mm00478374), Tnf- α (Mm00443258), Cyp7a1 (Mm00484150), Cyp8b1 (Mm00501637), Cyp27a1 (Mm00470430), Cyp2b10 (Mm01972453), Cyp3a11 (Mm00731567), Ugt1a1 (Mm02603337), Abcb11 (Mm00445168), Abcc2 (Mm00496899), Abcc3 (Mm00551550), Abcc4 (Mm01226381), Slc01b2 (Mm00451510), Slc10a1 (Mm00441421), and Gapdh (Mm99999915), were acquired from Invitrogen (Carlsbad, CA, USA).

Techniques: Knock-Out, Control, Staining, Real-time Polymerase Chain Reaction

Journal: Redox Biology

Article Title: Inhibition of NLRP3-mediated crosstalk between hepatocytes and liver macrophages by geniposidic acid alleviates cholestatic liver inflammatory injury

doi: 10.1016/j.redox.2022.102404

Figure Lengend Snippet: Liver-specific overexpression of NLRP3 antagonizes the hepatoprotective effect of GPA against ANIT-induced acute inflammatory liver injury. WT mice were intravenously injected with Adv-Con or Adv-Nlrp3 adenovirus (1 × 10 9 pf) via the tail vein for three days and then orally administered with MCC950 (20 mg/kg) or GPA (100 mg/kg). On day 8, they were orally administered with ANIT(100 mg/kg) to induce acute liver injury. (A) Protein levels of NLRP3 in the liver tissues. Actin was used as internal reference protein. Data are expressed as the mean ± SEM of 3 independent mouse samples ( n = 3). (B) Serum Il-1β and Tnf-α levels were detected by ELISA. Data are expressed as the mean ± SEM of 5 independent mouse samples ( n = 5). # P < 0.05, ## P < 0.01 versus vehicle-treated control group in WT and Nlrp3 oe mice; * P < 0.05, ** P < 0.01 compared to ANIT-treated alone group. (C) Serum biochemical indexes of cholestatic liver injury including ALT, AST, TBA and TBIL levels were evaluated. Data are expressed as the mean ± SE from 5 to 6 independent mice samples (n = 5–6). # P < 0.05, ## P < 0.01 versus vehicle-treated control group in WT and Nlrp3 oe mice; * P < 0.05, ** P < 0.01 compared to ANIT-treated alone group. (D) Representative images of haematoxylin and eosin staining in liver sections (magnification:200 × ). The circle indicated bleeding, inflammatory infiltration and hepatic necrosis. Data are expressed as the mean ± SE from 5 independent mice samples (n = 5). # P < 0.05, ## P < 0.01 versus vehicle-treated control group in WT and Nlrp3 oe mice; * P < 0.05, ** P < 0.01 compared to ANIT-treated alone group.

Article Snippet: TaqMan probes, including those for human-specific NLRP3 (Hs00918082), NF κ B1 (Hs00765730), IL-1 β (Hs00174097), IL-6 (Hs00174131 ) , ICAM-1 (Hs00164932), COX-2 (Hs00153133), TNF- α (Hs00174128), and GAPDH (Hs02786624); and mouse-specific Nlrp3 (Mm00840904), Nf κ b1 (Mm00476361), Il-1 β (Mm00434228), Il-6 (Mm00446190 ) , Icam-1 (Mm00516023), Cox-2 (Mm00478374), Tnf- α (Mm00443258), Cyp7a1 (Mm00484150), Cyp8b1 (Mm00501637), Cyp27a1 (Mm00470430), Cyp2b10 (Mm01972453), Cyp3a11 (Mm00731567), Ugt1a1 (Mm02603337), Abcb11 (Mm00445168), Abcc2 (Mm00496899), Abcc3 (Mm00551550), Abcc4 (Mm01226381), Slc01b2 (Mm00451510), Slc10a1 (Mm00441421), and Gapdh (Mm99999915), were acquired from Invitrogen (Carlsbad, CA, USA).

Techniques: Over Expression, Injection, Enzyme-linked Immunosorbent Assay, Control, Staining

Journal: Pulmonary Circulation

Article Title: Versican accumulates in vascular lesions in pulmonary arterial hypertension

doi: 10.1086/686994

Figure Lengend Snippet: Versican accumulates in the vascular lesions of individuals with pulmonary arterial hypertension (PAH). A, Immunohistochemistry for versican (in brown) in different vascular lesions. B, Representative images of versican staining in lungs from patients with PAH of different etiologies. A monoclonal antiversican antibody (clone 2B1) against the C-terminal G3 domain was used for immunostaining. Images were captured with a ×20 objective. One section per subject was analyzed (n = 7 for idiopathic PAH [IPAH], n = 7 for failed donor lung as control, n = 3 for atrial septal defect with PAH [ASD], and n = 3 for scleroderma with PAH [Scl]). Donor: healthy donor lungs unused for transplantation as controls; Neg: mouse immunoglobulin G used as negative control.

Article Snippet: After incubation with the blocking buffer (10% Aqua Block in Tris-buffered saline [TBS] containing 0.1% Tween-20) for 2 hours at room temperature, membranes were incubated with a goat polyclonal antibody against the human versican βGAG domain (1∶2000, AF 3054, R&D systems) overnight at 4°C in blocking buffer.

Techniques: Immunohistochemistry, Staining, Immunostaining, Control, Transplantation Assay, Negative Control

Journal: Pulmonary Circulation

Article Title: Versican accumulates in vascular lesions in pulmonary arterial hypertension

doi: 10.1086/686994

Figure Lengend Snippet: Western blots confirm increased levels of versican in the lungs of patients with pulmonary arterial hypertension (PAH). Proteoglycan extracts from lung tissue homogenates treated with the chondroitinase ABC were used. For each sample, 1.5 mg of protein was subjected to sodium dodecyl sulfate polyacrylamide gel electrophoresis under reducing conditions, and immunoblotting for versican was performed using two different antibodies, mAb 2B1 for the C-terminal G3 domain (A) and pAb 3054 for the βGAG domain (B). Samples from 4 patients with idiopathic PAH (IPAH) were compared with age-matched unused donor lungs (control). Increased versican fragments (lanes 5, 6, and 8) were observed in subjects with IPAH compared with age-matched control subjects. C, Schematic drawing of versican splice variants and recognition sites for mAb 2B1 and pAb 3054. Versican has two globular domains, the G1 domain at the amino terminus and the G3 domain at the carboxy terminus. The splice variants vary in the GAG-attachment domains (αGAG and βGAG). C: complement regulatory region; E: epidermal growth factor–like domain; HABR: hyaluronan-binding region; Ig: immunoglobulin-like domain; L: lectin-binding domain.

Article Snippet: After incubation with the blocking buffer (10% Aqua Block in Tris-buffered saline [TBS] containing 0.1% Tween-20) for 2 hours at room temperature, membranes were incubated with a goat polyclonal antibody against the human versican βGAG domain (1∶2000, AF 3054, R&D systems) overnight at 4°C in blocking buffer.

Techniques: Western Blot, Polyacrylamide Gel Electrophoresis, Control, Binding Assay

Journal: Pulmonary Circulation

Article Title: Versican accumulates in vascular lesions in pulmonary arterial hypertension

doi: 10.1086/686994

Figure Lengend Snippet: Versican and inflammation in pulmonary arterial hypertension (PAH). Representative images of double immunostaining for versican (in brown) and CD45 (leukocyte common antigen, in red). A and B, Pronounced perivascular inflammation with little versican staining. C, Infiltration of inflammatory cells in the adventitia without colocalization with versican. D–F, Versican and CD45 are rarely colocalized in plexiform lesions. G and H, Versican accumulates in the neointima, but there is almost no colocalization with inflammatory cells. I–K, Some degree of colocalization in neointima, but it is not consistent. Arrows indicate the colocalization of versican and CD45. The letter L indicates the lumen of pulmonary vessels. Mouse immunoglobulin G (IgG) was used as a negative control. The images were captured with a ×4 objective for K and a ×20 objective for A–J. There were 4–6 sections per slide, and 1 slide per subject was analyzed (n = 13 for PAH group; n = 7 for control group).

Article Snippet: After incubation with the blocking buffer (10% Aqua Block in Tris-buffered saline [TBS] containing 0.1% Tween-20) for 2 hours at room temperature, membranes were incubated with a goat polyclonal antibody against the human versican βGAG domain (1∶2000, AF 3054, R&D systems) overnight at 4°C in blocking buffer.

Techniques: Double Immunostaining, Staining, Negative Control, Control

Journal: Pulmonary Circulation

Article Title: Versican accumulates in vascular lesions in pulmonary arterial hypertension

doi: 10.1086/686994

Figure Lengend Snippet: Versican and smooth-muscle α-actin–positive cells. Representative images of double immunofluorescence staining for smooth-muscle α-actin (in green) and versican (in red). Nuclear staining was shown in blue. A, Smooth-muscle α-actin staining of neointimal lesion (idiopathic pulmonary arterial hypertension [IPAH]). B, Strong versican immunostaining in the same neointima. C, Merged image (A and B), which shows versican accumulation in areas with smooth-muscle α-actin–positive cells within the neointima. D, Smooth-muscle α-actin staining of plexiform lesion (IPAH). E, Prominent versican staining in the same plexiform lesion. F, Merged image (D and E). G and H, Double immunofluorescence staining of vessels from healthy control subjects, with very low levels of versican in areas with smooth-muscle α-actin–positive cells. I, Rabbit immunoglobulin G–negative control. The images were captured with a ×20 objective. One section per subject was analyzed (n = 4 for IPAH; n = 3 for healthy control subjects).

Article Snippet: After incubation with the blocking buffer (10% Aqua Block in Tris-buffered saline [TBS] containing 0.1% Tween-20) for 2 hours at room temperature, membranes were incubated with a goat polyclonal antibody against the human versican βGAG domain (1∶2000, AF 3054, R&D systems) overnight at 4°C in blocking buffer.

Techniques: Double Immunofluorescence Staining, Staining, Immunostaining, Control, Negative Control

Journal: Pulmonary Circulation

Article Title: Versican accumulates in vascular lesions in pulmonary arterial hypertension

doi: 10.1086/686994

Figure Lengend Snippet: Upregulation of versican messenger RNA and protein by mechanical strain in human aortic smooth-muscle cells (SMCs) but not in human pulmonary artery smooth-muscle cells (hPASMCs). Results of quantitative polymerase chain reaction (qPCR) for versican isoforms (V0, V1, V2, and V3; A) and a representative Western blot (B) for versican from cell cultures of stretched aortic SMCs and nonstretched controls. Samples were collected after 6 hours of cyclic stretch. Results of qPCR (C) for versican isoforms and a representative Western blot (D) for versican from cell cultures of stretched hPASMCs and nonstretched controls. Samples were collected for another 6 hours after 6 hours of cyclic stretch. For qPCR (A and C), fold changes were calculated using ∆∆Ct method compared with nonstretched controls. β2-microglobulin was used as reference gene (n = 3; asterisk indicates P < 0.05 by Student t test). For Western blots (B and D), equal amount of total proteins from conditioned medium and cell lysates, respectively, were applied to ion-exchange chromatography for proteoglycan purification. Chondroitinase ABC-treated samples were then subjected to sodium dodecyl sulfate polyacrylamide gel electrophoresis under reducing condition and probed with goat antihuman versican β glycosaminoglycan domain antibody for versican isoforms V0 and V1. N: nonstretched control group; S: stretched group; (+): positive control extracted from human aortic SMC–conditioned medium.

Article Snippet: After incubation with the blocking buffer (10% Aqua Block in Tris-buffered saline [TBS] containing 0.1% Tween-20) for 2 hours at room temperature, membranes were incubated with a goat polyclonal antibody against the human versican βGAG domain (1∶2000, AF 3054, R&D systems) overnight at 4°C in blocking buffer.

Techniques: Real-time Polymerase Chain Reaction, Western Blot, Ion Exchange Chromatography, Purification, Polyacrylamide Gel Electrophoresis, Control, Positive Control

Journal: Pulmonary Circulation

Article Title: Versican accumulates in vascular lesions in pulmonary arterial hypertension

doi: 10.1086/686994

Figure Lengend Snippet: Versican is regulated by hypoxia. A, quantitative polymerase chain reaction (qPCR) for versican isoforms (V0, V1, V2, and V3) from cell lysates of hypoxic human pulmonary artery smooth-muscle cells (hPASMCs). Data were analyzed using ∆∆Ct method compared with normoxic controls. β2-microglobulin was used as reference gene (n = 3; asterisk indicates P < 0.05 by Student t test). B, Representative Western blot for versican from cell cultures of hypoxic hPASMCs and normoxic controls. Purified, chondroitinase-treated samples were probed with goat antihuman versican β glycosaminoglycan domain antibody for versican isoforms V0 and V1. H: hypoxia; N: normoxia; (+): positive control extracted from human aortic smooth-muscle cell–conditioned medium.

Article Snippet: After incubation with the blocking buffer (10% Aqua Block in Tris-buffered saline [TBS] containing 0.1% Tween-20) for 2 hours at room temperature, membranes were incubated with a goat polyclonal antibody against the human versican βGAG domain (1∶2000, AF 3054, R&D systems) overnight at 4°C in blocking buffer.

Techniques: Real-time Polymerase Chain Reaction, Western Blot, Purification, Positive Control

Journal: International Journal of Biological Sciences

Article Title: Downregulation of m 6 A Reader YTHDC2 Promotes the Proliferation and Migration of Malignant Lung Cells via CYLD/NF-κB Pathway

doi: 10.7150/ijbs.58514

Figure Lengend Snippet: YTHDC2 gene and protein expression is downregulated in patients with lung cancer from The Cancer Gene Atlas, Gene Expression Omnibus and Human Protein Atlas databases, as well as in CS-exposed cells. (A) Differential analysis of YTHDC2 mRNA expression in lung cancer tissues based on the Gene Expression Profiling Interactive Analysis tool. * P<0.05 vs. normal tissues. Differential analysis of YTHDC2 mRNA expression in lung cancer tissues from (B) GSE32665 and (C) GSE19188 datasets. (D) Representative IHC images showed that YTHDC2 staining was found in the cell cytoplasm in lung cancer and normal lung tissues. High expression of YTHDC2 could be found in adjacent normal tissues, while its expression was decreased in the majority of lung cancer tissues. (E) Differential analysis of YTHDC2 staining positive ratio quantitated by IHC Profiler in lung cancer tissue arrays. YTHDC2 staining positive ratio in lung cancer tissues with different (F) maximum diameter, (G) pathological stage and (H) invasion depth. (I) Relative mRNA expression level of YTHDC2 in CS-exposed cells (S10, S20 and S30) and normal BEAS-2B cells. Western blot analysis (J) and quantitative results (K) of YTHDC2 protein expression in CS-exposed cells (S10, S20 and S30) and normal BEAS-2B cells. S10, S20 and S30 represent BEAS-2B cells exposed to CS for 10, 20 and 30 passages, respectively. **P<0.01 vs. normal BEAS-2B cells. IHC, immunohistochemistry; YTHDC2, YTH domain containing 2; CS, cigarette smoke.

Article Snippet: IHC assay was used to analyze the expression of YTHDC2 in lung cancer and paired normal tissues with an anti-YTHDC2 antibody (1:500 dilusion; ProteinTech Group, Inc.).

Techniques: Expressing, Gene Expression, Staining, Western Blot, Immunohistochemistry

Journal: International Journal of Biological Sciences

Article Title: Downregulation of m 6 A Reader YTHDC2 Promotes the Proliferation and Migration of Malignant Lung Cells via CYLD/NF-κB Pathway

doi: 10.7150/ijbs.58514

Figure Lengend Snippet: Proteomics analysis of YTHDC2-knockdown cells. Bubble chart showing the KEGG enrichment results of the genes associated with (A) LUAD and (B) LUSC. The larger the rich factor, the greater the degree of enrichment. The color gradient from red to green represents the P-value; the closer to green color, the lower the P-value and the higher the significance level corresponding to the enrichment. Volcano plots showing the tumor suppressor genes in YTHDC2-related genes in (C) LUAD and (D) LUSC. (E) Bar graph showing the number of upregulated and downregulated proteins in YTHDC2-knockdown cells. (F) Subcellular distribution of DEGs. (G) Biological process and (H) KEGG pathway analysis of DEGs. (I) The protein-protein interaction network of DEGs was constructed using Cytoscape. (J-L) Identification of the top 3 significant clusters using the plug-in Minimal Common Oncology Data Elements in Cytoscape, and biological process enrichment analysis of the proteins in these clusters. KEGG, Kyoto Encyclopedia of Genes and Genomes; YTHDC2, YTH domain containing 2; LUSC, lung squamous cell carcinoma; LUAD , lung adenocarcinoma; DEGs, differentially expressed genes.

Article Snippet: IHC assay was used to analyze the expression of YTHDC2 in lung cancer and paired normal tissues with an anti-YTHDC2 antibody (1:500 dilusion; ProteinTech Group, Inc.).

Techniques: Knockdown, Construct

Journal: International Journal of Biological Sciences

Article Title: Downregulation of m 6 A Reader YTHDC2 Promotes the Proliferation and Migration of Malignant Lung Cells via CYLD/NF-κB Pathway

doi: 10.7150/ijbs.58514

Figure Lengend Snippet: YTHDC2 downregulation promotes lung cancer cell proliferation. Representative images and quantification results of cell cycle of (A) BEAS-2B and (C) H1299 cells transfected with siYTHDC2 and NC. Representative images and quantification results of cell cycle of (B) S30 and (D) H1299 cells transfected with overexpression vector (pYTHDC2) and blank vector. (E) Cell proliferation was measured in BEAS-2B and H1299 cells transfected with siYTHDC2 and NC by EdU cell proliferation assay. (F) Cell proliferation were evaluated in S30 and H1299 cells transfected with pYTHDC2 and blank control by EdU cell proliferation assay. (G) Representative immunofluorescence images and (I) quantification results of Ki67 in BEAS-2B and H1299 cells transfected with siYTHDC2 and NC. (H) Representative immunofluorescence images and (J) quantification results of Ki67 in S30 and H1299 cells transfected with pYTHDC2 and blank control. Relative mRNA expression level of cyclin D1 in (K) BEAS-2B and H1299 cells transfected with siYTHDC2 and NC, as well as in (L) S30 and H1299 cells transfected with pYTHDC2 and blank control. * P<0.05 vs. NC or blank control group, ** P<0.01 vs. NC or blank control group. YTHDC2, YTH domain containing 2; siRNA, small interfering RNA; NC, negative control; EdU, 5-ethynyl-2'-deoxyuridine.

Article Snippet: IHC assay was used to analyze the expression of YTHDC2 in lung cancer and paired normal tissues with an anti-YTHDC2 antibody (1:500 dilusion; ProteinTech Group, Inc.).

Techniques: Transfection, Over Expression, Plasmid Preparation, Proliferation Assay, Control, Immunofluorescence, Expressing, Small Interfering RNA, Negative Control

Journal: International Journal of Biological Sciences

Article Title: Downregulation of m 6 A Reader YTHDC2 Promotes the Proliferation and Migration of Malignant Lung Cells via CYLD/NF-κB Pathway

doi: 10.7150/ijbs.58514

Figure Lengend Snippet: YTHDC2 downregulation promotes lung cancer cell migration. (A) Representative images and quantification of the wound healing assay showing that cell migration was significantly increased at 24 and 48 h after transfection with siYTHDC2 in BEAS-2B and H1299 cells, as well as following transfection with (B) the overexpressing vector pYTHDC2 and a blank vector. Representative images and quantification of the Transwell migration assay of BEAS-2B and H1299 cells transfected with (C) siYTHDC2 and (D) pYTHDC2. (E) Quantitative PCR analysis of CDH1 and CDH2 in normal BEAS-2B andH1299 cells transfected with siYTHDC2, (F) as well as in S30 and H1299 cells transfected with pYTHDC2. (G) Western blot analysis and (I) quantitative results of EMT markers in BEAS-2B and H1299 cells transfected with siYTHDC2. (H) Western blot analysis and (J) quantitative results of EMT markers in S30 and H1299 cells transfected with pYTHDC2. *P<0.05 vs. NC (blank vector) group, **P<0.01 vs. NC (blank vector) group. YTHDC2, YTH domain containing 2; siRNA, small interfering RNA; EMT, epithelial-mesenchymal transition; NC: negative control; CDH1: E-cadherin; CDH2: N-cadherin.

Article Snippet: IHC assay was used to analyze the expression of YTHDC2 in lung cancer and paired normal tissues with an anti-YTHDC2 antibody (1:500 dilusion; ProteinTech Group, Inc.).

Techniques: Migration, Wound Healing Assay, Transfection, Plasmid Preparation, Transwell Migration Assay, Real-time Polymerase Chain Reaction, Western Blot, Small Interfering RNA, Negative Control

Journal: International Journal of Biological Sciences

Article Title: Downregulation of m 6 A Reader YTHDC2 Promotes the Proliferation and Migration of Malignant Lung Cells via CYLD/NF-κB Pathway

doi: 10.7150/ijbs.58514

Figure Lengend Snippet: YTHDC2 overexpression suppresses H1299 cells growth in vivo . (A) Images of the xenograft tumors formed in nude mice injected with YTHDC2-overexpressing and control cells. (B) Volume and (C) weight of xenograft tumors isolated from nude mice. (D) Representative images and (E) quantification of the results of cell cycle analysis of single cell suspensions yielded from xenograft tumors. (F) Representative images of hematoxylin and eosin staining, and immunohistochemical staining of YTHDC2, Ki-67, cyclin D1, E-cadherin and N-cadherin in xenograft tumors derived from nude mice. Scale bar, 50 µm; **P<0.01 vs. blank group. YTHDC2, YTH domain containing 2.

Article Snippet: IHC assay was used to analyze the expression of YTHDC2 in lung cancer and paired normal tissues with an anti-YTHDC2 antibody (1:500 dilusion; ProteinTech Group, Inc.).

Techniques: Over Expression, In Vivo, Injection, Control, Isolation, Cell Cycle Assay, Staining, Immunohistochemical staining, Derivative Assay

Journal: International Journal of Biological Sciences

Article Title: Downregulation of m 6 A Reader YTHDC2 Promotes the Proliferation and Migration of Malignant Lung Cells via CYLD/NF-κB Pathway

doi: 10.7150/ijbs.58514

Figure Lengend Snippet: YTHDC2 mRNA expression was regulated by gene amplification. Distribution of patients with (A) LUAD and (D) LUSC with different YTHDC2 amplification status. YTHDC2 mRNA expression in (B) LUAD and (E) LUSC tissues with different YTHDC2 amplification status. Different letters (a, b, c and d) represent statistically significant group differences. Pearson's correlation analysis revealed a significant positive correlation between YTHDC2 mRNA expression and copy numbers in (C) LUAD and (F) LUSC. The line represents linear regression of data (LUAD: y=1.065x+9.177, R 2 =0.385; LUSC: y=0.965x+9.318, R 2 =0.198). (G) The Oncomine datasets for the corresponding YTHDC2 copy numbers in lung cancer were obtained with a threshold P=0.001 and ≥2 fold-change. The data in the graphic show significant downregulation (blue column) of YTHDC2 copy numbers in lung cancer versus normal tissue. The intensity of the blue color represents the respective levels of YTHDC2 copy number. (H) Copy number variation in LUAD samples with different smoking histories. Different letters (a, b, c and d) represent statistically significant group differences. (I) Copy number variation of YTHDC2 in BEAS-2B cells and cigarette smoke-exposed cells (grey block), as well as in two lung cancer cell lines (black block). The dotted line (copy number = 2) represents the copy number of the reference gene RNase P. * P<0.05, ** P<0.01 vs. BEAS-2B cells. YTHDC2, YTH domain containing 2; LUSC, lung squamous cell carcinoma; LUAD , lung adenocarcinoma.

Article Snippet: IHC assay was used to analyze the expression of YTHDC2 in lung cancer and paired normal tissues with an anti-YTHDC2 antibody (1:500 dilusion; ProteinTech Group, Inc.).

Techniques: Expressing, Amplification, Blocking Assay

Journal: International Journal of Biological Sciences

Article Title: Downregulation of m 6 A Reader YTHDC2 Promotes the Proliferation and Migration of Malignant Lung Cells via CYLD/NF-κB Pathway

doi: 10.7150/ijbs.58514

Figure Lengend Snippet: YTHDC2 promotes CYLD mRNA stability and inhibits NF-κB activity. (A) Scatter plot showing the mRNA transcripts identified by YTHDC2 RIP-sequencing, and transcripts that were significantly enriched are marked in red. (B) Kyoto Encyclopedia of Genes and Genomes pathway enrichment analysis of the mRNAs significantly enriched by YTHDC2 RIP. (C) Venn diagram showing the intersection of the YTHDC2 RIP-enriched mRNAs, YTHDC2-related mRNAs, upregulated differentially expressed proteins and tumor suppressor genes. (D) Relative mRNA expression level of CYLD in YTHDC2-overexpressing and knocked down H1299 cells. ** P<0.01 vs. blank group, ## P<0.01 vs. negative control group. (E) Relative protein expression level of CYLD in YTHDC2-overexpressing and knocked down H1299 cells. (F) RNA decay assay for CYLD mRNA stability upon YTHDC2 overexpression and knockdown in H1299 cells. (G) Immunostaining analysis of NF-κB p65 and CYLD in YTHDC2-overexpressing and -knockdown H1299 cells. (H) The DNA-binding activity of NF-κB in YTHDC2-overexpressing and -knockdown H1299 cells was measured by electrophoretic mobility shift assay using the biotin-labeled consensus NF-κB-binding sequence. (I) Immunohistochemistry staining of NF-κB p65 and CYLD in xenograft tumors derived from nude mice. RIP, RNA immunoprecipitation; YTHDC2, YTH domain containing 2; CYLD, cylindromatosis.

Article Snippet: IHC assay was used to analyze the expression of YTHDC2 in lung cancer and paired normal tissues with an anti-YTHDC2 antibody (1:500 dilusion; ProteinTech Group, Inc.).

Techniques: Activity Assay, Sequencing, Expressing, Negative Control, Over Expression, Knockdown, Immunostaining, Binding Assay, Electrophoretic Mobility Shift Assay, Labeling, Immunohistochemistry, Staining, Derivative Assay, RNA Immunoprecipitation

Journal: International Journal of Biological Sciences

Article Title: Downregulation of m 6 A Reader YTHDC2 Promotes the Proliferation and Migration of Malignant Lung Cells via CYLD/NF-κB Pathway

doi: 10.7150/ijbs.58514

Figure Lengend Snippet: YTHDC2 regulates the stability of CYLD through m 6 A modification. (A) Prediction score of m 6 A distribution in CYLD sequence according to the sequence-based RNA adenosine methylation site predictor online tool. (B) meRIP-quantitative PCR and (C) meRIP-PCR results showed amplification of sites 2 to 5, indicating m 6 A modification in these four segments. Anti-IgG antibody was used as control. (D and E) Western blot results showing the relative protein expression level of CYLD in METTL3 , METTL14 , FTO and ALKBH5 knocked down H1299 cells. (F and G) Western blot result showing the relative protein expression level of CYLD in H1299 cells treated with DAA, a global methylation inhibitor, and with meclofenamic acid, a FTO inhibitor. (H and I) Western blot results showing the relative protein expression level of CYLD in YTHDC2-overexpressing H1299 cells treated with or without 3-DAA. YTHDC2, YTH domain containing 2; CYLD, cylindromatosis; meRIP, m 6 A methylated RNA immunoprecipitation; m 6 A, N6-methyladenosine; METTL, methyltransferase-like; FTO, fat mass and obesity-associated protein; DAA, 3-deazaadenosine.

Article Snippet: IHC assay was used to analyze the expression of YTHDC2 in lung cancer and paired normal tissues with an anti-YTHDC2 antibody (1:500 dilusion; ProteinTech Group, Inc.).

Techniques: Modification, Sequencing, Methylation, Real-time Polymerase Chain Reaction, Amplification, Control, Western Blot, Expressing, RNA Immunoprecipitation

Journal: The Journal of Pharmacology and Experimental Therapeutics

Article Title: Clinically Advanced p38 Inhibitors Suppress DUX4 Expression in Cellular and Animal Models of Facioscapulohumeral Muscular Dystrophy

doi: 10.1124/jpet.119.259663

Figure Lengend Snippet: p38 Inhibitor PH-797804 reduces DUX4 and DUX4 target gene expression in FSHD patient-derived proliferating myoblasts and differentiating myotubes. (A) PH-797804 concentration-response curve. Differentiating MB200 (FSHD2) cells were treated with PH-797804 in an 11-point dilution series for 40 hours. RNA levels for DUX4 target MBD3L2 and differentiation markers MYOG and MYH2 were determined in cell lysates by quantitative real-time (qRT) PCR. (B) PH-797804 in FSHD myoblasts. Proliferating cultures of 54-2 (FSHD1) and MB200 (FSHD2) myoblasts were treated with 100 nM PH-797804 for 72 hours. RNA was isolated and analyzed for DUX4 targets MBD3L2, ZSCAN4, and LEUTX. (C and D) Differentiating cultures of 54-2 (FSHD1) and MB200 (FSHD2) muscle cells were treated with varying concentrations of PH-797804, as indicated, for 40 hours and the cultures were analyzed for DUX4, DUX4 target (MBD3L2, ZSCAN4, and LEUTX), and differentiation marker (MYOG and MYH2) RNA levels by qRT-PCR. Data are expressed as relative expression (mean with S.D.) with the expression in absence of inhibitor set to 1. *P < 0.01 vs. control (unpaired, two-tailed t test). **P < 0.01 vs. control (one-way ANOVA with Dunnett’s post test).

Article Snippet: Taqman assays were purchased from Applied Biosystems (Thermo Fisher Scientific): MBD3L2, Hs00544743_m1; LEUTX, Hs01028718_m1; MYH2, Hs00430042_m1; MAPK11, Hs00177101_m1; MAPK14, Hs01051152_m1; MYOG, Hs01072232_m1; RPL30, Hs00265497_m1; ZSCAN4, Hs00537549_m1; and DUX4, primers GCCGGCCCAGGTACCA and CAGCGAGCTCCCTTGCA with probe 6FAM-CAGTGCGCACCCCG-MGBNFQ.

Techniques: Targeted Gene Expression, Derivative Assay, Concentration Assay, Quantitative RT-PCR, Isolation, Marker, Expressing, Control, Two Tailed Test

Journal: The Journal of Pharmacology and Experimental Therapeutics

Article Title: Clinically Advanced p38 Inhibitors Suppress DUX4 Expression in Cellular and Animal Models of Facioscapulohumeral Muscular Dystrophy

doi: 10.1124/jpet.119.259663

Figure Lengend Snippet: DUX4 activity: p38 inhibitors do not block DUX4 transcriptional activation function. Non-DUX4 expressing 54-6 (normal) myoblasts were transfected with a DUX4 expression plasmid and treated with varying concentrations of control p300 inhibitor A-485 (A), p38 inhibitor PH-797804 (B) or p38 inhibitor pamapimod (C), as indicated, for 20 hours. Cultures were analyzed for DUX4 targets (MBD3L2, ZSCAN4, and LEUTX) RNA levels by quantitative real-time PCR. Data are expressed as relative expression with the expression in absence of inhibitor set to 100.

Article Snippet: Taqman assays were purchased from Applied Biosystems (Thermo Fisher Scientific): MBD3L2, Hs00544743_m1; LEUTX, Hs01028718_m1; MYH2, Hs00430042_m1; MAPK11, Hs00177101_m1; MAPK14, Hs01051152_m1; MYOG, Hs01072232_m1; RPL30, Hs00265497_m1; ZSCAN4, Hs00537549_m1; and DUX4, primers GCCGGCCCAGGTACCA and CAGCGAGCTCCCTTGCA with probe 6FAM-CAGTGCGCACCCCG-MGBNFQ.

Techniques: Activity Assay, Blocking Assay, Activation Assay, Expressing, Transfection, Plasmid Preparation, Control, Real-time Polymerase Chain Reaction

Journal: The Journal of Pharmacology and Experimental Therapeutics

Article Title: Clinically Advanced p38 Inhibitors Suppress DUX4 Expression in Cellular and Animal Models of Facioscapulohumeral Muscular Dystrophy

doi: 10.1124/jpet.119.259663

Figure Lengend Snippet: p38 siRNAs suppress DUX4 in FSHD myoblasts. 54-2 (FSHD) (A) or MB200 (FSHD2) (B) myoblasts were transfected with siRNAs targeting p38α (si-p38α), p38β (si-p38β), or control siRNAs (si-CTRL) twice starting 72 hours before harvest and again 24 hours before harvest to ensure efficient knockdown. Myoblasts were maintained in growth media before RNA was isolated and analyzed for p38 (left panels) and DUX4 target [MBD3L2, ZSCAN4, and LEUTX (right panels)] expression levels. Data are expressed as relative expression (mean with S.D.) with the expression in the presence of si-CTRL set to one. *P < 0.01 vs. si-CTRL (one-way ANOVA with Dunnett’s post test).

Article Snippet: Taqman assays were purchased from Applied Biosystems (Thermo Fisher Scientific): MBD3L2, Hs00544743_m1; LEUTX, Hs01028718_m1; MYH2, Hs00430042_m1; MAPK11, Hs00177101_m1; MAPK14, Hs01051152_m1; MYOG, Hs01072232_m1; RPL30, Hs00265497_m1; ZSCAN4, Hs00537549_m1; and DUX4, primers GCCGGCCCAGGTACCA and CAGCGAGCTCCCTTGCA with probe 6FAM-CAGTGCGCACCCCG-MGBNFQ.

Techniques: Transfection, Control, Knockdown, Isolation, Expressing

Journal: The Journal of Pharmacology and Experimental Therapeutics

Article Title: Clinically Advanced p38 Inhibitors Suppress DUX4 Expression in Cellular and Animal Models of Facioscapulohumeral Muscular Dystrophy

doi: 10.1124/jpet.119.259663

Figure Lengend Snippet: p38 siRNAs suppress DUX4 in FSHD myotubes. (A) 54-2 (FSHD1) myoblasts were transfected with siRNA targeting p38α (si-p38α), p38β (si-p38β), or control (si-CTRL) siRNAs, as indicated, 24 hours prior to induction of differentiation. Cells were induced to differentiate and harvested 40 hours later. RNA was isolated and analyzed for p38 (left panel) and DUX4, MBD3L2, ZSCAN4, and LEUTX (right panel) expression levels. (B) MB200 (FSHD2) myoblasts were transfected with the indicated siRNAs on two consecutive days prior to inducing differentiation. Twenty-four hours after the second transfection, cells were induced to differentiate and RNA was isolated 40 hours later and analyzed as in (A). Data are expressed as relative expression (mean with S.D.) with the expression in the presence of si-CTRL set to 1. *P < 0.01 vs. si-CTRL (one-way ANOVA with Dunnett’s post test).

Article Snippet: Taqman assays were purchased from Applied Biosystems (Thermo Fisher Scientific): MBD3L2, Hs00544743_m1; LEUTX, Hs01028718_m1; MYH2, Hs00430042_m1; MAPK11, Hs00177101_m1; MAPK14, Hs01051152_m1; MYOG, Hs01072232_m1; RPL30, Hs00265497_m1; ZSCAN4, Hs00537549_m1; and DUX4, primers GCCGGCCCAGGTACCA and CAGCGAGCTCCCTTGCA with probe 6FAM-CAGTGCGCACCCCG-MGBNFQ.

Techniques: Transfection, Control, Isolation, Expressing

Journal: The Journal of Pharmacology and Experimental Therapeutics

Article Title: Clinically Advanced p38 Inhibitors Suppress DUX4 Expression in Cellular and Animal Models of Facioscapulohumeral Muscular Dystrophy

doi: 10.1124/jpet.119.259663

Figure Lengend Snippet: p38 Inhibitors suppress DUX4 expression in a mouse xenograft pharmacology model of FSHD gene regulation. (A) PH-797804 was administered to xenograft mice twice daily by subcutaneous injections at the indicated doses for 4 days. RNA was isolated from excised tibialis anterior (TA) muscles and analyze for DUX4, MBD3L2, ZSCAN4, and LEUTX RNA levels. (B) Losmapimod was administered orally to xenograft mice twice daily at the indicated doses for 4 days. RNA was isolated and analyzed as in (A). (C) Losmapimod was administered orally to xenograft mice twice daily at 6 mg/kg for 14 days. RNA and DNA were isolated from excised TA muscles and analyzed for differentiation marker MYH2 RNA levels and two-copy gene (hTERT and mTfrc) DNA levels. Data are expressed as relative expression (mean with S.E.) with the expression in the vehicle groups set to 1. For MYH2 analysis, the 14-day levels were compared with levels in 4-day animals (day 4) not treated with drugs, which was set to 1. *P < 0.01 vs. vehicle (one-way ANOVA with Dunnett’s post test). #P = 0.030 vs. vehicle (unpaired two-tailed t test).

Article Snippet: Taqman assays were purchased from Applied Biosystems (Thermo Fisher Scientific): MBD3L2, Hs00544743_m1; LEUTX, Hs01028718_m1; MYH2, Hs00430042_m1; MAPK11, Hs00177101_m1; MAPK14, Hs01051152_m1; MYOG, Hs01072232_m1; RPL30, Hs00265497_m1; ZSCAN4, Hs00537549_m1; and DUX4, primers GCCGGCCCAGGTACCA and CAGCGAGCTCCCTTGCA with probe 6FAM-CAGTGCGCACCCCG-MGBNFQ.

Techniques: Expressing, Isolation, Muscles, Marker, Two Tailed Test