Journal: Brain, behavior, and immunity

Article Title: The role of the Toll like receptor 4 signaling in sex-specific persistency of depression-like behavior in response to chronic stress

doi: 10.1016/j.bbi.2023.10.006

Figure Lengend Snippet: (A) Experimental outline. Unstressed mice that were not exposed to chronic stress and age-matched (upper panel) and stressed mice that were exposed to chronic stress exposure for 28 days, followed by 28 days of post-stress period. (B and C) Bar graphs showing total immobility time in female (red bar) and male (blue bar) stressed mice compared to unstressed mice at D28 (B) and at D56(C). Statistical analyses were performed using Two-way ANOVA (stress effect, p < 0.0001; sex effect, p = 0.2065; interaction effect, p = 0.0030; * p < 0.05, **** p < 0.001 compared to unstressed male mice; #### p < 0.0001, compared to unstressed female mice, + p < 0.05, +++ p < 0.001, compared to stressed male mice, n = 7–9 mice for each group). (D) A graph showing depression-like behavior over the entire experimental, with female mice shown in red and male mice shown in blue. (E-N) Microglia activation and morphological analysis in female and male mice at D56. (E and J) Representative immunofluorescence images of IBA-1 + (red) and CD68 + (yellow) cells in female (E.1 and E.2) and male (J.1 and J.2) mice. (F and K) Bar graphs showing number of IBA-1 + cells in female (F) and male (K) mice. (G and L) Bar graphs showing quantification of CD68 + area per IBA-1 + microglia in female (G) and male (L) mice. (E and J) Representative images of three-dimensional (3D) reconstruction in female (E.3, E.4, E.5 and E.6) male (J.3, J.4, J.5 and J.6) mice. The outlined with a white box is magnified. (H-N) Sholl analysis based on 3D reconstruction in female and male mice. Bar graph showing soma size per microglia in female (H) and male (M) mice. Line graphs showing quantification of number of intersection between each circle (green, E5, E6, J5 and J6) in female (I) and male (N) mice. Statistical analyses were performed using t -test (* p < 0.05, compared to unstressed female mice). Scale bars indicate 20 (E and J.1–2) and 10 (E and J.3–6) um. Each dot represents an individual mouse. Results are expressed as the mean ± SEM.

Article Snippet: The membrane was then blocked for 1 h at room temperature and treated overnight with primary antibodies specific to p-NF-kB p65 (3033 s, Abcam), NF-kB p65 (51–0500, Thermo Fisher Scientific), caspase-1 (20B-0042, adipogen, CA, USA), IL-1β (6243S, Cell signaling, MA, USA), and α-tubulin (T9026, Sigma) at 4 °C.

Techniques: Activation Assay, Immunofluorescence

Journal: Brain, behavior, and immunity

Article Title: The role of the Toll like receptor 4 signaling in sex-specific persistency of depression-like behavior in response to chronic stress

doi: 10.1016/j.bbi.2023.10.006

Figure Lengend Snippet: (A) The experimental outline in Cx3Cr1 CreEr ; Tlr4 fl double transgenic mice. Unstressed female mice that were not exposed to chronic stress and age-matched and stressed female mice that were exposed to chronic stress for 28 days, followed by 28 days of post-stress period, and then treated with vehicle or tamoxifen for an additional 12 days. (B-D) Bar graphs showing total immobility time at D28 (B), D56 (C) and D68 (D). (E-G) Representative western blot of p-NF-KB 65 (E, upper panel), NF-kB p65 (E, upper panel), caspase-1 (F, upper panel) and IL-1β (G, upper panel) and bar graphs showing relative quantification of p-NF-KB p65 (E, bottom left panel) normalized to NF-kB p65, NF-kB p65 (E, bottom right panel), cleaved caspase-1 (F, bottom panel) and IL-1β (G, bottom panel) levels normalized to α-tubulin. (H) Bar graph showing concentration of IL-1β in PFC lysates. Statistical analyses were performed using t -test or One-way ANOVA (* p < 0.05, ** p < 0.01, **** p < 0.0001 compared to female unstressed mice; # p < 0.05, ## p < 0.01, ### p < 0.001, compared to female stressed mice, n = 4–5 mice for each group). Each dot represents an individual mouse. Results are expressed as the mean ± SEM.

Article Snippet: The membrane was then blocked for 1 h at room temperature and treated overnight with primary antibodies specific to p-NF-kB p65 (3033 s, Abcam), NF-kB p65 (51–0500, Thermo Fisher Scientific), caspase-1 (20B-0042, adipogen, CA, USA), IL-1β (6243S, Cell signaling, MA, USA), and α-tubulin (T9026, Sigma) at 4 °C.

Techniques: Transgenic Assay, Western Blot, Quantitative Proteomics, Concentration Assay

Journal: Brain, behavior, and immunity

Article Title: The role of the Toll like receptor 4 signaling in sex-specific persistency of depression-like behavior in response to chronic stress

doi: 10.1016/j.bbi.2023.10.006

Figure Lengend Snippet: (A) Schematic showing TRAP-sequencing. (B) Selected Gene ontology (GO) annotations enriched in GFP-bound (green, upper panel) and GFP-unbound group (gray, lower panel).The x-axis represents - log10 (p-value), with dotted line representing a p -value of 0.05. (C-D) Volcano plots in female (C) and male (D) mice. The x-axis represents the log 2 conversion of the fold change (log 2 FC) values, and the y-axis represents the corrected significance level after base log10 conversion ( p value). Red and blue dots in the volcano plot indicate all DEGs that were found to differ significantly (absolute value of log 2 FC > 1, * p < 0.05). (E) Heat map with hierarchical clustering distances shows the variation in the expression levels (VST-scored log 2 RPKM) between female stressed and unstressed mice. (F) A bar graph showing selected GO annotations enriched in female (red) and male (blue) mice. The x-axis represents -log10 ( p -value), with dotted line representing a p -value of 0.05. (G) A bar graph showing log 2 FC of significant DEGs in female stress (red) and male stress (blue) mice using a cut-off of p < 0.05.

Article Snippet: The membrane was then blocked for 1 h at room temperature and treated overnight with primary antibodies specific to p-NF-kB p65 (3033 s, Abcam), NF-kB p65 (51–0500, Thermo Fisher Scientific), caspase-1 (20B-0042, adipogen, CA, USA), IL-1β (6243S, Cell signaling, MA, USA), and α-tubulin (T9026, Sigma) at 4 °C.

Techniques: Sequencing, Expressing

Journal: Molecular cancer research : MCR

Article Title: Prostate tumor cell-derived IL-1β induces an inflammatory phenotype in bone marrow adipocytes and reduces sensitivity to docetaxel via lipolysis-dependent mechanisms

doi: 10.1158/1541-7786.MCR-19-0540

Figure Lengend Snippet: A: Gene expression of IL-1β in PC3 cells cultured alone or in Transwell with adipocytes under normoxic or hypoxic conditions; B: IL-1β protein levels in PC3 cells cultured alone or in Transwell and in the absence or presence of 5μM HIF-1α inhibitor CAY10585; representative blot is shown; C: IL-1β densitometry normalized to tubulin; data represent the mean+/− SD from 3 separate experiments; D: IL-1β gene expression in PC3 cells grown in Transwell cultures in the absence or presence of 5μM CAY10585. Gene expression of GLUT1 (E), HIF-1α (F) and COX-2 (G) in bone marrow adipocytes cultured alone or in Transwell with PC3 cells under normoxic (21% O2) or hypoxic (1% O2) conditions. Data are shown as the mean of 3 biological replicate experiments. Gene expression of GLUT1 (H) and HIF-1α (I) of adipocytes upon treatment with recombinant IL-1β. Gene expression of GLUT1 (J) and HIF-1α (K) of adipocytes upon treatment with media conditioned by PCa cells; +/− SD. * p < 0.05; ** p < 0.01; n.s. -not significant.

Article Snippet: Adipocyte cultures were fixed with 3.7% formaldehyde, stained with NFκB (p65) antibody and imaged on a Zeiss LSM 780 confocal microscope with a 40x water immersion objective. siRNA approaches For gene expression analyses, PC3 or ARCaP(M) cells were plated in 6-well plates or on Transwell filters and grown overnight, then a unique 27mer siRNA duplex targeting IL-1β transcripts (OriGene, Rockville, MD: SR302365, Locus ID 3553) or Trilencer-27 Universal scrambled negative control (Origene: SR30004) was added using RNAiMAX transfection reagent at a final concentration of 20μM (based on manufacturer’s protocol).

Techniques: Expressing, Cell Culture, Recombinant

Journal: Molecular cancer research : MCR

Article Title: Prostate tumor cell-derived IL-1β induces an inflammatory phenotype in bone marrow adipocytes and reduces sensitivity to docetaxel via lipolysis-dependent mechanisms

doi: 10.1158/1541-7786.MCR-19-0540

Figure Lengend Snippet: A: Protein levels of IL-1β in PC3 cells cultured alone or in Transwell with adipocytes in the absence or presence of lipolysis-inducing agent Isoproterenol; representative blot is shown; B: IL-1β densitometry normalized to β-actin; data represent the mean from 3 experiments; C: IL-1β gene expression in PC3 cells upon treatment with Isoproterenol; D: Protein levels of IL-1β in PC3 cells cultured alone or in Transwell with adipocytes in the absence or presence of lipolysis-inducing agent Forskolin; representative blot is shown; E: IL-1β densitometry normalized to tubulin; data represent the mean from 3 experiments; F: IL-1β gene expression in PC3 cells upon treatment with Forskolin; G: IL-1β gene expression in PC3 cells grown in Transwell co-cultures with adipocytes in the absence or presence of 5μM inhibitor of hormone sensitive lipase BAY59–9435 (BAY). H: COX-2 mRNA levels in marrow adipocytes cultured alone or in Transwell with PC3 cells in the absence or presence of Forskolin; three individual experiments are shown. I: COX-2 mRNA levels in marrow adipocytes grown in Transwell with PC3 cells and in the absence or presence of 5μM BAY. J: Gene expression of HSL and ATGL and free glycerol release (K) by adipocytes upon treatment with recombinant IL-1β. * p < 0.05; ** p < 0.01; ***p < 0.001.

Article Snippet: Adipocyte cultures were fixed with 3.7% formaldehyde, stained with NFκB (p65) antibody and imaged on a Zeiss LSM 780 confocal microscope with a 40x water immersion objective. siRNA approaches For gene expression analyses, PC3 or ARCaP(M) cells were plated in 6-well plates or on Transwell filters and grown overnight, then a unique 27mer siRNA duplex targeting IL-1β transcripts (OriGene, Rockville, MD: SR302365, Locus ID 3553) or Trilencer-27 Universal scrambled negative control (Origene: SR30004) was added using RNAiMAX transfection reagent at a final concentration of 20μM (based on manufacturer’s protocol).

Techniques: Cell Culture, Expressing, Recombinant

Journal: Molecular cancer research : MCR

Article Title: Prostate tumor cell-derived IL-1β induces an inflammatory phenotype in bone marrow adipocytes and reduces sensitivity to docetaxel via lipolysis-dependent mechanisms

doi: 10.1158/1541-7786.MCR-19-0540

Figure Lengend Snippet: PC3 (A-C) and ARCaP(M) (D-F) cells were grown alone or in Transwell co-culture with bone marrow adipocytes. Taqman RT-PCR results show highly induced mRNA levels of IL-1β in PC3 (A) and ARCaP(M) cells (D); graph representative of multiple experiments. Immunoblot analyses depicting increased levels of IL-1β (1:1000) protein in PC3 (B) and ARCaP(M) cells (E). Tubulin (1:2000) shown for equal loading control. C,F: ELISA assay results depicting levels of IL-1β secreted by PC3 cells (C) or ARCaP(M) cells (F) grown alone or in Transwell with adipocytes. G: COX-2 protein levels in the absence or presence of recombinant IL-1RA (200 ng/ml). H: Densitometry of COX-2 bands normalized to actin bands; data represent the mean of three experiments I: COX-2 gene expression in bone marrow adipocytes grown alone or in Transwell with ARCaP(M) cells in the absence or presence of IL-1RA; J: siRNA-mediated IL-1β knockdown in ARCaP(M) cells reduces COX-2 gene expression levels in marrow adipocytes grown in Transwell cultures with ARCaP(M) cells as compared to cells transfected with scrambled control; K: mPGES mRNA levels in marrow adipocytes treated with IL-1RA; L: mPGES gene expression in adipocytes upon siRNA-mediated knockdown of IL-1β in tumor cells; mRNA levels of COX-2 (M) and mPEGS (N) in marrow adipocytes treated with recombinant IL-1β. O: p65(NFκB) immunofluorescence in bone marrow adipocytes grown under control conditions or treated with recombinant IL-1β. NFκB activation is demonstrated by the nuclear p65 staining in response to IL-1β. P: Marrow adipocytes grown in Transwell with ARCaP(M) cells. Reduced nuclear p65 upon treatment with NFκB inhibitor BAY 11–0782. * p < 0.05; **p < 0.01. **** p < 0.0001.

Article Snippet: Adipocyte cultures were fixed with 3.7% formaldehyde, stained with NFκB (p65) antibody and imaged on a Zeiss LSM 780 confocal microscope with a 40x water immersion objective. siRNA approaches For gene expression analyses, PC3 or ARCaP(M) cells were plated in 6-well plates or on Transwell filters and grown overnight, then a unique 27mer siRNA duplex targeting IL-1β transcripts (OriGene, Rockville, MD: SR302365, Locus ID 3553) or Trilencer-27 Universal scrambled negative control (Origene: SR30004) was added using RNAiMAX transfection reagent at a final concentration of 20μM (based on manufacturer’s protocol).

Techniques: Co-Culture Assay, Reverse Transcription Polymerase Chain Reaction, Western Blot, Enzyme-linked Immunosorbent Assay, Recombinant, Expressing, Transfection, Immunofluorescence, Activation Assay, Staining

Journal: Molecular cancer research : MCR

Article Title: Prostate tumor cell-derived IL-1β induces an inflammatory phenotype in bone marrow adipocytes and reduces sensitivity to docetaxel via lipolysis-dependent mechanisms

doi: 10.1158/1541-7786.MCR-19-0540

Figure Lengend Snippet: A: IL-1β mRNA levels in ARCaP(M) cells upon treatment with two non-overlapping siRNAs. B-G: DIC tile images of 3D ARCaP(M) spheroids treated with scrambled control or IL-1β siRNA A and C and grown in the absence (B,D,F) or presence (C,E,G) of 10nM DCTx. IL-1β siRNA A and C have a visibly significant effect on spheroid size in the presence of DCTx. H-M: Live/Dead assay of control and IL-1β silenced 3D cultures treated with vehicle (H,J,L) or 10nM DCTx (I,K,M). Green: Calcein AM-positive live cells; Red: ethidium homodimer–positive dead cells; N: Quantification of spheroid volume in ARCaP(M) cells treated with scrambled control or IL-1β siRNA in the absence or presence of DCTx O: Quantification of ethidium–positive (dead) cells/total spheroid volume shown as percent control; P: 3D images from Live/Dead assay on 3D cultures of ARCaP(M) cells grown in culture with adipocytes and exposed to HSL and ATGL inhibitors; R: Quantification of spheroid volume upon treatment with HSL inhibitor BAY59–9435 and ATGL inhibitor, Atglistatin; S: Quantification of ethidium–positive (dead) cells/total spheroid volume shown as percent control; T: 3D images from Live/Dead assay on 3D cultures of ARCaP(M) cells grown in culture with adipocytes and exposed to 10nM DCTx in the absence or presence of HSL and ATGL inhibitors; U: Quantification of total 3D spheroid volume in response to treatment. * p<0.05; **p<0.01; ***p<0.001.

Article Snippet: Adipocyte cultures were fixed with 3.7% formaldehyde, stained with NFκB (p65) antibody and imaged on a Zeiss LSM 780 confocal microscope with a 40x water immersion objective. siRNA approaches For gene expression analyses, PC3 or ARCaP(M) cells were plated in 6-well plates or on Transwell filters and grown overnight, then a unique 27mer siRNA duplex targeting IL-1β transcripts (OriGene, Rockville, MD: SR302365, Locus ID 3553) or Trilencer-27 Universal scrambled negative control (Origene: SR30004) was added using RNAiMAX transfection reagent at a final concentration of 20μM (based on manufacturer’s protocol).

Techniques: Live Dead Assay

Journal: Molecular cancer research : MCR

Article Title: Prostate tumor cell-derived IL-1β induces an inflammatory phenotype in bone marrow adipocytes and reduces sensitivity to docetaxel via lipolysis-dependent mechanisms

doi: 10.1158/1541-7786.MCR-19-0540

Figure Lengend Snippet: A: Gene expression analysis of VEGF, Cyclin D and PPARD in PC3 cells cultured alone or in Transwell co-culture with adipocytes; B: Oncomine gene analysis comparing the expression of prostaglandin/EP receptor target genes (VEGF, MYC, PPARD, and CCND1) in patient samples collected from metastatic or primary sites. Data were ordered by “overexpression” and the threshold was adjusted to P-value < 1E-4; fold change, 2 and gene rank, top 10%. C: Immunoblot analysis of p-GSK-3β (1:1000), GSK-3β (1:1000), p-β-catenin (1:1000), β-catenin (1:1000) and Cyclin D (1:1000) proteins in PC3 cells grown alone or in Transwell with adipocytes. D: Proposed mechanism of adipocyte-tumor cell crosstalk involving IL-1β/COX-2/MCP-1 axis. Tumor cells stimulate adipocyte lipolysis. Lipolysis-mediated increase in IL-1β and COX-2 levels and augmented production of PGE2 lead to pro-survival effects on the tumor and therapy resistance.

Article Snippet: Adipocyte cultures were fixed with 3.7% formaldehyde, stained with NFκB (p65) antibody and imaged on a Zeiss LSM 780 confocal microscope with a 40x water immersion objective. siRNA approaches For gene expression analyses, PC3 or ARCaP(M) cells were plated in 6-well plates or on Transwell filters and grown overnight, then a unique 27mer siRNA duplex targeting IL-1β transcripts (OriGene, Rockville, MD: SR302365, Locus ID 3553) or Trilencer-27 Universal scrambled negative control (Origene: SR30004) was added using RNAiMAX transfection reagent at a final concentration of 20μM (based on manufacturer’s protocol).

Techniques: Expressing, Cell Culture, Co-Culture Assay, Over Expression, Western Blot

Journal: Molecular and Cellular Biology

Article Title: Uncoupling the Senescence-Associated Secretory Phenotype from Cell Cycle Exit via Interleukin-1 Inactivation Unveils Its Protumorigenic Role

doi: 10.1128/MCB.00586-18

Figure Lengend Snippet: IL-1α signals through IL-1R to induce the SASP. (A) Western blot analysis of IL-1α, IL-1β, and tubulin (Tub) levels in IMR90T cells expressing either a doxycycline-inducible scramble shRNA (shScr) or shRNA against IL-1α (shIL1α). Cells were treated with either ethanol (Et) as a control or tamoxifen (4OHT) to induce Ras activity and doxycycline to induce knockdown. (B) qRT-PCR analyses of the indicated genes in the indicated cells. Values are shown as fold change compared to levels in shScr treated with Et. (C) Secreted levels of IL-6 in the indicated cell lines as detected by ELISA. (D) Western blot analysis detecting IL-1α and Tub levels in wild-type (WT) and IL-1α-deleted (KO) IMR90T cells. WT1, parental cell line; WT2, a clone that failed to delete IL-1α; KO pool, total pool of clones, where KO1 and KO2 each represent an independent clone. (E) qRT-PCR detecting IL-8 mRNA levels in the indicated cells. (F) Secreted levels of IL-6 in the indicated cell lines as detected by ELISA. (G) qRT-PCR analyses of the indicated SASP factors found via transcriptome analysis to be affected by IL-1R knockdown. Data are represented as fold change compared to levels for WT1 treated with Et. (H) Western blot analysis of IL-1α in IMR90T cells expressing an empty vector control (EV), full-length IL-1α (IL-1α FL), the N-terminal propiece (ppIL-1α), or the C-terminal mature fragment (mIL-1α). The antibody used only recognizes the C-terminal portion of IL-1α. (I) qRT-PCR analyses of the indicated genes in the indicated cell lines. Primers for IL-1α anneal to the N terminus. Values are shown as fold change compared to levels in cells expressing EV. Each experiment was performed at least 3 times. ns, not significant; *, P < 0.05; **, P < 0.01.

Article Snippet: Inducible shRNAs against IL-1α and IL-1R were purchased from Dharmacon, and shRNA against IL-1β was purchased from Sigma.

Techniques: Western Blot, Expressing, shRNA, Activity Assay, Quantitative RT-PCR, Enzyme-linked Immunosorbent Assay, Clone Assay, Plasmid Preparation

Journal: Molecular and Cellular Biology

Article Title: Uncoupling the Senescence-Associated Secretory Phenotype from Cell Cycle Exit via Interleukin-1 Inactivation Unveils Its Protumorigenic Role

doi: 10.1128/MCB.00586-18

Figure Lengend Snippet: IL-1α can induce the SASP in the absence of IL-1β and vice versa. (A) Western blot analysis of IL-1α, IL-1β, and tubulin (Tub) levels in IMR90T cells expressing either an empty vector (EV), full-length IL-1α, or myc-tagged mature IL-1α (mIL-1α). IMR90T cells were also expressing either a scramble shRNA (shScr), a doxycycline-inducible shRNA against IL-1R (shIL-1R), or a constitutively active shRNA against IL-1β (shIL-1β). (B) mRNA expression levels of the indicated genes in the indicated cell lines as detected by qRT-PCR. Values are expressed as fold change compared to levels for shScr cells expressing EV. (C) Western blot analysis of IL-1β and Tub levels in IMR90T cells expressing either EV, full-length IL-1β, or mature IL-1β (mIL-1β). Cells were also expressing either a doxycycline-inducible scramble shRNA (shScr) or a doxycycline-inducible shRNA against IL-1R (shIL-1R) or IL-1α (shIL-1α). (D) mRNA expression levels of the indicated genes in the indicated cell lines as detected by qRT-PCR. Values are expressed as fold change compared to levels in shScr cells expressing EV. Each experiment was repeated at least 3 times. *, P < 0.05; **, P < 0.01; ***, P < 0.001.

Article Snippet: Inducible shRNAs against IL-1α and IL-1R were purchased from Dharmacon, and shRNA against IL-1β was purchased from Sigma.

Techniques: Western Blot, Expressing, Plasmid Preparation, shRNA, Quantitative RT-PCR

Journal: Molecular and Cellular Biology

Article Title: Uncoupling the Senescence-Associated Secretory Phenotype from Cell Cycle Exit via Interleukin-1 Inactivation Unveils Its Protumorigenic Role

doi: 10.1128/MCB.00586-18

Figure Lengend Snippet: IL-1β is also required for SASP expression but not cell cycle exit. (A) Western blot analysis of IL-1β, IL-1α, and tubulin (Tub) levels in IMR90T cells expressing either an empty vector (EV) or shRNA against IL-1β (shIL1β). Cells were treated with either ethanol (Et) as a control or tamoxifen (4OHT) to induce Ras activity. (B) mRNA expression levels of the indicated genes in the indicated cells as detected by qRT-PCR. Values are shown as fold change compared to the level for EV treated with Et. (C) Secreted levels of IL-6 in the indicated cell lines as detected by ELISA. (D to F) Representative images and quantitation of BrdU incorporation (D), SA-βgal positivity (E), and SAHF positivity (F) in the indicated cell lines. Scale bars for panels D and E, 200 µm; scale bar for panel F, 12 µm. Each experiment was performed at least 3 times. *, P < 0.05; **, P < 0.01.

Article Snippet: Inducible shRNAs against IL-1α and IL-1R were purchased from Dharmacon, and shRNA against IL-1β was purchased from Sigma.

Techniques: Expressing, Western Blot, Plasmid Preparation, shRNA, Activity Assay, Quantitative RT-PCR, Enzyme-linked Immunosorbent Assay, Quantitation Assay, BrdU Incorporation Assay

Journal: Molecular and Cellular Biology

Article Title: Uncoupling the Senescence-Associated Secretory Phenotype from Cell Cycle Exit via Interleukin-1 Inactivation Unveils Its Protumorigenic Role

doi: 10.1128/MCB.00586-18

Figure Lengend Snippet: Model of senescence-induced SASP activation under physiological conditions (A) or conditions where either IL-1α or IL-1β is depleted (B).

Article Snippet: Inducible shRNAs against IL-1α and IL-1R were purchased from Dharmacon, and shRNA against IL-1β was purchased from Sigma.

Techniques: Activation Assay