Journal: Nature cancer

Article Title: ALK peptide vaccination restores the immunogenicity of ALK-rearranged non-small cell lung cancer

doi: 10.1038/s43018-023-00591-2

Figure Lengend Snippet: (a) Eml4-Alk PGPGRVAKI−1 cells were injected subcutaneously in the flank of syngeneic BALB/c mice and spontaneous tumor growth was measured. Two independent experiments are shown (N=9 mice). (b) Eml4-Alk PGPGRVAKI−1 cells were injected as in a and mice treated as indicated with anti-PD1. Tumor growth was measured. Two independent experiments are shown (N=10 mice). (c-d) Eml4-Alk PGPGRVAKI−1 cells were injected as in a and mice treated as indicated (N=11 mice). Tumor-free mice were subcutaneously rechallenged at day 50 after initial tumor injection with either mEml4-Alk (N=5 mice) orEml4-Alk PGPGRVAKI−1 cells (N=5 mice) and tumor volume monitored. (e) Kaplan-Meier curves showing overall survival of mice described in a - d (non-significant, P=0.1091 ; P=0.0880 ; **** P <0.001 by Log-rank test). Rechallenged mice are not shown. (f) Kaplan-Meier curves showing overall survival of mice subjected to tumor rechallenge through intravenous injection with either mEml4-Alk or Eml4-Alk PGPGRVAKI−1 cells at day 100 post initial challenge (** P <0.01 by Log-rank test). (g) Representative H&E staining of lung adenocarcinomas from BALB/c mice injected intravenously with Eml4-Alk PGPGRVAKI−1 cell line and treated as indicated (N=4 mice per group). Black arrows indicate lung tumors. Scale bars indicate 1.5mm. (h) Kaplan-Meier curves showing overall survival of mice described in g (not significant, P=0.5395 by Log-rank test) (Untreated, N=4 mice; anti-PD-1, N=7 mice; anti-CTLA-4; n=7 mice; combo, N=7 mice). (i) IFN-γ-ELISPOT analysis of isolated splenocytes after 15 days post subcutaneous (flank) and intravenous (lung) injection of mice treated as in e and h. Data is shown as average number of spot forming units (SFU± SEM). Each dot represents a mouse (N=4 mice per group). Statistics were performed by unpaired two-tailed Student’s t test (not significant: P=0.0587 ; P=0.0665 ; * P=0.0134 ; * P=0.0301 ). (j) Dextramer staining PGPGRVAKI-specific CD8 + T-cells isolated splenocytes after 15 days post subcutaneous (flank; N=6 mice per group) and intravenous (lung; N=4 mice per group) injection of mice treated as in e and i . Data is displayed as percentage (% ± SEM). Each dot represents a mouse. Statistics were performed by unpaired two-tailed Student’s t test ( *P=0.022 ; *P=0.0162 ; *P=0.0113 ; ****P<0.0001 ).

Article Snippet: Human ALK-rearranged ALCL cell lines (DEL and Karpas-299) were obtained from DSMZ German cell collection; the murine ASB-XIV cell line, derived from BALB/c mice was purchased from Cell Lines Service (CLS, product number # 400120), and the murine KP1233 lung cancer cell line, immortalized from C57BL/6 KRAS G12D mice, was kindly gifted by Tyler Jacks (Koch Institute, Cambridge, MA).

Techniques: Injection, Staining, Enzyme-linked Immunospot, Isolation, Two Tailed Test

Journal: Nature cancer

Article Title: ALK peptide vaccination restores the immunogenicity of ALK-rearranged non-small cell lung cancer

doi: 10.1038/s43018-023-00591-2

Figure Lengend Snippet: (a) IFN-γ-ELISPOT analysis of isolated splenocytes from BALB/c mice intravenously injected with Eml4-Alk PGPGRVAKI−1 cells (N=21 mice) compared to tumor-free mice vaccinated with the ALK vaccine (N=6 mice). Naïve mice (N=5 mice) were used as negative control. Data is shown as average number of spot forming units (SFU± SEM). Each dot represents a mouse. Unpaired two-tailed Student’s t test, * P <0.05; **** P <0.0001. (b) Dextramer staining of PGPGRVAKI-specific CD8 + T-cells isolated from lung TILs of untreated lung tumor-bearing mice (N= 8 mice) and lung tumor-bearing mice vaccinated with PGPGRVAKI peptide with CDN adjuvant (N=6 mice) (% ± SEM). Unpaired two-tailed Student’s t test, ** P <0.005. ( c ) PD-1 staining displayed as percentage (% ± SEM) within the CD8 + /ALK-Dextramer + populations isolated from lung TILs of untreated (N= 8 mice) and ALK vaccinated mice (N= 6 mice). Unpaired two-tailed Student’s t test, *** P <0.001. (d) Schematic representation of treatment protocol in therapeutic experiment to combine the ALK vaccine (PGPGRVAKI peptide with CDN adjuvant) with ALK TKI and ICIs. DIE, once a day. (e) Kaplan-Meier curves showing the overall survival of mice treated as indicated in d . Data are combined from two independent experiments. Log-rank test, ** P <0.005; *** P <0.001; **** P <0.0001; n/s, not significant. (f) ALK-dextramer staining of PGPGRVAKI-specific CD8 + T cells isolated from the peripheral blood of surviving mice (for more than 250 days) from the experiment shown in e and rechallenged intravenously with Eml4-Alk PGPGRVAKI−1 cells. Each dot represents a mouse and data are displayed as percentage (% ± SEM; number of mice per group is shown in g ). (g) Kaplan-Meier curves showing overall survival of mice rechallenged intravenously with Eml4-Alk PGPGRVAKI−1 cells as in f . Log-rank test, * P <0.05; ** P <0.005; *** P <0.001.

Article Snippet: Human ALK-rearranged ALCL cell lines (DEL and Karpas-299) were obtained from DSMZ German cell collection; the murine ASB-XIV cell line, derived from BALB/c mice was purchased from Cell Lines Service (CLS, product number # 400120), and the murine KP1233 lung cancer cell line, immortalized from C57BL/6 KRAS G12D mice, was kindly gifted by Tyler Jacks (Koch Institute, Cambridge, MA).

Techniques: Enzyme-linked Immunospot, Isolation, Injection, Negative Control, Two Tailed Test, Staining, Adjuvant

Journal: Nature cancer

Article Title: ALK peptide vaccination restores the immunogenicity of ALK-rearranged non-small cell lung cancer

doi: 10.1038/s43018-023-00591-2

Figure Lengend Snippet: (a) Schematic representation of the metastatic assay to the CNS by intravenous injection of Eml4-Alk PGPGRVAKI−1 cells. (b) Representative H&E staining of the meninges and the brain tissue from BALB/c mice enrolled in the brain metastasis assay and treated as indicated. ALK vax: PGPGRVAKI peptide with CDN adjuvant. Lorlatinib and ICI were given as in Fig. 4d . Scale bars = 50μm. Number of mice per treatment group is shown in c . (c) Incidence of CNS metastases in mice treated with the indicated combination therapies. Number of mice per treatment group is shown on the graph.

Article Snippet: Human ALK-rearranged ALCL cell lines (DEL and Karpas-299) were obtained from DSMZ German cell collection; the murine ASB-XIV cell line, derived from BALB/c mice was purchased from Cell Lines Service (CLS, product number # 400120), and the murine KP1233 lung cancer cell line, immortalized from C57BL/6 KRAS G12D mice, was kindly gifted by Tyler Jacks (Koch Institute, Cambridge, MA).

Techniques: Injection, Staining, Adjuvant

Journal: Nature cancer

Article Title: ALK peptide vaccination restores the immunogenicity of ALK-rearranged non-small cell lung cancer

doi: 10.1038/s43018-023-00591-2

Figure Lengend Snippet: (a) Representative H&E staining of lung adenocarcinomas from syngeneic BALB/c mice treated as described in Fig. 4d . Black arrows indicate lung tumors (N=4 mice per group). Scale bars indicate 1.5mm. (b) H-2D d staining by flow cytometry of cell lines isolated from lung tumors that escaped the indicated treatment displayed as mean fluorescence intensity (MFI) (± SEM) from mice described in Fig. 4d . Each dot represents a cell line isolated from a different tumor (Untreated, n= 20; Lorlatinib, n= 10; Lorlatinib + anti-PD-1, n=10; Lorlatinib + anti-CTLA-4, n= 7; Lorlatinib + ALK vax, n=30; Lorlatinib + ALK vax + anti-PD1, n= 17 biological independent ex vivo cell lines). Unpaired two-tailed Student’s t test, **** P <0.0001. (c) H-2D d staining displayed as MFI of MHC-I low cell lines isolated from lung tumors that escaped the indicated treatment and treated for 24 hours with IFN-γ. Each dot represents a technical replicate and paired columns (±IFN-γ) represent individual cell lines (parental cell line; ex vivo cell lines isolated from mice treated with lorlatinib + ALK vax [N= 4 ex vivo cell lines], and treated with lorlatinib + ALK vax + anti-PD-1 [N=3 ex vivo cell lines]). (d-g) Growth curves of tumors generated by two independent MHC-I high ( d and e ) and MHC-I low (f and g) cell lines that were injected subcutaneously into naïve BALB/c mice and treated as indicated. Each line represents a mouse. (h) H-2D d staining of eight independent MHC-I low cell lines isolated from lung tumors that escaped the indicated treatment treated with a STING agonist (ADU-S100).

Article Snippet: Human ALK-rearranged ALCL cell lines (DEL and Karpas-299) were obtained from DSMZ German cell collection; the murine ASB-XIV cell line, derived from BALB/c mice was purchased from Cell Lines Service (CLS, product number # 400120), and the murine KP1233 lung cancer cell line, immortalized from C57BL/6 KRAS G12D mice, was kindly gifted by Tyler Jacks (Koch Institute, Cambridge, MA).

Techniques: Staining, Flow Cytometry, Isolation, Fluorescence, Ex Vivo, Two Tailed Test, Generated, Injection

Journal: Nature cancer

Article Title: ALK peptide vaccination restores the immunogenicity of ALK-rearranged non-small cell lung cancer

doi: 10.1038/s43018-023-00591-2

Figure Lengend Snippet: (a, b) IFN-γ intracellular staining of CD4 + -gated (a) and CD8 + -gated (b) peripheral mononuclear cells (PBMCs) isolated from mice vaccinated with the indicated peptides and stimulated in vitro with the same peptide (%± SEM). Each dot represents a mouse (ALK 1058–1066 , n = 3; remaining groups, N = 4 mice). (c) IFN-γ intracellular staining in CD4 + and CD8 + splenocytes isolated from a representative mouse (N = 3 independent mice) vaccinated with SLP7 and pulsed with 10 μg/mL of the same peptide. (d) Representative flow cytometric analysis of H-2K d and H-2D d expression on ASB-XIV and ASB-XIV TAP2KO cells. Experiment performed three times. (e) H-2D d and H-2K d staining of ASB XIV TAP2KO cells incubated with increasing concentrations of PGPGRVAKI peptide displayed as mean fluorescence intensity (MFI) ( ± SEM) (n = 3 independent experiments). (f) Schematic representation of the generation of the mEml4-Alk immortalized cell lines. (g) Sanger sequencing chromatogram showing the mElm4-Alk inversion. The mouse Eml4-Alk inversion involves exon 14 of Eml4 and exon 20 of mouse Alk . (h) Representative immunoblot analysis (N = 3 independent experiments with similar results) showing mEML4-ALK protein expression in two mEml4-Alk (mEml4-Alk 1 and mEml4-Alk 2 ) immortalized cell lines. The K-Ras G12D KP1233 cell line was used as a negative control; ALK SP8 antibody recognizes the murine EML4-ALK (arrow); ALK D5F3 antibody recognizes the human EML4-ALK in NCI-H3122. *Indicates a non-specific band recognized by the SP8 antibody. (i) Dose response curves of mElm4-Alk 1 cells to different ALK TKIs (crizotinib, N = 2 independent experiments; alectinib, N = 1; ceritinib, N = 1; brigatinib, N = 2 independent experiments; and lorlatinib, N = 2 independent experiments). ( j) Representative immunoblot for the indicated proteins in mEml4-Alk 1 cells treated with crizotinib and lorlatinib at the indicated concentrations for 6 h (N = 2 independent experiments with similar results). (k) Subcutaneous tumor growth (mm 3 ± SEM) of mEml4-Alk −1 and mEml4-Alk −2 immortalized cell lines in NSG and syngeneic BALB/c mice (N = 4 mice per group). (l) Quantification of volume changes compared with baseline tumor volume (change from baseline, % ± SEM) in syngeneic BALB/c mice injected subcutaneously with a mEml4-Alk cell line and treated as indicated. Red arrow indicates the end of treatment (N = 5 mice per group).

Article Snippet: Human ALK-rearranged ALCL cell lines (DEL and Karpas-299) were obtained from DSMZ German cell collection; the murine ASB-XIV cell line, derived from BALB/c mice was purchased from Cell Lines Service (CLS, product number # 400120), and the murine KP1233 lung cancer cell line, immortalized from C57BL/6 KRAS G12D mice, was kindly gifted by Tyler Jacks (Koch Institute, Cambridge, MA).

Techniques: Staining, Isolation, In Vitro, Expressing, Incubation, Fluorescence, Sequencing, Western Blot, Negative Control, Injection

Journal: Nature cancer

Article Title: ALK peptide vaccination restores the immunogenicity of ALK-rearranged non-small cell lung cancer

doi: 10.1038/s43018-023-00591-2

Figure Lengend Snippet: (a) Representative H&E image of lung tumors generated in syngeneic BALB/c mice injected intravenously with mEml4-Alk. 2x magnification (left panel); 40x magnification (right panel) (n = 3 independent mice). Scale bar left = 1.5 mm; Scale bar right= 100 μm. (b) Schematic illustration of mouse and human ALK short peptide 7 sequences differences and CRISPR/Cas9-edited DNA bases (Red). NetMHC4.0 showing peptide affinity (predicted IC50 values in nM) for H-2D d and NetMHCpan4.1 showing the rank of the elution ligand likelihood (%Rank_EL) for H-2D d . (c) Sanger sequencing chromatogram showing the cDNA sequence of mEml4-Alk (upper panel), Eml4-Alk PGPGRVAKI-1 (middle panel), and Eml4-Alk PGPGRVAKI-2 (lower panel) of PGPGRVAKI peptide. (d,e) Dose response curves of Eml4-Alk PGPGRVAKI-1 (d) and Eml4-Alk PGPGRVAKI-2 (e) cell lines to different ALK inhibitors (crizotinib, alectinib, ceritinib, brigatinib, and lorlatinib). Two independent experiments were performed for each ALK inhibitor in each cell line. (f) Representative immunoblot for the indicated proteins in Eml4-Alk PGPGRVAKI-1 cells treated with lorlatinib at the indicated concentrations for 6 h (N = 2 independent experiments with similar results). (g) Representative H&E of lung tumors generated in syngeneic BALB/c mice injected intravenously with Eml4-Alk PGPGRVAKI-1 . 2x magnification (left panel); 40x magnification (right panel) (n = 3 independent mice). Scale bar left = 1.5 mm; Scale bar right= 100 μm. (h) IFN-γ-ELISPOT analysis of freshly isolated splenocytes from tumor-bearing BALB/c mice 15 days after been injected either subcutaneously (flank) or intravenously (lung) with mEml4-Alk (left panel), Eml4-Alk PGPGRVAKI-1 (middle panel), and Eml4-Alk PGPGRVAKI-2 (right panel) cells. Data is shown as average number of spot forming units (SFU ± SEM). Unpaired two-tailed Student’s t test, not significant, P = 0.2038 ; ** P < 0.005; *** P < 0.001. The N shown on the figure corresponds to the number of mice per group. (i,j) Dextramer staining of PGPGRVAKI-specific CD8 + T cells isolated from splenocytes (i) and PGPGRVAKI-specific tumor infiltrating T lymphocytes (TILs) isolated from lung tumors ( j) at day 15 post subcutaneous (flank) and intravenous (lung) injection. Mice were injected with mElm4-Alk (left panel), with Eml4-Alk PGPGRVAKI-1 (middle panel), and with Eml4-Alk PGPGRVAKI-2 (right panel) cells (%±SEM). Unpaired two-tailed Student’s t test. The N shown on the figure corresponds to the number of mice per group. (k) Tumor growth of mice injected subcutis in the flank with mEml4-Alk 1 cells and treated as indicated. Data are shown as average tumor volume (mm 3 ± SEM). (l) Kaplan-Meier curves showing overall survival of mice injected intravenously with Eml4-Alk PGPGRVAKI-1 and treated as indicated. Log-rank test), not significant, P = 0.6040 ).

Article Snippet: Human ALK-rearranged ALCL cell lines (DEL and Karpas-299) were obtained from DSMZ German cell collection; the murine ASB-XIV cell line, derived from BALB/c mice was purchased from Cell Lines Service (CLS, product number # 400120), and the murine KP1233 lung cancer cell line, immortalized from C57BL/6 KRAS G12D mice, was kindly gifted by Tyler Jacks (Koch Institute, Cambridge, MA).

Techniques: Immunopeptidomics, Generated, Injection, CRISPR, Sequencing, Western Blot, Enzyme-linked Immunospot, Isolation, Two Tailed Test, Staining

Journal: International journal of molecular sciences

Article Title: Interactions of CFTR and Arylsulfatase B (ARSB; N-acetylgalactosamine-4-sulfatase) in Prostate Carcinoma.

doi: 10.3390/ijms26094350

Figure Lengend Snippet: Figure 2. Effects of ARSB and CFTR inhibition. (A). Knockdown of ARSB by siRNA had no effect on CFTR mRNA expression in the normal PEC (n = 6). (B). Inhibition of CFTR by either the chemical inhibitor CFTRinh-172 or CFTR siRNA reduced ARSB expression (p < 0.01, n = 3), but had no impact on expression of GALNS (galactose 6-sulfate sulfatase) and significantly increased expression of Type 1 collagen (Collagen 1; COL1A2) (p = 0.008, p = 0.002). (C). When PC3 cells were treated with CFTR siRNA, ARSB activity declined (p = 0.006, n = 3). The effect of ARSB overexpression was inhibited by CFTR knockdown (p = 0.004, n = 3). (D). ARSB silencing increased total sulfated glycosaminoglycans (GAGs) and chondroitin 4-sulfate (C4S) (p = 0.002, 0.0001, n = 3) in the normal prostate epithelial cells. Similarly, following CFTR silencing, total sulfated (GAG) and C4S increased (p = 0.03, p = 0.0004, n = 3), consistent with inhibition of ARSB when CFTR is silenced. * represents p ≤0.05; ** is for p ≤0.01, *** for p ≤0.001, and **** for p ≤0.0001. [ARSBox = arylsulfatase B overexpression; GAGs = glycosaminoglycans; N.D. = no difference; PEC = prostate epithelial cells; Vcn = vector control].

Article Snippet: Overexpression of ARSB (NCBI NM_000046, transcript variant 1; TrueClone, OriGene, Rockville, MD, USA) was performed by transfection of specific untagged plasmids in pCMV6-XL4 vector using 2 μg of the plasmid and LipofectamineTM 2000 (Invitrogen, Carlsbad, CA, USA) [12].

Techniques: Inhibition, Knockdown, Expressing, Activity Assay, Over Expression, Plasmid Preparation, Control

Journal: International journal of molecular sciences

Article Title: Interactions of CFTR and Arylsulfatase B (ARSB; N-acetylgalactosamine-4-sulfatase) in Prostate Carcinoma.

doi: 10.3390/ijms26094350

Figure Lengend Snippet: Figure 3. Inhibition of CFTR replicates effects of ARSB knockdown on MMP2, MMP9, and invasive- ness. (A). Expression of MMP2 and MMP9 is significantly increased in malignant prostate tissue, compared to adjacent normal tissue (p = 0.002, p = 0.002; n = 3). (B). Expression of MMP2 and MMP9 is greater in the PC3 cells than in the normal PEC (p = 0.015, p = 0.013; n = 3). (C). Inhibition of CFTR by either CFTRinh-172 or siRNA in the PC3 cells increased the expression of MMP2 (p = 0.005, p = 0.003; n = 3) and MMP9 (p = 0.002, p = 0.002; n = 3). (D,E) ARSB silencing significantly increased MMP2 and MMP9 expression in the PEC cells (p = 0.002, p = 0.05; n = 3) and PC3 cells (p < 0.0001, p = 0.01; n = 3). (F). When ARSB was overexpressed in the PC3 cells, MMP2 and MMP9 expression declined (p = 0.0017, p = 0.026; n = 3). ARSB overexpression reduced the impact of CFTR silencing on MMP9 and MMP2 expression (p = 0.0016, p = 0.0005; n = 3). (G). Invasiveness of the PC3 cells increased when CFTR or ARSB was silenced (p = 0.0014, p = 0.004; n = 3). ARSB overexpression reduced the in- vasiveness to less than the baseline level in the PC3 cells (p = 0.0005; n = 3). The CFTR siRNA-induced increase in invasiveness was reduced when ARSB was overexpressed (p = 0.0014; n = 3). * represents p ≤0.05; ** is for p ≤0.01, *** for p ≤0.001, and **** for p < 0.0001. [ARSBox = ARSB overexpression; MMP = matrix metalloproteinase; PEC = prostate epithelial cells; si = siRNA; Vcn = vector control].

Article Snippet: Overexpression of ARSB (NCBI NM_000046, transcript variant 1; TrueClone, OriGene, Rockville, MD, USA) was performed by transfection of specific untagged plasmids in pCMV6-XL4 vector using 2 μg of the plasmid and LipofectamineTM 2000 (Invitrogen, Carlsbad, CA, USA) [12].

Techniques: Inhibition, Knockdown, Expressing, Over Expression, Plasmid Preparation, Control

Journal: International journal of molecular sciences

Article Title: Interactions of CFTR and Arylsulfatase B (ARSB; N-acetylgalactosamine-4-sulfatase) in Prostate Carcinoma.

doi: 10.3390/ijms26094350

Figure Lengend Snippet: Figure 4. Effects of ARSB and CFTR on c-Myc and EGFR and BrdU incorporation. (A). EGF (10 ng/mL × 24 h) increased BrdU incorporation in the PC3 cells (p = 0.0004; p = 3). Both ARSB silencing and CFTR silencing further increased the response (p = 0.004, p = 0.0008; n = 3). ARSB overexpression reduced the BrdU incorporation (p = 0.0003; n = 3) and inhibited the effect of CFTR knockdown (p = 0.0009; n = 3). (B). In PC3 cells, mRNA expression of c-Myc and EGFR was greater than in normal PEC (p = 0.004, p = 0.00013; n = 3). (C). In the PEC, silencing ARSB increased mRNA expression of c-Myc (p = 0.004; n = 3) and EGFR (p = 0.00013; n = 3). (D). In PC3 cells, ARSB siRNA increased c-Myc (p = 0.008; n = 3) and EGFR (p = 0.02; n = 3). (E). In PC3 cells, CFTR inhibition by CFTRinh-172 or by siRNA significantly increased c-Myc (p = 0.0002, p = 0.0004; n = 3) and EGFR (p = 0.007, p = 0.0004; n = 3) expression. (F). In PC3 cells, CFTR siRNA significantly increased c-Myc (p = 0.005; n = 3) and EGFR (p = 0.0008; n = 3). ARSB overexpression reduced their expression (p = 0.0025, p = 0.0022; n = 3). In combination with CFTR silencing, ARSB overexpression reduced the CFTR knockdown-induced increases in c-Myc and EGFR (p = 0.066, p = 0.003; n = 3). (G). EGFR protein was significantly increased in the malignant compared to the adjacent non-malignant human prostate tissues (p < 0.0001; n = 6). * represents p ≤0.05; ** is for p ≤0.01, *** for p ≤0.001, and **** for p ≤0.0001. [ARSBox = ARSB overexpression; ARSBsi = ARSB siRNA; CFTRsi = CFTR siRNA; controlsi = control siRNA; EGF = epidermal growth factor; EGFR = epidermal growth factor receptor; PEC = prostate epithelial cells; Vcn = vector control].

Article Snippet: Overexpression of ARSB (NCBI NM_000046, transcript variant 1; TrueClone, OriGene, Rockville, MD, USA) was performed by transfection of specific untagged plasmids in pCMV6-XL4 vector using 2 μg of the plasmid and LipofectamineTM 2000 (Invitrogen, Carlsbad, CA, USA) [12].

Techniques: BrdU Incorporation Assay, Over Expression, Knockdown, Expressing, Inhibition, Control, Plasmid Preparation

Journal: bioRxiv

Article Title: “Patient-specific Alzheimer-like pathology in trisomy 21 cerebral organoids reveals BACE2 as a gene-dose-sensitive AD-suppressor in human brain”

doi: 10.1101/2020.01.29.918037

Figure Lengend Snippet: a For Fluorescence In Situ Hybridisation (FISH), organoid slices were processed and probed with a MetaSystems Probes for chromosome 21 (red) and chromosome 13 (green). Quantification was performed automatically using IMARIS software (nuclei were scored for 1, 2, 3 or >3 spots n>500 nuclei for each line). Scale bar: 3μm. b APP-4G8 antibody (green), BACE2 (red), MAP2 (magenta), DAPI (blue). Scale bar: 10μm. c Aβx-34 neo-epitope specific antibody (green), BACE2 (red), MAP2 (magenta), DAPI (blue). Scale bar: 10μm. d Quantification was performed blinded to the genotype, on 5 independent images representing three individual organoids per genotype, and containing 3,000-4,000 cells per image. Only images within the “cortical” part of the organoid were considered for the analysis. Graphs show total fluorescence intensity of positive signals for each wavelength for a given antibody, normalised by the total fluorescence intensity of MAP2 as a pan-neuronal marker. Additional staining (not shown) was analysed for Aβx-40 neo-epitope specific antibody. Image analysis was performed using IMARIS software. Error bars: standard error, and p-values: calculated after Holm-Bonferroni correction (α=0.05) of sequential two-tailed student t-test comparisons.

Article Snippet: Fig. 2: The FRET assay positive control was performed using recombinant human BACE2 at 370C, pH=3.5 for 2h in the R&D systems assay buffer, as specified in the manufacturer’s protocol, using the R&D systems FRET control peptide (ES010).

Techniques: Fluorescence, In Situ, Hybridization, Software, Marker, Staining, Two Tailed Test

Journal: bioRxiv

Article Title: “Patient-specific Alzheimer-like pathology in trisomy 21 cerebral organoids reveals BACE2 as a gene-dose-sensitive AD-suppressor in human brain”

doi: 10.1101/2020.01.29.918037

Figure Lengend Snippet: The amino acid cleavage site for each secretase is noted in parentheses, and for sites in Aβ the respective peptide position is also noted. Secretases for each cleavage site are η: MT5-MMP, δ: AEP, β: BACE1, α: ADAM10, θ: BACE2, γ: gamma secretase complex. In addition to it’s role as a θ-secretase, in which C99 is cleaved to preclude Aβ formation, in this report we highlight the role of BACE2 to act on Aβ as substrate, as an Aβ-degrading protease (AβDP).

Article Snippet: Fig. 2: The FRET assay positive control was performed using recombinant human BACE2 at 370C, pH=3.5 for 2h in the R&D systems assay buffer, as specified in the manufacturer’s protocol, using the R&D systems FRET control peptide (ES010).

Techniques:

Journal: bioRxiv

Article Title: “Patient-specific Alzheimer-like pathology in trisomy 21 cerebral organoids reveals BACE2 as a gene-dose-sensitive AD-suppressor in human brain”

doi: 10.1101/2020.01.29.918037

Figure Lengend Snippet: a Using Aβ IP-MS spectra from organoid (see Extended Data Figs. 1, 2, 4) conditioned media (CM), ratios were calculated of areas under the peak between the non-amyloidogenic and amyloidogenic peptides within a single mass-spectrogram. IP-MS spectra were produced for 3 timepoints (4 timepoints in exp3) for each iPSC-derived organoid line, in each of 3 independent experiments (each experiment starting at the point of undifferentiated iPSC). The team performing the IP-MS analysis was blinded to the genotypes in all experiments. BACE2-AβDP (clearance) products=[1-20&1-34], total BACE2=[1-19&1-20&1-34], amyloidogenic peptides=[1-38&1-39&1-40&1-42], α-site products=[1-16&1-17]. Exp1 and Exp2 p-values: Holm-Bonferroni sequential corrections (α=0.05) of 2-tailed student t-test comparisons. Exp3: Holm-corrected p-values after one way ANOVA. Error bars: standard error. Combined data for the isogenic iPSC lines for all 3 experiments passed the Holm-Bonferroni correction (α=0.05) of sequential 2-tailed student t-test comparisons of each peptide ratio shown in Fig1a (available on request). T21 and D21: isogenic iPCS derived from a single mosaic individual with DS published previously (Murray A et al. 2015), QM-DS1 and QM-DS2: unrelated DS iPSC, DupAPP: FEOAD iPSC. b All 3 experiments in Fig. 1a were combined to calculate the ratios of BACE2-related non-amyloidogenic peptides (1-19 or 1-34) to BACE2-unrelated non-amyloidogenic peptides (1-16 or 1-17) in organoid CM. Holm-corrected p-values after one way ANOVA are shown. Error bars: standard error. c Same ratios as in part A were calculated on IP-MS spectra obtained from cerebrospinal fluid samples of people with DS and age-matched normal controls. Data are presented as mean ± 1SD.

Article Snippet: Fig. 2: The FRET assay positive control was performed using recombinant human BACE2 at 370C, pH=3.5 for 2h in the R&D systems assay buffer, as specified in the manufacturer’s protocol, using the R&D systems FRET control peptide (ES010).

Techniques: Protein-Protein interactions, Produced, Derivative Assay

Journal: bioRxiv

Article Title: “Patient-specific Alzheimer-like pathology in trisomy 21 cerebral organoids reveals BACE2 as a gene-dose-sensitive AD-suppressor in human brain”

doi: 10.1101/2020.01.29.918037

Figure Lengend Snippet: Note: two new SNPs (rs746064 and rs9983496 new data in this paper) correlating (with nominal statistical significance) with the age of dementia onset found by the genetic analysis of the LonDownS cohort of people with DS (n=554) (purple font) cluster in close proximity with one previously published SNP (red font), and all 3 SNPs are contained within a 12 kb intron1 deletion (blue line) that on its own causes EOAD in a non-DS patient. Genomic position of SNPs which have been implicated previously in sporadic Alzheimer’s disease onset in Finnish population (black font). UCSC browser snapshot of human Chr21 region 41,100,000-41,345,000 (GRCh38/hg38) containing the BACE2 locus using Gencode v24 and curated RefSeq transcript annotation is given. Underneath, the layered H3K27ac track (ENCODE) indicates putative promoter and enhancer elements present in a panel human cell lines (see legend on the right). DNase clusters (ENCODE) mark accessible chromatin.

Article Snippet: Fig. 2: The FRET assay positive control was performed using recombinant human BACE2 at 370C, pH=3.5 for 2h in the R&D systems assay buffer, as specified in the manufacturer’s protocol, using the R&D systems FRET control peptide (ES010).

Techniques:

Journal: bioRxiv

Article Title: “Patient-specific Alzheimer-like pathology in trisomy 21 cerebral organoids reveals BACE2 as a gene-dose-sensitive AD-suppressor in human brain”

doi: 10.1101/2020.01.29.918037

Figure Lengend Snippet: A newly custom designed FRET reagent (spanning the Aβ34 site) was digested at pH=3.5 by the human BACE2 in presence or absence of the stated inhibitors for 2h. Enzyme activity was defined by measuring the fluorescence increase before and after the incubation. Blank-subtracted fluorescence units were normalized to the control digest and a one-way ANOVA was performed. P-values were calculated with a post-hoc Bonferroni multiple comparison (only pairs relative to the untreated control simultaneously compared). Error bars: standard error.

Article Snippet: Fig. 2: The FRET assay positive control was performed using recombinant human BACE2 at 370C, pH=3.5 for 2h in the R&D systems assay buffer, as specified in the manufacturer’s protocol, using the R&D systems FRET control peptide (ES010).

Techniques: Activity Assay, Fluorescence, Incubation, Control, Comparison

Journal: bioRxiv

Article Title: “Patient-specific Alzheimer-like pathology in trisomy 21 cerebral organoids reveals BACE2 as a gene-dose-sensitive AD-suppressor in human brain”

doi: 10.1101/2020.01.29.918037

Figure Lengend Snippet: Pairwise Pearson’s coefficient of colocalisation for a pair of co-stained antibodies: Aβx-34, and a specific marker for the sub-cellular vesicle compartment: lipid rafts (Flotillin1), lysosomes (LAMP1), macro-autophagosomes (LC3A), early endosomes (EEA1), macro-autophagosome-lysosome fusion/exosomes (Sortilin), late endosomes (Rab7), specific sub-sets of lysosomes (LAMP2, LAMP2A) and CMA-chaperone (HSC70). In the final two columns of the histogram, the Pearson’s colocalisation level was shown between Aβx-34 and BACE1 or BACE2, respectively (repeated in more detail in Supplementary Figure 8). Representative images of the organoid stainings from which the coefficients were calculated are shown in the panels. Last column in the bottom right panel is the zoomed-in inset from the previous column. Images were captured using Airy-Scan Zeiss confocal microscopy, and single 0.16μm slices are shown (from 20μm full z-stack analysed). Error bars: standard error, p-values: after standard one-way ANOVA using post-hoc Bonferroni multiple comparison calculation. Scale bar: 5μm.

Article Snippet: Fig. 2: The FRET assay positive control was performed using recombinant human BACE2 at 370C, pH=3.5 for 2h in the R&D systems assay buffer, as specified in the manufacturer’s protocol, using the R&D systems FRET control peptide (ES010).

Techniques: Staining, Marker, Confocal Microscopy, Comparison

Journal: bioRxiv

Article Title: “Patient-specific Alzheimer-like pathology in trisomy 21 cerebral organoids reveals BACE2 as a gene-dose-sensitive AD-suppressor in human brain”

doi: 10.1101/2020.01.29.918037

Figure Lengend Snippet: a Pairwise Pearson’s coefficient of colocalised volume for a pair of co-stained antibodies: Aβx-34 in all combinations, either BACE1 or BACE2 as a second antibody, and a marker of the sub-cellular vesicle compartment (shown at the bottom of each 3-columns histogram) as a third antibody. Error bars: standard error, and p-values: standard one-way ANOVA using post-hoc Bonferroni correction for multiple comparisons calculation. Representative individual z-slice images for the calculations performed in a are shown in b and c. b BACE1 (green), c BACE2 (green), with either Aβx-34, Sortilin or LAMP2 (red) and DAPI (blue). Scale bar: 10μm.

Article Snippet: Fig. 2: The FRET assay positive control was performed using recombinant human BACE2 at 370C, pH=3.5 for 2h in the R&D systems assay buffer, as specified in the manufacturer’s protocol, using the R&D systems FRET control peptide (ES010).

Techniques: Staining, Marker

Journal: bioRxiv

Article Title: “Patient-specific Alzheimer-like pathology in trisomy 21 cerebral organoids reveals BACE2 as a gene-dose-sensitive AD-suppressor in human brain”

doi: 10.1101/2020.01.29.918037

Figure Lengend Snippet: a Immunofluorescence analysis of the brain of DS-AD-1 co-stained for Aβx-34, BACE2 and GFAP. A typical near-circular neuritic plaque is shown (in which DAPI faintly stains the fibrillar amyloid deposits). Arrows indicate 3 categories of objects in which the colocalisation of BACE2 and the AβDP product Aβx-34 is observed. White arrows: intra-neuronal fine-vesicular pattern; White arrowheads: large intra-neuronal spherical granules (lipofuscin); Black arrows with white arrowheads: amorphous extra-cellular aggregates. See Methods and FigS6 for experiments controlling the extent of lipofuscin autofluorescence effects. b DS-AD1 brain co-stained for Aβx-34 and BACE1, or Aβx-40, BACE2 and GFAP, and Pearson’s coefficient of colocalisation for proteins stained in parts A and B, with the addition of the staining for Aβx-42 neo-epitope (not shown). Error bars: SEM. c Same I.F. staining combinations as in part A (except for GFAP) were used in 3 additional brain samples: DS-AD-2, 3 and 4. d brain sample co-stained for BACE2 and Aβx-34 of a 28yrs old person with Down syndrome without dementia, and euploid non-demented (ND) controls aged 42 and 84. Scale bar: 20μm.

Article Snippet: Fig. 2: The FRET assay positive control was performed using recombinant human BACE2 at 370C, pH=3.5 for 2h in the R&D systems assay buffer, as specified in the manufacturer’s protocol, using the R&D systems FRET control peptide (ES010).

Techniques: Immunofluorescence, Staining

Journal: bioRxiv

Article Title: “Patient-specific Alzheimer-like pathology in trisomy 21 cerebral organoids reveals BACE2 as a gene-dose-sensitive AD-suppressor in human brain”

doi: 10.1101/2020.01.29.918037

Figure Lengend Snippet: a Sudan black B was used to confirm the specificity of the Aβx-34 and BACE2 antibodies in human brain sections, and to eliminate lipofuscin autofluorescence. Three different human brain samples were used: DS-AD1, DS (28 yrs) pre-AD and euploid sporadic AD (73 yrs). Both antibodies show the same pattern of expression and colocalisation after Sudan black B staining (white arrows: intraneuronal fine-vesicular pattern and black arrows with white arrowhead: amorphous extra-cellular aggregates) except for a loss of the large intraneuronal spherical granules (white arrowheads, Fig. 3), which are likely lipofuscin. Scale bar: 5μm. b and c Chromogenic, immunohistochemical analysis of the human brain sections of DS-AD1, stained using polymer-HRP/AP double-staining kit. b The primary antibody against BACE2 was labelled with DAB (brown) and primary antibody against Aβx-34 neo-epitope was labelled with GBI-permanent-red (red); b(i) is a zoomed-in inset of the rectangle in B. c same as b, but both antibodies were pre-absorbed for 12 hours, and incubated overnight, with the excess of immunogenic peptide for the BACE2 antibody; c(i) is a zoomed-in inset of the rectangle in c. d and e: BACE2 antibody specificity control for immunofluorescence on T21 organoids (100DIV). Scale bar: 10μm. d Immunofluorescent staining with Aβx-34 and BACE2, e same as b and d , but both antibodies were pre-absorbed for 12 hours, and incubated overnight, with the excess of immunogenic peptide for the BACE2 antibody. The specificity for the neo-epitope specific antibody against Aβx-34 was extensively proven in a previous report (Cabrera et al., 2018). f and g In order to distinguish the contribution of lipofuscin auto-fluorescence to the colocalised signals, specificity of primary antibodies (Aβx-34 and BACE2) has been validated using Lambda (λ) scan function on confocal microscope (see Methods). f Aβx-34 shows specific peak in different ROI and uniform pattern on the three different human brain samples: DS-AD1 (59 yrs), DS (28 yrs) pre-AD and DS (8 months). As negative control of staining, DAPI and secondary antibody alone were used. g BACE2 also shows specific peak in different ROI and uniform pattern in human brain. h secondary antibody alone control. Scale bar: 20μm.

Article Snippet: Fig. 2: The FRET assay positive control was performed using recombinant human BACE2 at 370C, pH=3.5 for 2h in the R&D systems assay buffer, as specified in the manufacturer’s protocol, using the R&D systems FRET control peptide (ES010).

Techniques: Expressing, Staining, Immunohistochemical staining, Polymer, Double Staining, Incubation, Control, Immunofluorescence, Fluorescence, Microscopy, Negative Control

Journal: bioRxiv

Article Title: “Patient-specific Alzheimer-like pathology in trisomy 21 cerebral organoids reveals BACE2 as a gene-dose-sensitive AD-suppressor in human brain”

doi: 10.1101/2020.01.29.918037

Figure Lengend Snippet: a BACE2 exon3 sequence with 7bp deletion (yellow) provoked by the CRISPR/SpCas9-HF1 is shown. Red: restriction endonuclease HpyCH4IV sites (a de novo HpyCH4IV site is generated by the 7bp deletion). b agarose gel electrophoresis of the 733 bp PCR product containing the targeted site before (uncut) and after digestion with HpyCH4IV(cut), for the initial clone 2.5, and its colony-purified sub-clone 2.3.5 (renamed further below as “Δ7”). The 294bp fragment in 2.3.5 is reduced to 65% of the wt value (normalized to the 439bp band), and a de novo 255 bp fragment appears in CRSPR targeted line (red asterisk). c Western blot stained with anti-BACE2 antibody of the lysates of the iPSC line Δ7 compared to the wt T21C5 iPSC line. Quantification of the total actin-normalised BACE2 signal showed a significant reduction in Δ7 compared to T21 unedited line. Error bars: standard error, p-value: student’s t-test. d BACE2 AβDP/amyloidogenic peptides ratio after IP-MS analysis of CM produced by the 48DIV organoids derived from the iPSC line Δ7 compared to the T21C5wt control were significantly decreased. Error bars: standard error, p-values: two-tailed t-test comparison.

Article Snippet: Fig. 2: The FRET assay positive control was performed using recombinant human BACE2 at 370C, pH=3.5 for 2h in the R&D systems assay buffer, as specified in the manufacturer’s protocol, using the R&D systems FRET control peptide (ES010).

Techniques: Sequencing, CRISPR, Generated, Agarose Gel Electrophoresis, Purification, Western Blot, Staining, Protein-Protein interactions, Produced, Derivative Assay, Control, Two Tailed Test, Comparison

Journal: bioRxiv

Article Title: “Patient-specific Alzheimer-like pathology in trisomy 21 cerebral organoids reveals BACE2 as a gene-dose-sensitive AD-suppressor in human brain”

doi: 10.1101/2020.01.29.918037

Figure Lengend Snippet: a Following CRISPR editing, C5Δ7 iPSC line was assessed by SNP array and no genomic alternations were detected compared to the parental C5 iPSC line. B-allele frequency and LogR ratio plots comparing these two lines are shown for chromosome 21. Data for the whole genome available on request. b To further confirm retention of trisomy after BACE2 CRISPR editing, selected other genes on chromosome 21 were validated as trisomic using quantitative paralogous amplification/pyrosequencing method. GABPA and ITSN allele number were quantified relative to paralogous sequence mismatches on other chromosomes. GABPA and ITSN both show approximate 60:40 ratios for trisomic cells, and 50:50 ratios for disomic cells as expected. Quantified nucleotides are shown on the pyrogram in shaded grey boxes and the relative values for the corresponding peaks are shown.

Article Snippet: Fig. 2: The FRET assay positive control was performed using recombinant human BACE2 at 370C, pH=3.5 for 2h in the R&D systems assay buffer, as specified in the manufacturer’s protocol, using the R&D systems FRET control peptide (ES010).

Techniques: CRISPR, Amplification, Sequencing

Journal: bioRxiv

Article Title: “Patient-specific Alzheimer-like pathology in trisomy 21 cerebral organoids reveals BACE2 as a gene-dose-sensitive AD-suppressor in human brain”

doi: 10.1101/2020.01.29.918037

Figure Lengend Snippet: Western blot stained with anti-BACE2 antibody or anti-APP antibody of the lysates of the iPSC line Δ7 compared to the wt T21C5, and D21C3 iPSC lines. Quantification of the total actin-normalised BACE2 signal showed a 27% reduction in Δ7 compared to T21 unedited line, and no significant difference compared to D21 control. Quantification of the total actin-normalised APP signal showed no significant difference between Δ7 and unedited T21 line, whereas they both had significantly higher APP protein levels compared to the disomic control line. Error bars: standard error, p-values after standard one way ANOVA and Tukey’s multiple comparisons test.

Article Snippet: Fig. 2: The FRET assay positive control was performed using recombinant human BACE2 at 370C, pH=3.5 for 2h in the R&D systems assay buffer, as specified in the manufacturer’s protocol, using the R&D systems FRET control peptide (ES010).

Techniques: Western Blot, Staining, Control

Journal: Frontiers in Physiology

Article Title: Murine Myocardial Transcriptome Analysis Reveals a Critical Role of COPS8 in the Gene Expression of Cullin-RING Ligase Substrate Receptors and Redox and Vesicle Trafficking Pathways

doi: 10.3389/fphys.2017.00594

Figure Lengend Snippet: Cardiac CSN8 deficiency causes significant changes in the mRNA levels of myocardial CRL substrate receptors in mice. Shown are microarray-derived mRNA expression levels of the indicated CRL substrate receptors in CTL, Het-CKO, and Hom-CKO littermate mice at 2- and 3-weeks-of-age. (A) Fbxo31 , F-box only protein 31; (B) Klhl41 , Kelch like 41; (C) Klhdc1 , Kelch domain containing 1; (D) Fbxo14 , F-box only protein 14; (E) Klhl31 , Kelch like 31; (F) Asb14 , ankyrin repeat and SOCS box-containing protein 14. Mean ± SEM, n = 3 mice/group; * p < 0.05, one way ANOVA followed by Tukey's modified Student's t -test

Article Snippet: To probe for FBXO31, 60 μg of total proteins, and to probe for Asb-14 and TTLL1, 100 μg of total proteins were separated by SDS-PAGE, transferred to PVDF membrane, and blotted with rabbit anti-FBXO31 antibody (1:500, Abcam), Anti-Asb14 antibody (1:200, Santa Cruz), and anti-TTLL1 antibody (Santa Cruz, 1:1,000).

Techniques: Microarray, Derivative Assay, Expressing, Modification

Journal: Frontiers in Physiology

Article Title: Murine Myocardial Transcriptome Analysis Reveals a Critical Role of COPS8 in the Gene Expression of Cullin-RING Ligase Substrate Receptors and Redox and Vesicle Trafficking Pathways

doi: 10.3389/fphys.2017.00594

Figure Lengend Snippet: Western blot analyses for myocardial ASB14 in control and homozygous Csn8-CKO (Hom-CKO) mice at 2 weeks (A,B) and 3 weeks (C,D) of age. α-Actinin was probed as loading control. Mean ± SEM, n = 3 mice/group, Student's t -test.

Article Snippet: To probe for FBXO31, 60 μg of total proteins, and to probe for Asb-14 and TTLL1, 100 μg of total proteins were separated by SDS-PAGE, transferred to PVDF membrane, and blotted with rabbit anti-FBXO31 antibody (1:500, Abcam), Anti-Asb14 antibody (1:200, Santa Cruz), and anti-TTLL1 antibody (Santa Cruz, 1:1,000).

Techniques: Western Blot, Control

Journal: Journal of animal science and biotechnology

Article Title: CLOCK inhibits the proliferation of porcine ovarian granulosa cells by targeting ASB9.

doi: 10.1186/s40104-023-00884-7

Figure Lengend Snippet: Fig. 4 Transcriptomic profiling of GCs with CLOCK overexpression treatment. A A volcano plot of the expressed genes. B Heatmap of the differentially expressed genes in GCs overexpressing CLOCK according to RNA-seq. C Gene ontology analysis. BP: biological process; CC: cellular component; MF: molecular function. D Kyoto Encyclopedia of Genes and Genomes pathway analysis. E RT-qPCR detected the expression levels of ASB9. Data are expressed as mean ± SEM (n = 3), **P < 0.01. F Quantitative statistics of ASB9. Data are expressed as mean ± SEM (n = 3), *P < 0.05. G Western blotting revealed the expression levels of ASB9

Article Snippet: Table 1 The information of antibodies Reagent type Designation Source Catalog No. Dilution rate/concentration Antibody GAPDH Abways AB0036 WB(1:5,000) Antibody CLOCK Abways CY6972 WB(1:1,000), IF(1:100) Antibody CCNB1 Abways CY5378 WB(1:1,000) Antibody CCND1 Abways CY5404 WB(1:1,000) Antibody CCNE1 Abways CY1028 WB(1:1,000) Antibody CDK4 Abways CY5836 WB(1:1,000) Antibody CDKN1A Abways CY5088 WB(1:1,000) Antibody ASB9 Santa Cruz sc-166723 WB(1:1,000) Antibody HRP conjugated AffiniPure goat anti-mouse IgG (H + L) Boster BA1051 WB(1:5,000) Antibody HRP conjugated AffiniPure goat anti-rabbit IgG (H + L) Boster BA1054 WB(1:5,000) Antibody CY3 conjugated AffiniPure goat anti-rabbit IgG (H + L) Boster BA1032 IF(1:100) Antibody Anti-mouse IgG goat monoclonal antibody Boster M04575-3 ChIP (1μg) Antibody CLOCK Santa Cruz sc-271603 ChIP (1μg)

Techniques: Over Expression, RNA Sequencing, Quantitative RT-PCR, Expressing, Western Blot

Journal: Journal of animal science and biotechnology

Article Title: CLOCK inhibits the proliferation of porcine ovarian granulosa cells by targeting ASB9.

doi: 10.1186/s40104-023-00884-7

Figure Lengend Snippet: Fig. 6 ASB9 interference promotes GCs proliferation. A RT-qPCR detected the interference efficiency of ASB9. Data are expressed as mean ± SEM (n = 6), **P < 0.01. B Western blotting reveals the expression levels of ASB9. C Quantification of the western blot analysis. Data are expressed as mean ± SEM (n = 3), *P < 0.05. D EdU staining was used to detect the number of proliferating cells. RED, EdU-positive cells; BLUE, Hoechst staining for total nuclei. Data are expressed as mean ± SEM (n = 4), **P < 0.01. E CCK-8 assay detecting cell viability at 24 h after transfection. Data are expressed as mean ± SEM (n = 16), ****P < 0.0001. F RT-qPCR analysis of proliferation-related genes, including CCNB1, CCND1, CCNE1, CDK1, and CDK4. Data are expressed as mean ± SEM (n = 5), *P < 0.05, **P < 0.01. G Western blot analysis of proliferation-related gene protein level (ASB9, CCNB1, CCNE1, CDK4, and CDKN1A). H Quantifying the western blot analysis of CLOCK, CCNB1, CCNE1, CDK4, and CDKN1A. Data are expressed as mean ± SEM (n = 3), *P < 0.05, **P < 0.01. I RNA expression of ASB9 in GCs. ZT: zone time

Article Snippet: Table 1 The information of antibodies Reagent type Designation Source Catalog No. Dilution rate/concentration Antibody GAPDH Abways AB0036 WB(1:5,000) Antibody CLOCK Abways CY6972 WB(1:1,000), IF(1:100) Antibody CCNB1 Abways CY5378 WB(1:1,000) Antibody CCND1 Abways CY5404 WB(1:1,000) Antibody CCNE1 Abways CY1028 WB(1:1,000) Antibody CDK4 Abways CY5836 WB(1:1,000) Antibody CDKN1A Abways CY5088 WB(1:1,000) Antibody ASB9 Santa Cruz sc-166723 WB(1:1,000) Antibody HRP conjugated AffiniPure goat anti-mouse IgG (H + L) Boster BA1051 WB(1:5,000) Antibody HRP conjugated AffiniPure goat anti-rabbit IgG (H + L) Boster BA1054 WB(1:5,000) Antibody CY3 conjugated AffiniPure goat anti-rabbit IgG (H + L) Boster BA1032 IF(1:100) Antibody Anti-mouse IgG goat monoclonal antibody Boster M04575-3 ChIP (1μg) Antibody CLOCK Santa Cruz sc-271603 ChIP (1μg)

Techniques: Quantitative RT-PCR, Western Blot, Expressing, Staining, CCK-8 Assay, Transfection, RNA Expression

Journal: Journal of animal science and biotechnology

Article Title: CLOCK inhibits the proliferation of porcine ovarian granulosa cells by targeting ASB9.

doi: 10.1186/s40104-023-00884-7

Figure Lengend Snippet: Fig. 8 Schematic diagram of CLOCK regulation on porcine GC proliferation. CLOCK inhibits cell proliferation by promoting ASB9 expression in porcine ovarian GCs. Specifically, CLOCK and BMAL1 complex binds to the E-box element of ASB9 promoter to increase the level of ASB9. Then, ASB9 inhibits GC proliferation by regulating cell cycle

Article Snippet: Table 1 The information of antibodies Reagent type Designation Source Catalog No. Dilution rate/concentration Antibody GAPDH Abways AB0036 WB(1:5,000) Antibody CLOCK Abways CY6972 WB(1:1,000), IF(1:100) Antibody CCNB1 Abways CY5378 WB(1:1,000) Antibody CCND1 Abways CY5404 WB(1:1,000) Antibody CCNE1 Abways CY1028 WB(1:1,000) Antibody CDK4 Abways CY5836 WB(1:1,000) Antibody CDKN1A Abways CY5088 WB(1:1,000) Antibody ASB9 Santa Cruz sc-166723 WB(1:1,000) Antibody HRP conjugated AffiniPure goat anti-mouse IgG (H + L) Boster BA1051 WB(1:5,000) Antibody HRP conjugated AffiniPure goat anti-rabbit IgG (H + L) Boster BA1054 WB(1:5,000) Antibody CY3 conjugated AffiniPure goat anti-rabbit IgG (H + L) Boster BA1032 IF(1:100) Antibody Anti-mouse IgG goat monoclonal antibody Boster M04575-3 ChIP (1μg) Antibody CLOCK Santa Cruz sc-271603 ChIP (1μg)

Techniques: Expressing