Journal: bioRxiv

Article Title: PRC1 catalytic activity is central to Polycomb system function

doi: 10.1101/667667

Figure Lengend Snippet: (A) A schematic of the endogenous Ring1b allele in PRC1 CPM cell line before and after addition of OHT, showing positions of the primers used for RT-qPCR quantification of conversion from Ring1b WT to Ring1b I53A/D56K . In untreated cells, incorporation of wild-type exon 3 into Ring1b mRNA gives RT-qPCR signal from the primer pair RT1-RT3, but not RT1-RT2. Following OHT treatment and flipping of the exon 3 cassette, incorporation of I53A/D56K version of exon 3 into Ring1b mRNA gives RT-qPCR signal from the primer pair RT2-RT3, but not RT1-RT3. (B) RT-qPCR validation of PRC1 CPM line using primers described in (A). Error bars represent SEM (n=4). (C) DNA sequencing traces of Ring1b mRNA in untreated and OHT-treated PRC1 CPM cells, showing complete conversion from RING1B WT to RING1B I53A/D56K . Vertical dashed lines indicate boundaries between exons. Amino acid sequences are shown above corresponding DNA sequence, with I53A and D56K positions highlighted in red. Wild-type and I53A/D56K mutant versions of exon 3 were engineered to be different at wobble position of each triplet codon, to allow each exon to be easily distinguished and minimize formation of secondary RNA structures that could impact on splicing. (D) A chromosome density plot showing H2AK119ub1 cChIP-seq across chromosome 18 in untreated (blue) and OHT-treated (red) PRC1 CPM cells. (E) A schematic of the endogenous Ring1b allele in PRC1 CKO cell line before and after addition of OHT, showing parallel LoxP sites flanking exon 2 (the first coding exon). OHT treatment causes CRE-mediated deletion of exon 2 which puts the rest of the Ring1b coding sequence out of frame, resulting in no functional protein being produced. (F) A chromosome density plot showing H2AK119ub1 cChIP-seq data across chromosome 18 in untreated (blue) and OHT-treated (red) PRC1 CKO cells.

Article Snippet: The following day, ChIP samples and Inputs were incubated with Proteinase K (Sigma) for 1.5 hours at 56°C and purified using ChIP DNA Clean and Concentrator Kit (Zymo Research). cChIP-seq libraries for both ChIP and Input samples were prepared using NEBNext Ultra DNA Library Prep Kit for Illumina, following manufacturer’s guidelines.

Techniques: Quantitative RT-PCR, DNA Sequencing, Sequencing, Mutagenesis, Functional Assay, Produced

Journal: bioRxiv

Article Title: PRC1 catalytic activity is central to Polycomb system function

doi: 10.1101/667667

Figure Lengend Snippet: (A) Heatmap analyses of cChIP-seq for H3K27me3, H2AK119ub1, RING1B, PCGF2, PCGF1 and PCGF6 at RING1B-bound sites in PRC1 CPM cells (untreated and OHT-treated). Also shown is BioCAP-seq (measure of non-methylated CpG-rich DNA) and ChIP-seq for MAX and H3K4me3 in wild-type ESCs. The genomic intervals were sorted based on the log2 fold change in RING1B occupancy following tamoxifen treatment in PRC1 CPM ESCs. (B) Maximum intensity projections of RING1B-Halo-JF 549 signal in PRC1 CPM ESCs (untreated and OHT-treated). Examples of RING1B nuclear foci (Polycomb bodies) are indicated by arrowheads. Scale bar is 5 µm. (C) Box plots comparing the number of all ( left panel ) or top 25% highest intensity RING1B-Halo-JF 549 foci per nucleus ( right panel ) in PRC1 CPM ESCs before (n cells = 69) and after OHT treatment (n cells = 83). P-values denote statistical significance calculated by a Student’s t-test. (D) Genomic snapshots of two genes, showing cChIP-seq for RING1B, PCGF1, PCGF6 and PCGF2 in PRC1 CPM ESCs (untreated and OHT-treated). H2AK119ub1 cChIP-seq in untreated cells and H3K4me3 ChIP-seq in wild-type ESCs is also shown. Cbln2 is a lowly transcribed gene with high-level RING1B occupancy which relies on PRC1 catalysis and Ptges3 is a more highly transcribed gene at which low-level RING1B binding is independent of catalysis. (E) Correlation of RING1B cChIP-seq signal in untreated and OHT-treated PRC1 CPM ESCs with cChIP-seq signal of PRC2 (SUZ12 and H3K27me3) and PRC1 (PCGF2, PCGF1, PCGF6, and H2AK119ub1) in untreated and OHT-treated PRC1 CPM ESCs. Correlation with BioCAP-seq and ChIP-seq for MAX and H3K4me3 in wild-type ESCs is also shown. This reveals a switch from chromatin-based to DNA-based target site selection by PRC1 following loss of PRC1 catalysis. (F) Relative enrichment of RING1B, PCGF1, PCGF6 and PCGF2 cChIP-seq signal in PRC1 CPM ESCs (untreated and OHT-treated) across promoter-proximal RING1B-bound sites divided into percentiles based on the expression level of the associated gene in untreated PRC1 CPM cells. For each factor, enrichment is shown relative to the fiftieth percentile. Lines represent smoothed conditional means based on loess local regression fitting. See also .

Article Snippet: The following day, ChIP samples and Inputs were incubated with Proteinase K (Sigma) for 1.5 hours at 56°C and purified using ChIP DNA Clean and Concentrator Kit (Zymo Research). cChIP-seq libraries for both ChIP and Input samples were prepared using NEBNext Ultra DNA Library Prep Kit for Illumina, following manufacturer’s guidelines.

Techniques: Methylation, ChIP-sequencing, Binding Assay, Selection, Expressing

Journal: The Journal of Biological Chemistry

Article Title: FK506-binding protein-like and FK506-binding protein 8 regulate dual leucine zipper kinase degradation and neuronal responses to axon injury

doi: 10.1016/j.jbc.2022.101647

Figure Lengend Snippet: FKBPL and FKBP8 induced lysosome-dependent DLK degradation. A , comparative analysis of relative expression levels in mouse L4,5 dorsal root ganglion (DRG) tissues, sciatic nerve tissues, and cultured embryonic DRG neurons ( , , , ). Red and blue circles indicate Illumina short-read sequencing and Oxford Nanopore direct RNA long-read sequencing, respectively. Circle sizes indicate relative levels of microarray data from cultured embryonic DRG neurons. B , Western blot analysis for the expression of DLK with FKBPs (N; null vector, L; FKBPL, 3; FKBP3, 4; FKBP4, 8; FKBP8, 12; FKBP12, 14; FKBP14, 15; FKBP15). The number indicates normalized relative intensity. Dual leucine zipper kinase and FLAG-epitope-tagged FKBP protein family members were expressed in HEK293T cells and subjected to SDS-PAGE. C , Western blot analysis for the immunoprecipitation of DLK with mouse (m) and human (h) FKBP4/8 that was overexpressed in HEK293T cells. Empty arrowhead , non-specific band; blue arrowhead , FKBP4; red arrowhead , FKBP8. D , Western blot analysis for the expression of DLK and FKBPL/4/8 expressed in HEK293T cells with or without bafilomycin A1 treatment. The numbers indicate the normalized relative intensity. E , Western blot analysis of DLK protein levels under Fkbp8 knockdown (sh Fkbp8 ) by lentiviral delivery in primary cultured embryonic DRG neurons. The numbers indicate the normalized relative intensity. F , statistical analysis of ( E ) (FC, fold change; n = 3 for each condition; ∗ p < 0.05 by t test; mean ± S.E.M.). DLK, dual leucine zipper kinase.

Article Snippet: An expression plasmid for full-length Myc-DDK-tagged Mouse Fkbp3 (MR202616), Fkbp4 (MR227193), Fkbp8 (MR220865), Fkbp12 (MR200405), Fkbp14 (MR202290), and Fkbp15 (MR220579) were purchased from Origene.

Techniques: Expressing, Cell Culture, Sequencing, Microarray, Western Blot, Plasmid Preparation, FLAG-tag, SDS Page, Immunoprecipitation

Journal: Age

Article Title: Global gene expression profile of normal and regenerating liver in young and old mice

doi: 10.1007/s11357-015-9796-7

Figure Lengend Snippet: YAP expression in old and young livers after PH. a Microarray analysis of Yap mRNA. mRNA expression was determined in old and young mice after PH. Gene expression is reported as fold difference relative to age-matched controls. b Western blot analysis of YAP and phospho-YAP proteins. Old and young mice were sacrificed 12, 24, and 36 h after 2/3 PH. Total extracts (100 to 150 mg/lane) were prepared as described in “Materials and methods” section. Appropriate loading was confirmed by staining the gel with Coomassie Blue and using anti-actin antibody as loading control. Each lane represents a pool of three livers. CO controls. c Western blot analysis of cyclin D1, PCNA, YAP, and phospho-YAP in livers from mice subjected to PH with or without TCPBOP pretreatment (TCP + PH and PH, respectively). Young mice were subjected to PH 3 days after treatment with the mitogen TCP, as described in “Materials and methods” section. Animals were sacrificed 72 h after PH. Total extracts (100 to 150 mg/lane) were prepared from the livers, and Western analysis was performed as described in “Materials and methods” section. Appropriate loading was confirmed by staining the gel with Coomassie Blue and using anti-actin antibody as loading control. Each lane represents a pool of three livers. CO controls. d Western blot analysis of nuclear YAP in TCP + PH or PH livers. Nuclear extracts for YAP (100 to 150 mg/lane) were prepared, and Western analysis was performed as described in “Materials and methods” section. Appropriate loading was confirmed by staining the gel with Coomassie Blue and using anti-albumin antibody as loading control. Each lane represents a pool of three livers. CO controls

Article Snippet: The following antibodies were used: mouse monoclonal antibodies against cyclin D1 (72–13), cyclin D2, cyclin D3, and proliferating cell nuclear antigen (PCNA) (Santa Cruz Biotechnology, CA); actin (clone AC-40) (Sigma Chem.

Techniques: Expressing, Microarray, Gene Expression, Western Blot, Staining, Control

Journal: Biosensors & bioelectronics

Article Title: Microarray method to monitor 40 intestinal bacterial species in the study of azo dye reduction

doi: 10.1016/j.bios.2004.04.011

Figure Lengend Snippet: Bacteria and the probe numbers in the microarray

Article Snippet: Anaerobic bacteria were cultured at 35 °C in either prereduced anaerobically sterilized (PRAS) Brain Heart Infusion (BIH) broth supplemented with vitamin K and hemin (Remel, Lenexa, KS, USA), inoculated under an oxygen-free cannula using 85% nitrogen, 10% hydrogen and 5% carbon dioxide, or on PRAS brucella blood agar plates supplemented with vitamin K and hemin (Remel). table ft1 table-wrap mode="anchored" t5 caption a7 Number Bacterial species and strain Probe number 1 B. thetaiotaomicron ATCC 29148 1, 2, 3 2 B. vulgatus ATCC 8482 4, 5, 6 3 B. fragilis ATCC 23745 7, 8, 9 4 B. distasonis ATCC 8503 10, 11, 12 5 C. clostridioforme ATCC 29084 13, 14, 15 6 C. leptum ATCC 29065 16, 17, 18 7 F. prausnitzii ATCC 27768 19, 20, 21 8 P. productus ATCC 27340 22, 23, 24 9 R. obeum ATCC 29174 25, 26, 27 10 R. bromii ATCC 27255 28, 29, 30 11 R. callidus ATCC 27760 31, 32, 33 12 R. albus ATCC 27210 34, 35, 36 13 B. longum ATCC 15707 37, 38, 39 14 B. adolescentis ATCC 15703 40, 41, 42 15 B. infantis ATCC 15697 43, 44, 45 16 E. biforme ATCC 27806 46, 47, 48 17 E. aerofaciens ATCC 25986 49, 50, 51 18 L. acidophilus ATCC 4356 52, 53, 54 19 E. coli ATCC 25922 55, 56, 57 20 E. faecium ATCC 19434 58, 59, 60 21 B. uniformis ATCC 8492 61, 62, 63 22 B. ovatus ATCC 8483 64, 65, 66 23 B. caccae ATCC 43185 67, 68, 69 24 C. perfringens ATCC 13124 70, 71, 72 25 C. butyricum ATCC 19398 73, 74, 75 26 C. ramosum ATCC 25582 76, 77, 78 27 C. difficile ATCC 9689 79, 80, 81 28 C. indolis ATCC 25771 82, 83, 84 29 F. russii ATCC 25533 85, 86, 87 30 F. nucleatum ATCC 25586 88, 89, 90 31 B. catenulatum ATCC 27539 91, 92, 93 32 B. angulatum ATCC 27535 94, 95, 96 33 E. rectale ATCC 33656 97, 98, 99 34 E. eligens ATCC 27750 100, 101, 102 35 E. limosum ATCC 8486 103, 104, 105 36 E. lentum ATCC 25553 106, 107, 108 37 L. fermentum ATCC 9338 109, 110, 111 38 E. faecalis ATCC 27274 112, 113, 114 39 P. magnus ATCC 14955 115, 116, 117 40 R. gnavus ATCC 291492 118, 119, 120 Open in a separate window Bacteria and the probe numbers in the microarray

Techniques: Bacteria

Journal: Biosensors & bioelectronics

Article Title: Microarray method to monitor 40 intestinal bacterial species in the study of azo dye reduction

doi: 10.1016/j.bios.2004.04.011

Figure Lengend Snippet: Microarray test results read from

Article Snippet: Anaerobic bacteria were cultured at 35 °C in either prereduced anaerobically sterilized (PRAS) Brain Heart Infusion (BIH) broth supplemented with vitamin K and hemin (Remel, Lenexa, KS, USA), inoculated under an oxygen-free cannula using 85% nitrogen, 10% hydrogen and 5% carbon dioxide, or on PRAS brucella blood agar plates supplemented with vitamin K and hemin (Remel). table ft1 table-wrap mode="anchored" t5 caption a7 Number Bacterial species and strain Probe number 1 B. thetaiotaomicron ATCC 29148 1, 2, 3 2 B. vulgatus ATCC 8482 4, 5, 6 3 B. fragilis ATCC 23745 7, 8, 9 4 B. distasonis ATCC 8503 10, 11, 12 5 C. clostridioforme ATCC 29084 13, 14, 15 6 C. leptum ATCC 29065 16, 17, 18 7 F. prausnitzii ATCC 27768 19, 20, 21 8 P. productus ATCC 27340 22, 23, 24 9 R. obeum ATCC 29174 25, 26, 27 10 R. bromii ATCC 27255 28, 29, 30 11 R. callidus ATCC 27760 31, 32, 33 12 R. albus ATCC 27210 34, 35, 36 13 B. longum ATCC 15707 37, 38, 39 14 B. adolescentis ATCC 15703 40, 41, 42 15 B. infantis ATCC 15697 43, 44, 45 16 E. biforme ATCC 27806 46, 47, 48 17 E. aerofaciens ATCC 25986 49, 50, 51 18 L. acidophilus ATCC 4356 52, 53, 54 19 E. coli ATCC 25922 55, 56, 57 20 E. faecium ATCC 19434 58, 59, 60 21 B. uniformis ATCC 8492 61, 62, 63 22 B. ovatus ATCC 8483 64, 65, 66 23 B. caccae ATCC 43185 67, 68, 69 24 C. perfringens ATCC 13124 70, 71, 72 25 C. butyricum ATCC 19398 73, 74, 75 26 C. ramosum ATCC 25582 76, 77, 78 27 C. difficile ATCC 9689 79, 80, 81 28 C. indolis ATCC 25771 82, 83, 84 29 F. russii ATCC 25533 85, 86, 87 30 F. nucleatum ATCC 25586 88, 89, 90 31 B. catenulatum ATCC 27539 91, 92, 93 32 B. angulatum ATCC 27535 94, 95, 96 33 E. rectale ATCC 33656 97, 98, 99 34 E. eligens ATCC 27750 100, 101, 102 35 E. limosum ATCC 8486 103, 104, 105 36 E. lentum ATCC 25553 106, 107, 108 37 L. fermentum ATCC 9338 109, 110, 111 38 E. faecalis ATCC 27274 112, 113, 114 39 P. magnus ATCC 14955 115, 116, 117 40 R. gnavus ATCC 291492 118, 119, 120 Open in a separate window Bacteria and the probe numbers in the microarray

Techniques: Microarray