Journal: The Journal of Experimental Medicine

Article Title: Regulation of amyloid-β dynamics and pathology by the circadian clock

doi: 10.1084/jem.20172347

Figure Lengend Snippet: Intrinsic hippocampal molecular circadian clock function does not influence the daily rhythm of hippocampal ISF Aβ. (A) Schematic depicting tissue-specific AAV-Cre–mediated excision of Bmal1 within the hippocampus of Bmal1 f/f ; APPPS1-21 transgenic mice. (B) Diagram depicting Bmal1 deletion abrogating circadian clock function. In addition to Per and Cry genes, BMAL1 regulates transcription of many other clock-controlled genes in a tissue-specific manner. (C) Schematic illustrating the in vivo microdialysis method used to collect soluble Aβ in the hippocampus subsequent to viral injections. This technique was performed in awake, behaving mice. (D) Representative images of microdialysis probe placement (dashed lines) within the hippocampus of mice injected with either AAV-GFP (labeled Cre − ; APP, left) with normal BMAL1 expression (bottom left) or injected with AAV-Cre–IRES-GFP (labeled Cre + ; APP, right), resulting in a loss of local hippocampal BMAL1 expression (bottom right). Bar, 1 mm. (E) Graph showing that hourly ISF Aβ levels are not influenced by the targeted hippocampal excision of Bmal1 (green, AAV-Cre–labeled Cre + ; APP) compared with APP mice with normal BMAL1 expression (black, AAV-GFP–labeled Cre − ; APP). Dots represent normalized mean hourly ISF Aβ levels in 12-h:12-h L:D conditions (shaded gray) ± SEM ( n = 4–5 mice per genotype), and curves represent cosinor method fits. (F) Bar graph showing the effect of targeted hippocampal Bmal1 deletion on the rhythmicity of ISF Aβ as indicated by cosinor circadian amplitudes. Means were NS by two-tailed t test (P = 0.23). Error bars indicate SEM.

Article Snippet: The following antibodies were used: BMAL1 rabbit polyclonal (1:2,000; 2288; Novus Biologicals) and a monoclonal Aβ 1–13 (1:1,000; HJ3.4) made in-house ( ).

Techniques: Transgenic Assay, In Vivo, Injection, Labeling, Expressing, Two Tailed Test

Journal: The Journal of Experimental Medicine

Article Title: Regulation of amyloid-β dynamics and pathology by the circadian clock

doi: 10.1084/jem.20172347

Figure Lengend Snippet: Bmal1 deletion throughout in the brain but sparing the SCN does not markedly affect Aβ levels or hippocampal amyloid pathology. (A) Representative images showing normal BMAL1 expression in the hippocampus and SCN regions from a control mouse ( Cre − ). (B) Representative raw tracing of core body temperature recordings over 7 d from a Cre − mouse (top). Mean body core temperature recordings over 6 d in 12-h:12-h L:D (bottom right), with a χ 2 periodogram (bottom left) showing a dominant 24-h rhythm present in a control Cre − mouse ( n = 3). (C) Representative images of a Nestin-Cre + ;Bmal1 f/f ( Nes-Bmal1 KO ) mouse showing loss of hippocampal but not SCN BMAL1 expression. OC, optic chiasm. (D) Representative raw tracing of core body temperature recordings over 7 d from a Nes-Bmal1 KO mouse (top). Mean body core temperature recordings over 6 d in 12-h:12-h L:D (bottom right), with a χ 2 periodogram (bottom left) showing a dominant 24-h rhythm present in a Nes-Bmal1 KO mouse ( n = 3). (E) Graph showing hourly hippocampal ISF-soluble Aβ levels in Cre − ;Bmal1 f/f ;APPPS1-21 (black) and Nestin-Cre + ; Bmal1 f/f ;APPPS1-21 ( Nes-Bmal1 KO;APP, orange) mice. Squares represent the normalized mean hourly ISF Aβ level in 12h:12–h L:D conditions (shaded gray) ± SEM ( n = 5 mice per genotype), and curves represent cosinor method fits. (F) Bar graph showing rhythmicity of ISF Aβ as indicated by cosinor circadian amplitudes in Cre − ;APPPS1-21 (black) and Nes-Bmal1 KO;APP (orange) mice. Means were NS by two-tailed t test (P = 0.26). (G) Representative images showing X34 staining of fibrillar Aβ plaques in the hippocampus of 4-mo Cre − ;APPPS1-21 (top) and Nes-Bmal1 KO;APP mice (bottom). The hippocampus is outlined by dashed gray lines. Bars: (A and C, top, and G) 500 µm; (A and C, bottom) 250 µm. (H) Bar graph showing quantification of immunoreactive Aβ (HJ3.4 antibody staining, left) and fibrillar Aβ (X34 staining, right) plaque burden within the hippocampus of Cre − APPPS1-21 (black) and Nes-Bmal1 KO;APP (orange) mice ( n = 9–14 mice per genotype). Bars represent means ± SEM (two-tailed t test; P = 0.88 for the immunoreactive Aβ comparison, and P = 0.064 for the fibrillar Aβ comparison).

Article Snippet: The following antibodies were used: BMAL1 rabbit polyclonal (1:2,000; 2288; Novus Biologicals) and a monoclonal Aβ 1–13 (1:1,000; HJ3.4) made in-house ( ).

Techniques: Expressing, Control, Two Tailed Test, Staining, Comparison

Journal: The Journal of Experimental Medicine

Article Title: Regulation of amyloid-β dynamics and pathology by the circadian clock

doi: 10.1084/jem.20172347

Figure Lengend Snippet: Global Bmal1 deletion disrupts Aβ rhythmicity. (A) Representative images showing decreased BMAL1 expression in the hippocampus and SCN regions from a global Bmal1 KO mouse. (B) Representative raw tracing of core body temperature recordings over 6 d from a Bmal1 KO mouse. Mean body core temperature recordings over 6 d in 12-h:12-h L:D (bottom right), with a χ 2 periodogram (bottom left) showing a loss of the dominant 24-h rhythm in a global Bmal1 KO mouse ( n = 3). (C) Graph showing hippocampal ISF-soluble Aβ levels in WT (labeled control, black) and global Bmal1 KO mice (red). Squares represent the normalized mean murine ISF Aβ level pooled every 3 h in 12h:12–h L:D conditions (shaded gray) ± SEM ( n = 7 mice per genotype), and curves represent cosinor method fits. (D) Bar graph showing rhythmicity of ISF Aβ as indicated by cosinor circadian amplitudes in WT (labeled control, black) and Bmal1 KO (red) mice. *, P = 0.002 by two-tailed t test. (E) Bar graph depicting daily mean hippocampal ISF Aβ levels in WT (black) and Bmal1 KO mice (red). Means were NS by two-tailed t test (P = 0.71). Means + SEM are shown; n = 7 mice/group.

Article Snippet: The following antibodies were used: BMAL1 rabbit polyclonal (1:2,000; 2288; Novus Biologicals) and a monoclonal Aβ 1–13 (1:1,000; HJ3.4) made in-house ( ).

Techniques: Expressing, Labeling, Control, Two Tailed Test

Journal: The Journal of Experimental Medicine

Article Title: Regulation of amyloid-β dynamics and pathology by the circadian clock

doi: 10.1084/jem.20172347

Figure Lengend Snippet: Global Bmal1 deletion accelerates amyloid plaque deposition. (A) Representative images showing normal BMAL1 expression in the hippocampus and SCN regions from a tamoxifen-treated Cre − mouse. (B) Representative raw tracing of core body temperature recordings over 7 d from a Cre − mouse (top). Mean body core temperature recordings over 6 d in 12-h:12-h L:D (bottom right), with a χ 2 periodogram (bottom left) showing a dominant 24-h rhythm present in a Cre- control mouse ( n = 3). (C) Representative images of a tamoxifen-treated CAG-Cre ERT2+ ;Bmal1 f/f ( Bmal1 iKO ) mouse showing a loss of both hippocampal and SCN BMAL1 expression. OC, optic chiasm. (D) Representative raw tracing of core body temperature recordings over 7 d from an inducible Bmal1 KO mouse (top). Mean body core temperature recordings over 6 d in 12-h:12-h L:D are arrhythmic (bottom right), with a χ 2 periodogram (bottom left) showing a loss of the dominant 24-h rhythm ( n = 3). (E) Representative images showing X34 staining of fibrillar Aβ plaques in the hippocampus of 4-mo Cre − ;Bmal1 f/f ;APP ( Cre - ;APP , top) and Bmal1 iKO ; APP (red) mice (bottom). The hippocampus is outlined by dashed gray lines. Bars: (A and C, top, and E) 500 µm; (A and C, bottom) 300 µm. (F) Bar graph showing quantification of immunoreactive Aβ (HJ3.4 antibody staining, left) and fibrillar Aβ (X34 staining, right) plaque burden within the hippocampus of Cre − ;APP (black) and Bmal1 iKO ; APP (red) mice ( n = 8–13 mice per genotype). Bars represent means ± SEM (two-tailed t test; P = 0.001 for the immunoreactive Aβ comparison, and P = 0.001 for the fibrillar Aβ comparison). *, P < 0.05.

Article Snippet: The following antibodies were used: BMAL1 rabbit polyclonal (1:2,000; 2288; Novus Biologicals) and a monoclonal Aβ 1–13 (1:1,000; HJ3.4) made in-house ( ).

Techniques: Expressing, Control, Staining, Two Tailed Test, Comparison

Journal: Genes & Cancer

Article Title: CD133-positive cancer stem cells from Colo205 human colon adenocarcinoma cell line show resistance to chemotherapy and display a specific metabolomic profile

doi:

Figure Lengend Snippet: A-B. Cytometry analyses of Nestin, CK20, CD133, Oct3/4, CD44 and CD44v9 on 10% FBS Colo205 growing cells (control) and serum-free growing cultures (week 1 to week 5).

Article Snippet: The purity of sorted cells was evaluated by flow cytometry using FACSCalibur (BD Biosciences) after labeling with anti-human CD133/2 or CD44 antibody (Miltenyi Biotec).

Techniques: Cytometry, Control

Journal: Genes & Cancer

Article Title: CD133-positive cancer stem cells from Colo205 human colon adenocarcinoma cell line show resistance to chemotherapy and display a specific metabolomic profile

doi:

Figure Lengend Snippet: A. Tumorsphere evaluation of CD133+ and CD133- sorted cells. B. Invasiveness of different cell fractions measured using a collagen-based invasion kit (relative DO measured at 560 nm). C. Survival assay after chemotherapy treatment with cisplatin and 5-FU. D. Metabolite quantification after CE-TOF-MS experiments. Only metabolites of interest for Colo205 cells cultured in 10% FBS (control; green bar), CD133+ sorted cells (red bar) and CD44+ sorted cells (yellow bar) are reported here.

Article Snippet: The purity of sorted cells was evaluated by flow cytometry using FACSCalibur (BD Biosciences) after labeling with anti-human CD133/2 or CD44 antibody (Miltenyi Biotec).

Techniques: Clonogenic Cell Survival Assay, Cell Culture, Control