Journal: bioRxiv

Article Title: ATP-binding cassette protein ABCF1 couples gene transcription with maintenance of genome integrity in embryonic stem cells

doi: 10.1101/2020.05.28.122184

Figure Lengend Snippet: (A) Coomassie staining of Mono S fraction 14 demonstrates purification to homogeneity. The breakdown products observed in silver staining in are minor compared to the full-length product. Mass spectrometry analysis of Mono S peak activity fractions (#14-16) in identifies the ~110 kDa polypeptide as ABCF1. (B) ABCF1 is enriched in human ES cells. Downregulation of ABCF1 in human ES cell line H9 upon exit of pluripotency. H9 human ES cells are induced to differentiate in differentiation medium (DMEM/F12 with FBS). Cells are collected at day 7 and day 14 post induction. mRNA levels of ABCF1 are analyzed by quantitative PCR (qPCR) and normalized to β-actin ( ACTB ). Western blot analyses of whole cell extracts (WCEs) prepared from H9 cells collected at indicated days are performed using antibodies against ABCF1, pluripotency marker OCT4, and ACTB as loading control. (C) ABCF1 is enriched in mouse ES cells. mRNA and protein levels of ABCF1 are analyzed in pluripotent D3 mouse ES cells carrying V5-epitope tagged Abcf1 alleles (V5-ABCF1 knock-in), and in the same ES cell line induced to differentiate by retinoic acid (RA) treatment for 7 days (RA d7) by qPCR and western blotting, respectively. Error bars present SEM. n = 3. (*) P < 0.05, calculated by two-sided Student’s t-test. The following figure supplement is available for : .

Article Snippet: Polyclonal antibodies against ABCF1 (13950-1-AP) was purchased from ProteinTech, XPC (A301-122A) and mouse Nanog (A300-397A) from Bethyl Laboratories, DKC1 (H-300), OCT4 (N-19), and Pol II (N-20) from Santa Cruz Biotechnology, SOX2 (AB5603) from EMD Millipore, V5 ChIP grade (ab15828) from Abcam.

Techniques: Staining, Purification, Silver Staining, Mass Spectrometry, Activity Assay, Real-time Polymerase Chain Reaction, Western Blot, Marker, Control, Knock-In

Journal: bioRxiv

Article Title: ATP-binding cassette protein ABCF1 couples gene transcription with maintenance of genome integrity in embryonic stem cells

doi: 10.1101/2020.05.28.122184

Figure Lengend Snippet: ABCF1 is highly enriched in the transcriptionally active, partially purified nuclear extract fractions. Comparative western blot analysis of NT2 nuclear extract (NE), phosphocellulose 0.3 M, 0.5 M, 1 M KCl fractions (P0.3, P0.5, P1M, respectively), and Ni-NTA flowthrough (Ni-FT) using an antibody against ABCF1. P0.3 and P0.5 fractions do not contain SCCs . Each lane contains 5 μ g of protein as determined by Bradford protein assay.

Article Snippet: Polyclonal antibodies against ABCF1 (13950-1-AP) was purchased from ProteinTech, XPC (A301-122A) and mouse Nanog (A300-397A) from Bethyl Laboratories, DKC1 (H-300), OCT4 (N-19), and Pol II (N-20) from Santa Cruz Biotechnology, SOX2 (AB5603) from EMD Millipore, V5 ChIP grade (ab15828) from Abcam.

Techniques: Purification, Western Blot, Bradford Protein Assay

Journal: bioRxiv

Article Title: ATP-binding cassette protein ABCF1 couples gene transcription with maintenance of genome integrity in embryonic stem cells

doi: 10.1101/2020.05.28.122184

Figure Lengend Snippet: (A) shRNA-mediated knockdown of ABCF1 in mouse ES cells. WCEs of D3 cells transduced with lentiviruses expressing control non-targeting shRNA (shNT) or two independent shRNAs against ABCF1 (shABCF1-1 or shABCF1-2) are analyzed by western blotting to confirm knockdown efficiency. ACTB is used as loading control. Asterisk denotes a non-specific band. (B) shRNA-mediated depletion of ABCF1 in D3 cells leads to colony collapse with flattened cell morphology and reduced alkaline phosphatase (AP) staining, indicating spontaneous differentiation. (C) Loss of ABCF1 in mouse ES cells compromises pluripotency gene expression. Quantification of mRNA levels of pluripotency genes are analyzed by qPCR and normalized to Actb . mRNA level in each sample is expressed relative to its respective control set as 1. (D) Depletion of ABCF1 induces expression of differentiation-associated genes. Lineage-specific genes representing the three embryonic germ layers and the trophectoderm are analyzed by qPCR as in (C). Error bars present SEM. n = 3. (*) P < 0.05, calculated by two-sided Student’s t-test. The following figure supplement is available for : .

Article Snippet: Polyclonal antibodies against ABCF1 (13950-1-AP) was purchased from ProteinTech, XPC (A301-122A) and mouse Nanog (A300-397A) from Bethyl Laboratories, DKC1 (H-300), OCT4 (N-19), and Pol II (N-20) from Santa Cruz Biotechnology, SOX2 (AB5603) from EMD Millipore, V5 ChIP grade (ab15828) from Abcam.

Techniques: shRNA, Knockdown, Transduction, Expressing, Control, Western Blot, Staining, Gene Expression

Journal: bioRxiv

Article Title: ATP-binding cassette protein ABCF1 couples gene transcription with maintenance of genome integrity in embryonic stem cells

doi: 10.1101/2020.05.28.122184

Figure Lengend Snippet: (A) Efficiency of shRNA-mediated knockdown of ABCF1 in D3 mouse ES cells is determined by qPCR. Abcf1 mRNA levels are normalized to Actb and expressed as fraction of control (=1). (B) H9 human ES cells are transfected with non-targeting siRNA (siNT) or siRNAs against ABCF1 (siABCF1). Knockdown efficiency is analyzed by qPCR as in (A). Downregulation of ABCF1 in human ES cells compromises stem cell self-renewal. Phase contrast images of human H9 ES cells transfected with siNT or siABCF1. (C) Depletion of ABCF1 blocks somatic cell reprogramming. CF-1 MEFs are transduced with lentiviruses expressing either a control shRNA (NT) or shRNAs targeting ABCF1 (shABCF1-1 and shABCF1-2), together with lentiviruses expressing OCT4, KLF4, SOX2, and c-MYC. Cells are plated at the indicated number in 24-well plates; cellular reprogramming is initiated by the addition of doxycycline (dox). Cells are stained for alkaline phosphatase (AP) activity (left) and counted (right) after 14 days (11 days with dox followed by 3 days of dox withdrawal) post induction (dpi). (D) Single cells suspensions of 14 dpi reprogrammed CF-1 MEFs as described in (C) are stained with anti-mouse SSEA-1 and analyzed by flow cytometry. Error bars present SEM. n = 3. (*) P < 0.05, calculated by two-sided Student’s t-test.

Article Snippet: Polyclonal antibodies against ABCF1 (13950-1-AP) was purchased from ProteinTech, XPC (A301-122A) and mouse Nanog (A300-397A) from Bethyl Laboratories, DKC1 (H-300), OCT4 (N-19), and Pol II (N-20) from Santa Cruz Biotechnology, SOX2 (AB5603) from EMD Millipore, V5 ChIP grade (ab15828) from Abcam.

Techniques: shRNA, Knockdown, Control, Transfection, Transduction, Expressing, Staining, Activity Assay, Flow Cytometry

Journal: bioRxiv

Article Title: ATP-binding cassette protein ABCF1 couples gene transcription with maintenance of genome integrity in embryonic stem cells

doi: 10.1101/2020.05.28.122184

Figure Lengend Snippet: (A) Expression of V5-tagged ABCF1 (V5-ABCF1) in two independent knock-in (KI) D3 mouse ES cell clones are verified by western blotting using antibodies against V5 and ABCF1. ACTB is used as loading control. (B) V5 tagging of ABCF1 in mouse ES cells (D3 KI-1, −2) does not compromise ABCF1 function. mRNA levels of Oct4 , Sox2 , Nanog , Klf4 , and Fgf4 are quantified by qPCR and normalized to Actb . V5-ABCF1 knock-in clones display low expression level of differentiation-associated gene, Fgf5 , comparable to WT cells. (C) ChIP-qPCR analysis of ABCF1 enrichment on housekeeping gene Dhfr as described in . (D) MNase-digested chromatin from EGS/formaldehyde crosslinked V5-ABCF1 KI mouse ES cells are purified and separated on an agarose gel. (E) MNase-ChIP analysis of ABCF1 on the Oct4, Sox2, and Actb genes by qPCR as described in . Error bars present SEM. n = 3. (*) P < 0.05, calculated by two-sided Student’s t-test.

Article Snippet: Polyclonal antibodies against ABCF1 (13950-1-AP) was purchased from ProteinTech, XPC (A301-122A) and mouse Nanog (A300-397A) from Bethyl Laboratories, DKC1 (H-300), OCT4 (N-19), and Pol II (N-20) from Santa Cruz Biotechnology, SOX2 (AB5603) from EMD Millipore, V5 ChIP grade (ab15828) from Abcam.

Techniques: Expressing, Knock-In, Clone Assay, Western Blot, Control, ChIP-qPCR, Purification, Agarose Gel Electrophoresis

Journal: bioRxiv

Article Title: ATP-binding cassette protein ABCF1 couples gene transcription with maintenance of genome integrity in embryonic stem cells

doi: 10.1101/2020.05.28.122184

Figure Lengend Snippet: (A) Chromatin immunoprecipitation (ChIP) analysis of ABCF1 occupancy on control and enhancer regions of Oct4 and Sox2 gene loci in V5-ABCF1 knock-in D3 mouse ES cells. Representative data showing the enrichment of V5-ABCF1 (grey bars) compared to control IgGs (white bars) are analyzed by qPCR and expressed as percentage of input chromatin. (B) ABCF1 is recruited to strong SOX2-bound genomic regions. ABCF1 enrichment at the enhancer regions of Lefty1 and Trim28 genes is analyzed as described in (A). (C) ABCF1 is not recruited to the promoter of housekeeping gene Actb . Enrichment of ABCF1 is analyzed as in (A). (D) MNase-ChIP analysis of ABCF1 occupancy on the Nanog gene promoter. Enrichment of ABCF1 is analyzed as in (A). Schematic diagrams of OCT4/SOX2 binding sites of each gene and the relative positions of the amplicons are shown at the bottom. Error bars present SEM. n = 3. (*) P < 0.05, calculated by two-sided Student’s t-test. The following figure supplement is available for :

Article Snippet: Polyclonal antibodies against ABCF1 (13950-1-AP) was purchased from ProteinTech, XPC (A301-122A) and mouse Nanog (A300-397A) from Bethyl Laboratories, DKC1 (H-300), OCT4 (N-19), and Pol II (N-20) from Santa Cruz Biotechnology, SOX2 (AB5603) from EMD Millipore, V5 ChIP grade (ab15828) from Abcam.

Techniques: Chromatin Immunoprecipitation, Control, Knock-In, Binding Assay

Journal: bioRxiv

Article Title: ATP-binding cassette protein ABCF1 couples gene transcription with maintenance of genome integrity in embryonic stem cells

doi: 10.1101/2020.05.28.122184

Figure Lengend Snippet: (A) Intrinsically disordered protein prediction tool, IUPred2 ( https://iupred2a.elte.hu/ ) , is used to identify unstructured regions in both human ABCF1 and yeast GCN20. X-axis indicates position of the amino acids and Y-axis shows the probability of disordered sequences. Regions that are above the value of 0.5 are predicted to be unstructured and those below 0.5 are more likely structured. Relative positions of the protein domains are depicted below. Similar results are also obtained using a different prediction program DISOPRED3 (data not shown) . (B) Comparison of distribution of glutamine (Q), glutamic acid (E), and lysine (K) residues in ABCF1 from indicated organisms, analyzed as described in . (C) SDS-PAGE and Coomassie staining of endogenous ABCF1 purified from NT2 nuclear extracts (NT2), purified recombinant full-length and truncated ABCF1 proteins described in . (D) Amino acid composition of yeast GCN20, analyzed as described in . The relative positions of NBDs and the non-conserved N-terminal region of GCN20 are indicated. One-letter abbreviations for amino acids are indicated (Left): C, Cys; W, Trp; Y, Tyr; F, Phe; M, Met; L, Leu; I, Ile; V, Val; A, Ala; G, Gly; P, Pro; Q, Gln; N, Asn; T, Thr; S, Ser; E, Glu; D, Asp; K, Lys; H, His; R, Arg. (E) SDS-PAGE and Coomassie staining of recombinant human, mouse, and yeast homolog of ABCF1 (GCN20) as well as the human-yeast hybrid protein (H/Y).

Article Snippet: Polyclonal antibodies against ABCF1 (13950-1-AP) was purchased from ProteinTech, XPC (A301-122A) and mouse Nanog (A300-397A) from Bethyl Laboratories, DKC1 (H-300), OCT4 (N-19), and Pol II (N-20) from Santa Cruz Biotechnology, SOX2 (AB5603) from EMD Millipore, V5 ChIP grade (ab15828) from Abcam.

Techniques: Comparison, SDS Page, Staining, Purification, Recombinant

Journal: bioRxiv

Article Title: ATP-binding cassette protein ABCF1 couples gene transcription with maintenance of genome integrity in embryonic stem cells

doi: 10.1101/2020.05.28.122184

Figure Lengend Snippet: (A) Amino acid composition of human ABCF1. Each of the 20 amino acids is counted and marked as a black bar at that position in ABCF1. The schematic diagram (top) denotes protein domains of ABCF1: intrinsically disordered region (IDR, yellow, amino acids 1-302); two nucleotide-binding domains (NBDs, blue). One-letter abbreviations for amino acids are indicated (Left): C, Cys; W, Trp; Y, Tyr; F, Phe; M, Met; L, Leu; I, Ile; V, Val; A, Ala; G, Gly; P, Pro; Q, Gln; N, Asn; T, Thr; S, Ser; E, Glu; D, Asp; K, Lys; H, His; R, Arg. (B) Schematic diagram of full-length, wild-type (WT) ABCF1 protein depicting a predicted N-terminal IDR (yellow) containing a polyglutamine (polyQ) tract and lysine (K)/glutamic acid (E)-rich regions. The two conserved lysine residues (K324, K664) in the Walker A motif of each of the two NBDs (blue) in ABCF1 are highlighted. Full-length WT ABCF1, ATP-binding defective lysine-to-methionine mutant (2KM), various truncated ABCF1 proteins lacking part (Δ248, Δ115) or all of the IDR (Δ302) as well as the IDR by itself (1-302) are purified from E. coli . (C) Transcriptional activities of the various recombinant ABCF1 proteins shown in (B) are assayed (over a two-fold concentration range) together with recombinant XPC and DKC1 complexes in in vitro transcription as described in . (D) Schematic representation of the human and mouse ABCF1, and yeast homolog GCN20. The percentage of sequence similarity among human, mouse, and yeast homolog is indicated. Domain-swapped hybrid protein between the human IDR and yeast NBDs (H/Y) is generated and purified from E. coli . (E) Titration over a two-fold concentration range of human and mouse ABCF1, yeast GCN20, and human-yeast hybrid (H/Y) proteins are assayed in in vitro transcription reactions. The following figure supplement is available for : .

Article Snippet: Polyclonal antibodies against ABCF1 (13950-1-AP) was purchased from ProteinTech, XPC (A301-122A) and mouse Nanog (A300-397A) from Bethyl Laboratories, DKC1 (H-300), OCT4 (N-19), and Pol II (N-20) from Santa Cruz Biotechnology, SOX2 (AB5603) from EMD Millipore, V5 ChIP grade (ab15828) from Abcam.

Techniques: Binding Assay, Mutagenesis, Purification, Recombinant, Concentration Assay, In Vitro, Sequencing, Generated, Titration

Journal: bioRxiv

Article Title: ATP-binding cassette protein ABCF1 couples gene transcription with maintenance of genome integrity in embryonic stem cells

doi: 10.1101/2020.05.28.122184

Figure Lengend Snippet: (A) GST-fusion proteins containing the IDR of human ABCF1 (1-302), the N-terminal domain of yeast GCN20 (1-197), and the transactivation domain of human transcription factor SREBP1a (1-50) are incubated with buffer only (−) or NT2 nuclear extracts (NE, +). Input NE (IN) and bound proteins are analyzed by western blotting using antibodies against the largest subunit of RNA polymerase II (Pol II, RBP1), XPC, and DKC1. (B) WCEs from 293T cells co-transfected with plasmid expressing V5-ABCF1 together with either empty plasmid (−) or plasmids expressing FLAG-tagged OCT4 (O) or SOX2 (S) are immunoprecipitated with anti-FLAG antibody. Input WCEs (IN) and bound proteins are detected by western blotting. (C) Input V5-ABCF1 KI ES cell WCEs (IN) and IPs by IgG and anti-SOX2 antibodies are analyzed by western blotting using anti-V5 and anti-SOX2 antibodies. (D) HA IPs from 293T cells overexpressing HA-tagged SOX2 (SOX2-HA) with V5-tagged full-length (FL) or IDR-truncated human ABCF1 (Δ302). SOX2-bound V5-ABCF1 proteins are detected by western blotting. (E) ABCF1 knockdown-rescue assay. mRNAs from control D3 ES cells (shNT) overexpressing RFP, and ABCF1 knockdown ES cells (sh1) overexpressing RFP, V5-tagged full-length or IDR-truncated human ABCF1 (Δ302), or mouse NANOG are analyzed for Nanog , Fgf4 , and Klf4 mRNA levels by qPCR, normalized to Actb . Error bars present SEM. n = 3. (*) P < 0.05, calculated by two-sided Student’s t-test. The following figure supplement is available for : .

Article Snippet: Polyclonal antibodies against ABCF1 (13950-1-AP) was purchased from ProteinTech, XPC (A301-122A) and mouse Nanog (A300-397A) from Bethyl Laboratories, DKC1 (H-300), OCT4 (N-19), and Pol II (N-20) from Santa Cruz Biotechnology, SOX2 (AB5603) from EMD Millipore, V5 ChIP grade (ab15828) from Abcam.

Techniques: Incubation, Western Blot, Transfection, Plasmid Preparation, Expressing, Immunoprecipitation, Knockdown, Rescue Assay, Control

Journal: bioRxiv

Article Title: ATP-binding cassette protein ABCF1 couples gene transcription with maintenance of genome integrity in embryonic stem cells

doi: 10.1101/2020.05.28.122184

Figure Lengend Snippet: (A) SDS-PAGE and Coomassie staining of purified GST-fusion proteins containing N-terminal domain of ABCF1 (1-302), GCN20 (1-197), and SREBP1a (1-50). Comparison of distribution of glutamine (Q), glutamic acid (E), and lysine (K) residues in ABCF1 from indicated organisms, analyzed as described in . (B) Expression of control RFP and V5-tagged human ABCF1 proteins described in is confirmed by western blotting using antibodies against RFP and V5. Filled and white arrowheads denote V5-tagged full-length and IDR-truncated human ABCF1, respectively. ACTB is used as loading control.

Article Snippet: Polyclonal antibodies against ABCF1 (13950-1-AP) was purchased from ProteinTech, XPC (A301-122A) and mouse Nanog (A300-397A) from Bethyl Laboratories, DKC1 (H-300), OCT4 (N-19), and Pol II (N-20) from Santa Cruz Biotechnology, SOX2 (AB5603) from EMD Millipore, V5 ChIP grade (ab15828) from Abcam.

Techniques: SDS Page, Staining, Purification, Comparison, Expressing, Control, Western Blot

Journal: bioRxiv

Article Title: ATP-binding cassette protein ABCF1 couples gene transcription with maintenance of genome integrity in embryonic stem cells

doi: 10.1101/2020.05.28.122184

Figure Lengend Snippet: (A) V5-ABCF1 D3 mouse ES cell WCEs are incubated with three different 5’ biotinylated 98mer oligonucleotides: single-stranded (ss), double-stranded (ds) with SOX2-binding motif (Matched, ds-M), or ds without the motif (Unmatched, ds-UM). These DNA sequences are derived from Listeria monocytogenes genome. Input WCEs (IN) and streptavidin-beads captured, DNA-bound ABCF1 proteins are analyzed by western blotting. α-tubulin (TUBA) is used as control for binding specificity. (B) Genomic DNA purified from nuclear extracts prepared from DMSO and etoposide-treated (ETO, 20 μM) V5-ABCF1 knock-in (KI) D3 ES cells were analyzed on agarose gel and stained with ethidium bromide. (C) WCEs prepared from ETO-treated (20 μM) V5-ABCF1 KI D3 ES cells are incubated with IgGs or anti-V5 antibodies. Co-purified nucleic acids are treated with RNase A, separated on urea-PAGE, and stained with SYBR Gold. Vertical bar denotes DNAs specifically bound by ABCF1. (D) DNA damage disrupts ABCF1-SOX2 interaction. Input (IN) and SOX2 IPs from WCEs of DMSO or ETO-treated (20 μM) V5-ABCF1 KI D3 ES cells are analyzed by western blotting. (E) MNase-ChIP of ABCF1 in DMSO and ETO-treated (80 μM) V5-ABCF1 KI D3 ES cells. Enrichment of ABCF1 on OCT4/SOX2-targeted regions of Oct4 , Sox2 , and Nanog gene promoters is analyzed by qPCR as in . (F) Colony formation assays in control and ABCF1 gain-of-function D3 cells. 200 D3 ES cells stably expressing RFP or V5-ABCF1 are plated on 24-well plates, treated with DMSO (left) or ETO (1 μM, right) for indicated period of time (hr), and let recover for 6 days before staining for AP activity. AP-positive colonies are counted. Error bars represent SEM of three independent experiments. n = 3. (*) P < 0.05, calculated by two-sided Student’s t-test. (G) Model depicting mechanisms whereby ABCF1 couples pluripotency gene transcription with intracellular DNA sensing. ABCF1 IDR (red wavy line) promotes specific clustering and formation of a hub comprising of SOX2 (S), XPC (X), DKC1 (D), and Pol II molecules at target gene promoter to stimulate transcription by increasing local concentration of these factors. ABCF1 proteins available for transcription are diverted to bind intracellular dsDNAs generated from genome instability or pathogen infection. Decrease in ABCF1 at gene promoters destabilizes the multivalent interactions between SOX2, XPC, DKC1, and Pol II. This leads to disruption of the protein hub and decrease in gene transcription by Pol II. Downregulation of pluripotency-associated genes promotes differentiation of compromised ES cells and their elimination from the self-renewing population. The following figure supplement is available for : .

Article Snippet: Polyclonal antibodies against ABCF1 (13950-1-AP) was purchased from ProteinTech, XPC (A301-122A) and mouse Nanog (A300-397A) from Bethyl Laboratories, DKC1 (H-300), OCT4 (N-19), and Pol II (N-20) from Santa Cruz Biotechnology, SOX2 (AB5603) from EMD Millipore, V5 ChIP grade (ab15828) from Abcam.

Techniques: Incubation, Binding Assay, Derivative Assay, Western Blot, Control, Purification, Knock-In, Agarose Gel Electrophoresis, Staining, Stable Transfection, Expressing, Activity Assay, Concentration Assay, Generated, Infection, Disruption

Journal: bioRxiv

Article Title: ATP-binding cassette protein ABCF1 couples gene transcription with maintenance of genome integrity in embryonic stem cells

doi: 10.1101/2020.05.28.122184

Figure Lengend Snippet: (A) Single cell suspensions of D3 mouse ES cells transfected with 5’ 6-carboxyfluorescein (6-FAM) labeled ss, ds-M, or ds-UM on the sense strand are analyzed by flow cytometry. 6-FAM-positive cells (blue) are gated by comparing to untransfected cells (Negative). 6-FAM-positive cells are purified for further analyses. (B) Expression levels of pluripotency and differentiation genes in mouse ES cells as sorted in (A) are analyzed by qPCR. (C) Western blot analysis of nuclear and cytoplasmic fractions prepared from V5-ABCF1 knock-in mouse ES cells using antibodies against OCT4, SOX2, and TUBA. Effective nuclear-cytoplasmic fractionation is demonstrated by enrichment of OCT4 and SOX2 in nuclear extracts and TUBA in cytoplasmic fraction. (D) DNAs purified from DMSO or ETO-treated (80 μM) V5-ABCF1 KI mouse ES cells grown in 2i/LIF medium are separated on an agarose gel and visualized by ethidium bromide staining. (E) Crosslinked nuclear chromatin from DMSO or ETO-treated (80 μM) mouse cells are fragmented by MNase digestion. Digested DNAs are purified, separated on an agarose gel, and stained with ethidium bromide. Both DMSO and ETO-treated chromatins are digested to a similar degree. (F) Expression levels of pluripotency and differentiation genes in DMSO or ETO-treated (80 μM) V5-ABCF1 KI mouse ES cells are analyzed by qPCR, normalized to Actb . Error bars present SEM. n = 3. (*) P < 0.05, calculated by two-sided Student’s t-test. (G) Western blot analysis showing stable overexpression of V5-ABCF1 in wild-type D3 mouse ES cells transduced with lentiviruses expressing V5-ABCF1 compared to control mouse ES cells expressing RFP.

Article Snippet: Polyclonal antibodies against ABCF1 (13950-1-AP) was purchased from ProteinTech, XPC (A301-122A) and mouse Nanog (A300-397A) from Bethyl Laboratories, DKC1 (H-300), OCT4 (N-19), and Pol II (N-20) from Santa Cruz Biotechnology, SOX2 (AB5603) from EMD Millipore, V5 ChIP grade (ab15828) from Abcam.

Techniques: Transfection, Labeling, Flow Cytometry, Purification, Expressing, Western Blot, Knock-In, Fractionation, Agarose Gel Electrophoresis, Staining, Over Expression, Transduction, Control

Journal: bioRxiv

Article Title: An ATP-Binding Cassette Transporter Gene Links Innate and Adaptive Immune Responses

doi: 10.1101/2023.12.31.573785

Figure Lengend Snippet: ABCF1 promoter activity in CD8+ T cells from the thymus and the spleen. Spleens and thymi were dissected from threeABCF1+/- (top panels) and three of their wild-type littermate controls (bottom panels). Cells were labelled with the αnti-CD8 antibody and αnti-CD4 antibody. We depict the staining of two ABCF1+/- and one wild-type littermate controls. Data is representative of two separate experiments.

Article Snippet: Specifically, knockdown of the ABCF1 gene was accomplished using Mouse Abcf1 siRNA obtained from Santa Cruz Biotechnology (catalog number sc-140760), consistently resulting in a reduction of 79-81% compared to control samples treated with scrambled siRNA.

Techniques: Activity Assay, Staining

Journal: bioRxiv

Article Title: An ATP-Binding Cassette Transporter Gene Links Innate and Adaptive Immune Responses

doi: 10.1101/2023.12.31.573785

Figure Lengend Snippet: ABCF1 promoter activity in CD8+ T cells from the thymus and the spleen. Spleens and thymi were dissected out of three ABCF1+/- (top panels) and three of their wild-type littermate controls (bottom panels). Cells were labelled with the PE-αCD8 antibody and then stained with fluorescein by heat shock. Data is representative of two separate experiments.

Article Snippet: Specifically, knockdown of the ABCF1 gene was accomplished using Mouse Abcf1 siRNA obtained from Santa Cruz Biotechnology (catalog number sc-140760), consistently resulting in a reduction of 79-81% compared to control samples treated with scrambled siRNA.

Techniques: Activity Assay, Staining

Journal: bioRxiv

Article Title: An ATP-Binding Cassette Transporter Gene Links Innate and Adaptive Immune Responses

doi: 10.1101/2023.12.31.573785

Figure Lengend Snippet: ABCF1 +/- mice produce fewer IFN γ positive CD8+ T lymphocytes after infection with VSV. ABCF1+/- splenocytes showed fewer IFNγ positive CD8+ T lymphocytes. Figure shown represents the results of one experiment, which was later repeated three times with similar results.

Article Snippet: Specifically, knockdown of the ABCF1 gene was accomplished using Mouse Abcf1 siRNA obtained from Santa Cruz Biotechnology (catalog number sc-140760), consistently resulting in a reduction of 79-81% compared to control samples treated with scrambled siRNA.

Techniques: Infection

Journal: bioRxiv

Article Title: An ATP-Binding Cassette Transporter Gene Links Innate and Adaptive Immune Responses

doi: 10.1101/2023.12.31.573785

Figure Lengend Snippet: ABCF1+/- CTLs were as efficient as ABCF1 +/+ at killing of chromium. loaded VSV specific target lymphocytes. CTL activity was measured by the release of 51 Cr into the supernatant. Figure shown represents the results of one experiment, which was later repeated three times with similar results.

Article Snippet: Specifically, knockdown of the ABCF1 gene was accomplished using Mouse Abcf1 siRNA obtained from Santa Cruz Biotechnology (catalog number sc-140760), consistently resulting in a reduction of 79-81% compared to control samples treated with scrambled siRNA.

Techniques: Activity Assay

Journal: bioRxiv

Article Title: An ATP-Binding Cassette Transporter Gene Links Innate and Adaptive Immune Responses

doi: 10.1101/2023.12.31.573785

Figure Lengend Snippet: ABCF1 is essential for the production of ISG (Interferon-Stimulated Gene) specific cytokines expression of INF- α and β after Poly(I:C) stimulation. A bar graph illustrating the fold change in the levels of INF-α and β cytokines detected in the cell culture supernatants. These findings provide insights into the impact of Abcf1 -specific siRNA treatment, both with and without Poly(I:C), on the secretion of cytokines and chemokines as well as the activation of key signaling pathways and transcription factors within BMDMs. Expression levels of the protein was assessed by western blotting analysis.

Article Snippet: Specifically, knockdown of the ABCF1 gene was accomplished using Mouse Abcf1 siRNA obtained from Santa Cruz Biotechnology (catalog number sc-140760), consistently resulting in a reduction of 79-81% compared to control samples treated with scrambled siRNA.

Techniques: Expressing, Cell Culture, Activation Assay, Protein-Protein interactions, Western Blot

Journal: bioRxiv

Article Title: An ATP-Binding Cassette Transporter Gene Links Innate and Adaptive Immune Responses

doi: 10.1101/2023.12.31.573785

Figure Lengend Snippet: ABCF1 plays a role in regulating the phosphorylation levels of NF- κ B p65 and IRF3. A) Cytoplasmic and nuclear fractions obtained from BMDMs under different conditions were subjected to western blot analysis to assess the levels of transcription factors associated with Poly(I:C) stimulation confirming our hypothesis; Western Blot band sizes are as follows: anti- p65 (65 kD); anti p-p65 (65 kD); anti IRF3 (47 kD); anti p-IRF3 (36 kD); anti ABCF1 (110 kD); anti GAPDH (36 kD); anti histone H3 (15 kD); Dimerized-IRF3 (73 kD); IRF3 (36 kD) B) Whole-cell lysates were separated using native PAGE gel electrophoresis, and the analysis focused on examining the dimerization of IRF3, a transcription factor. Expression levels of the protein was assessed by western blotting analysis. Fold change calculations, as specified in the materials and methods section, were employed to quantify changes in the data. The bar graphs displayed in the figures represent the mean values along with standard deviations. The data presented here is representative of three separate experiments.

Article Snippet: Specifically, knockdown of the ABCF1 gene was accomplished using Mouse Abcf1 siRNA obtained from Santa Cruz Biotechnology (catalog number sc-140760), consistently resulting in a reduction of 79-81% compared to control samples treated with scrambled siRNA.

Techniques: Phospho-proteomics, Western Blot, Clear Native PAGE, Nucleic Acid Electrophoresis, Expressing

Journal: bioRxiv

Article Title: An ATP-Binding Cassette Transporter Gene Links Innate and Adaptive Immune Responses

doi: 10.1101/2023.12.31.573785

Figure Lengend Snippet: ABCF1 Parallels OAS1a Expression and Controls 2-5A Activity A) The immunoprecipitates were immunoblotted with anti-ABCF1 and anti-OAS1a antibodies. B) ABCF1 was over-expressed (Over Exp.) in BMDMs, an equal amount of whole cell lysates was immunoprecipitated with anti-ABCF1 antibody and the immunoprecipitates were detected with anti-ABCF1 and anti-OAS1a antibodies using western blots. C) Protein levels of OAS1a, ABCE1 and RNase L were analyzed from whole cell lysates from Abcf1 siRNA-treated BMDMs in presence or absence of Poly(I:C). Expression levels of the protein was assessed by western blotting analysis. D) Increased pyrophosphate production (a readout of 2-5A activity) was measured in scrambled and Abcf1 siRNA treated BMDMs in presence and absence of recombinant OAS1. E) Total RNA was extracted from Abcf1 siRNA treated BMDMs in presence or absence of Poly(I:C) and was run on a Agilent 2100 Bioanalyzer. 28S and 18S RNA have been shown and RIN score was calculated. Bars indicate the mean ±standard deviation. The data is representative of 3 separate experiments. The expression of ABCF1 follows a pattern that parallels the expression of OAS1a, and it also exerts control over the activity of 2-5A. This suggests a regulatory relationship where ABCF1’s expression correlates with OAS1a expression and influences the activity of 2-5A.

Article Snippet: Specifically, knockdown of the ABCF1 gene was accomplished using Mouse Abcf1 siRNA obtained from Santa Cruz Biotechnology (catalog number sc-140760), consistently resulting in a reduction of 79-81% compared to control samples treated with scrambled siRNA.

Techniques: Expressing, Activity Assay, Immunoprecipitation, Western Blot, Recombinant, Standard Deviation, Control