Journal: bioRxiv

Article Title: JUN mediates senescence and immune cell recruitment to prevent prostate cancer progression

doi: 10.1101/2023.11.29.569178

Figure Lengend Snippet: a) Top: Schematic representation of mouse models used in the study. Homozygous loss of Pten or Jun was achieved by a Probasin promoter-controlled Cre recombinase ( PbCre )-mediated ablation of floxed exons 4 and 5 ( Pten ) or exon 1 ( Jun ). Bottom: established and investigated genetic models. Wildtype ( PbCre +/+ ; wt ) and mice with single knockout of Pten ( PbCre tg/+ ; Pten PE Δ/Δ ) and Jun ( PbCre tg/+ ; Jun PE Δ/Δ ) were compared with double knockout ( PbCre tg/+ ; Jun PEΔ/Δ ;Pten PEΔ/Δ ). PE= prostate epithelium; tg = transgene; Δ = knockout. b) Western blot analysis of phosphorylated (pJUN S73 and pAKT S473 ) and total JUN and AKT. β-TUBULIN served as loading control. Protein lysates of entire organs (n=3 biological replicates) from 19-week-old wt , Pten PE Δ/Δ , Jun PEΔ/Δ and Jun PEΔ/Δ ;Pten PEΔ/Δ were investigated. c) Top row: H&E stainings of 19-week-old wt , Pten PE Δ/Δ , Jun PEΔ/Δ and Jun PEΔ/Δ ;Pten PEΔ/Δ prostates. Scale bars indicate 60 µm (top row) and 2 µm (second row), images are presented in 40.0x (top row) and 600.0x magnification (second row). Black rectangles represent the area used for the zoom image below. Bottom row: IHC with an antibody against JUN in 19-week-old prostates of all four experimental groups. Scale bars indicate 30 µm; images are presented in 100.0x magnification. d) Macroscopic images of 19-week-old dissected prostates of wt , Pten PE Δ/Δ , Jun PEΔ/Δ and Jun PEΔ/Δ ;Pten PEΔ/Δ . e) Box plot showing the weights of prostates in grams between wt , Pten PE Δ/Δ , Jun PEΔ/Δ and Jun PEΔ/Δ ;Pten PEΔ/Δ 19-week-old animals (n=20). Significance was determined with an unpaired, two-sided t-test. f) Kaplan-Meier survival analysis of wt , Pten PE Δ/Δ , Jun PEΔ/Δ and Jun PEΔ/Δ ;Pten PEΔ/Δ animals. Biological replicates are indicated and the cumulative survival (%) is shown. Statistical significance was calculated with a logrank test.

Article Snippet: Membranes were incubated with primary antibodies against pJUN S73 (CST 9164), JUN (CST 9165), pAKT S473 (CST 4060), AKT (CST 4691), EpCAM (Elab Science, E-AB-70132), β-ACTIN (CST 4967), NLRP3 (CST 15101), Pro-IL-1β (R&D Systems, AF-401-NA) and β-TUBULIN (CST 2146 and CST 2128) at 4°C overnight.

Techniques: Knock-Out, Double Knockout, Western Blot, Control

Journal:

Article Title: The Herpes Simplex Virus Type 2 R1 Protein Kinase (ICP10 PK) Functions as a Dominant Regulator of Apoptosis in Hippocampal Neurons Involving Activation of the ERK Survival Pathway and Upregulation of the Antiapoptotic Protein Bag-1

doi: 10.1128/JVI.77.2.1292-1305.2003

Figure Lengend Snippet: JNK and c-Jun are activated by HSV-1 and ICP10ΔPK. (A) Extracts of hippocampal cultures mock infected or infected with 10 PFU of HSV-2 or HSV-1 per cell for 24 h were immunoblotted with phosphorylated JNK antibody, followed by antibodies to JNK and actin. Barely visible levels of phosphorylated JNK1/2 were rarely seen in the mock-infected cultures. (B) Cultures were mock infected or infected with HSV-2 or HSV-1 (multiplicity of infection, 10) and stained with phosphorylated JNK antibody at 0.5 h postinfection Results (average of three independent experiments) are mean percent phosphorylated JNK-positive cells ± standard error of the mean. *, P < 0.05 versus mock-infected or HSV-2-infected cultures by analysis of variance. Normal rabbit serum was negative. (C) Extracts of cells infected with HSV-1, HSV-2, ICP10ΔPK, or ICP10ΔRR (multiplicity of infection, 10) or mock infected for 24 h were immunoblotted with antibodies specific for P-Jun(Ser73), followed by P-Jun(Ser63), c-Jun, and actin. (D) Extracts of cells infected with HSV-1, ICP6Δ, or hrR3 or mock infected for 24 h were immunoblotted as in C.

Article Snippet: The following polyclonal antibodies were purchased and used according to the manufacturer's instructions: antibodies to cleaved caspase-3 (D175; recognizes the 17- to 20-kDa fragment of the active caspase ( 63 , 66 , 67 ) (caspase3p20) (Cell Signaling Technology, Beverly, Mass.); cleaved poly(ADP-ribose) polymerase [recognizes the 85-kDa fragment of caspase-cleaved poly(ADP-ribose) polymerase (p85PARP) ( 17 , 66 , 69 ) as confirmed in our laboratory (data not shown) (Promega, Madison, Wis.)]; phosphorylated JNK [recognizes the dually phosphorylated form of JNK1/2/3 (Promega), actin, Bcl-2 (ΔC21), and Bag-1 (FL-274) (Santa Cruz Biotechnology, Santa Cruz, Calif.)]; antibodies to c-Jun phosphorylated on Ser 63 [P-Jun(Ser63)] or Ser 73 [P-Jun(Ser73)] and nonphosphorylated c-Jun [part of the PhosphoPlus c-Jun(Ser63) II c-Jun(Ser73) antibody kit]; antibodies to CREB phosphorylated on Ser 133 and nonphosphorylated CREB [part of the PhosphoPlus CREB(Ser133) antibody kit]; and antibody to ATF-2 phosphorylated on Thr 69/71 (phosphorylated ATF-2) were purchased from Cell Signaling Technology.

Techniques: Infection, Staining

Journal:

Article Title: The Herpes Simplex Virus Type 2 R1 Protein Kinase (ICP10 PK) Functions as a Dominant Regulator of Apoptosis in Hippocampal Neurons Involving Activation of the ERK Survival Pathway and Upregulation of the Antiapoptotic Protein Bag-1

doi: 10.1128/JVI.77.2.1292-1305.2003

Figure Lengend Snippet: ICP10 PK stabilizes Bcl-2 and inhibits BAD expression. (A) Extracts of hippocampal cultures mock infected or infected with HSV-1 or HSV-2 for the indicated times were immunoblotted with antibodies to Bcl-2 followed by actin. The apparent flaw in lane 5 is an artifact not seen in the other blots. (B) Extracts of hippocampal cultures mock infected or infected with HSV-2, ICP10ΔPK, ICP10ΔRR, or HSV-1 (multiplicity of infection, 10) for 0.5 h were immunoblotted with BAD and actin antibodies.

Article Snippet: The following polyclonal antibodies were purchased and used according to the manufacturer's instructions: antibodies to cleaved caspase-3 (D175; recognizes the 17- to 20-kDa fragment of the active caspase ( 63 , 66 , 67 ) (caspase3p20) (Cell Signaling Technology, Beverly, Mass.); cleaved poly(ADP-ribose) polymerase [recognizes the 85-kDa fragment of caspase-cleaved poly(ADP-ribose) polymerase (p85PARP) ( 17 , 66 , 69 ) as confirmed in our laboratory (data not shown) (Promega, Madison, Wis.)]; phosphorylated JNK [recognizes the dually phosphorylated form of JNK1/2/3 (Promega), actin, Bcl-2 (ΔC21), and Bag-1 (FL-274) (Santa Cruz Biotechnology, Santa Cruz, Calif.)]; antibodies to c-Jun phosphorylated on Ser 63 [P-Jun(Ser63)] or Ser 73 [P-Jun(Ser73)] and nonphosphorylated c-Jun [part of the PhosphoPlus c-Jun(Ser63) II c-Jun(Ser73) antibody kit]; antibodies to CREB phosphorylated on Ser 133 and nonphosphorylated CREB [part of the PhosphoPlus CREB(Ser133) antibody kit]; and antibody to ATF-2 phosphorylated on Thr 69/71 (phosphorylated ATF-2) were purchased from Cell Signaling Technology.

Techniques: Expressing, Infection