Journal: bioRxiv

Article Title: A palmitate-rich metastatic niche enables metastasis growth via p65 acetylation

doi: 10.1101/2022.10.24.513556

Figure Lengend Snippet: A . Intracellular levels of acetyl-CoA in 4T1 and MCF10A H-Ras V12 cells incubated in 2D monolayer and 3D spheroids cultures for 5 days in medium containing extra palmitate. Intracellular acetyl-CoA measurements in additional cell lines (EMT6.5 and MCF7) are shown in . Two-tailed unpaired student’s T-test (n = 4). B . Relatives changes in acetyl-CoA abundance in EMT6.5 (m.f.) breast primary tumors and lung metastases. Data are shown as fold changes compared with the acetyl-CoA abundance in the primary tumors. Two-tailed unpaired student’s T-test (n = 4). C . Relatives changes in acetyl-CoA abundance in 4T1 (m.f.) breast primary tumors and lung metastases upon acute inhibition of CPT1A using the inhibitor etomoxir (30 mg/kg i.p.) or vehicle (water). Data are shown as fold changes compared with the acetyl-CoA abundance in the primary tumors of the group of mice treated with the vehicle. Data points represented as zero were below the detection limit. One-way ANOVA with Dunnett’s multiple comparison test (n ≥4). D . Relatives changes in acetyl-CoA abundance in mouse 4T1 breast cancer spheroids upon treatment with the CPT1A inhibitor etomoxir (50 μM), transduced with a lentiviral vector with shRNA against Cpt1a (knockdown), sgRNA Cpt1a (knockout) or scrambled control sequences in the presence of extra palmitate. Intracellular acetyl-CoA measurements in additional human cell lines (MCF10A H-Ras V12 and MCF7) are shown in . One-way ANOVA with Dunnett’s multiple comparison test or two-tailed unpaired student’s T-test are shown (n = 4). E and F . 3D spheroids growth upon genetic inhibition of either Cpt1a or CPT1A together with pharmacologic ALCY inhibition using BMS-303141 (20 μM) in 4T1 ( A .) and MCF10A H-Ras V12 ( B ) cells with or without extra palmitate and in the presence of the acetate as metabolic rescue (5 mM). 3D spheroid growth is represented by the average spheroids area of >100 spheroids. One-way ANOVA with Turkey’s multiple comparison test (n ≥4).

Article Snippet: The ATP Citrate lyase (ACLY) inhibitor BMS-303141 was purchased from MedChemExpress, dissolved in DMSO and used at 20 μM.

Techniques: Incubation, Two Tailed Test, Inhibition, Comparison, Transduction, Plasmid Preparation, shRNA, Knockdown, Knock-Out, Control

Journal: Molecular cell

Article Title: Nuclear acetyl-CoA production by ACLY promotes homologous recombination

doi: 10.1016/j.molcel.2017.06.008

Figure Lengend Snippet: (A) U2OS reporter cell line transfected with indicated siRNA SMARTpools was treated with Shield-1 and 4-OHT for 5 h to induce DSBs by mCherry-Lac1-Fok1. Colocalization of BRCA1 or 53BP1 with Fok1 was imaged by IF, scale, 200 M. Representative experiment from n=3 each for 53BP1 and BRCA1. Quantitation of 15–20 fields from each sample is depicted, ****, p<0.0001, n.d.- not detected (lower left panel); A.U.- Arbitrary units. Re-drawn schematic of the reporter assay, previously published (Tang et al., 2013) is depicted (lower right panel) (B) BRCA1 colocalization with Fok1 was examined in 53BP1-deficient U2OS reporter cells. (See also Figure S2B.) n.s.- not significant; A.U.- Arbitrary units. (C) AcH4 ChIP using different primer sets for endogenous loci was performed in a U2OS-TRF1-Fok1 reporter cell line that induces DSBs within telomeres (Tang et al., 2013). DSBs were induced by treatment with 4-OHT for 5 h; mean +/− SEM for 5 h, *, p<0.05, **, p<0.01, n.s.- not significant, n.d.- not detected. See Figure S3 for primer locations. (D) HR assay performed in the DR-GFP U2OS reporter line (Pierce et al., 2001). Cells were transfected with individual siRNAs for 24 hours (siACLY A or siACLY B), followed by transfection of the endonuclease Sce-1 for an additional 48 h. GFP positivity, indicating HR efficiency, was analyzed by flow cytometer, mean +/− SEM, **, p<0.01; ***, p<0.001. (E) To assay HR, cells were transduced with EV or ACLY-expressing lentivirus. Cells were transfected with siRNA SMARTpools, followed by Sce-1 as in (D). Data is representative of 2 independent experiments. Mean +/− SEM is graphed, ***, p<0.001. (F) ACLY was silenced using two independent shRNAs in HeLa cells expressing TRF2ΔB/ΔM. NHEJ was assessed by scoring end-to-end chromosomal fusions. Mean +/− SEM **, p<0.01, ***,p<0.001. See also Figure S2 and Figure S3.

Article Snippet: HR assay To examine HR, DR-GFP reporter cells were plated at 30,000–50,000 cells/well in a 12 well plate and reverse transfected with either control hairpin or individual siRNAs targeting ACLY (Origene) with RNAiMax (Thermo Fischer Scientific) using standard protocol.

Techniques: Transfection, Quantitation Assay, Reporter Assay, Flow Cytometry, Transduction, Expressing

Journal: PLoS ONE

Article Title: Anoctamin Calcium-Activated Chloride Channels May Modulate Inhibitory Transmission in the Cerebellar Cortex

doi: 10.1371/journal.pone.0142160

Figure Lengend Snippet: PCR primer pairs used to characterize cerebellar ANO1 and ANO2.

Article Snippet: Antibodies used for immunoblotting were guinea-pig anti-ANO1 (dilution 1:400; C-terminus encoding amino acids 962–1040; marked "ANO1 in " in the text) [ ], and a rabbit ANO2 antiserum directed against the extracellular loop that connects TMDs 5 and 6 in ANO2 (Alomone Labs, ACL012, dilution 1:1000, marked “ANO2 ex ” in the text).

Techniques: Cloning

Journal: PLoS ONE

Article Title: Anoctamin Calcium-Activated Chloride Channels May Modulate Inhibitory Transmission in the Cerebellar Cortex

doi: 10.1371/journal.pone.0142160

Figure Lengend Snippet: (A) Membrane topology model for anoctamin Ca 2+ -activated Cl - channels based on the X-ray structure of a fungal TMEM16 protein . The transmembrane domains 5 and 6 are thought to provide the pore-lining region in the homodimeric channel . Five negatively charged amino-acid residues (E , D) and an asparagine residue (N) in transmembrane domains 6–8 serve as Ca 2+ -binding sites involved in channel gating [ – ]. Four alternatively spliced segments (a—d) determine the apparent Ca 2+ -sensitivity of the ANO1 channel . ANO2 has two isoforms A and B and a regulatory motif at a position homologous to segment c in ANO1 . (B) RT-PCR analysis from mouse olfactory epithelium (OE) and mouse cerebellum (CB) yield similarly strong ANO1 signals in cerebellum but weaker signals for ANO2. (C) Immunoblots obtained from lysates of cerebellum (CB) and main olfactory epithelium (OE) from wild-type and Ano2 -/- mice show an ANO1-specific signal at ~120 kDa with the ANO1 in antiserum. (D) Rabbit anti-ANO2 ex serum stains ANO2-specific bands (asterisks) in immunoblots obtained from lysates of main olfactory epithelium (OE) and eye, as well as in membrane-protein preparations of main olfactory bulb (OB) and cerebellum (CB) . ANO2 bands are not present in immunoblots from Ano2 -/- mice.

Article Snippet: Antibodies used for immunoblotting were guinea-pig anti-ANO1 (dilution 1:400; C-terminus encoding amino acids 962–1040; marked "ANO1 in " in the text) [ ], and a rabbit ANO2 antiserum directed against the extracellular loop that connects TMDs 5 and 6 in ANO2 (Alomone Labs, ACL012, dilution 1:1000, marked “ANO2 ex ” in the text).

Techniques: Membrane, Residue, Binding Assay, Reverse Transcription Polymerase Chain Reaction, Western Blot

Journal: PLoS ONE

Article Title: Anoctamin Calcium-Activated Chloride Channels May Modulate Inhibitory Transmission in the Cerebellar Cortex

doi: 10.1371/journal.pone.0142160

Figure Lengend Snippet: (A) ANO2 immunosignals from the cerebellar cortex are discernible in the dendrites of Purkinje cells. The signals are weak but stronger than the background signals emanating from the granule cell layer (GL) . (B) In the Purkinje cell, the ANO2 immunosignal (green) is visible in dendrites and the perinuclear region, but only weakly in the plasma membrane of the cell body. In contrast, ANO1 signals (red) label the entire Purkinje cell soma, but are not detectable in dendrites. (C) The ANO2 antiserum does not stain the cerebellar cortex of the Ano2 -/- mouse. (D) ANO2 immunosignals in the glomeruli of the olfactory bulb serving as positive control for ANO2 in brain tissue; gl : glomerular layer, opl : outer plexiform layer. (E) Absence of ANO2 immunosignals from the olfactory bulb of the Ano2 -/- mouse. Blue in B-E represents DAPI nuclear stain. All calibrations bars: 20 μm.

Article Snippet: Antibodies used for immunoblotting were guinea-pig anti-ANO1 (dilution 1:400; C-terminus encoding amino acids 962–1040; marked "ANO1 in " in the text) [ ], and a rabbit ANO2 antiserum directed against the extracellular loop that connects TMDs 5 and 6 in ANO2 (Alomone Labs, ACL012, dilution 1:1000, marked “ANO2 ex ” in the text).

Techniques: Clinical Proteomics, Membrane, Staining, Positive Control

Journal: PLoS ONE

Article Title: Anoctamin Calcium-Activated Chloride Channels May Modulate Inhibitory Transmission in the Cerebellar Cortex

doi: 10.1371/journal.pone.0142160

Figure Lengend Snippet: (A) A cerebellar Purkinje cell loaded with the fluorescent dye Alexa Fluor 568. Scale bar: 10 μm. (B) Spontaneous postsynaptic currents in a Purkinje cell with E Cl near 0 mV and V hold = -69 mV. Overlay of 764 current traces showing similar time courses but differing amplitudes, probably reflecting distinct positions of GABAergic synapses on the Purkinje cell dendritic tree. (C) Postsynaptic currents were completely blocked by 50 μM picrotoxin, an inhibitor of GABA A -receptor chloride channels. (D) Protocol for activation of climbing fibers: Ten 0.1-ms current pulses were applied to the area near the proximal dendrite of a Purkinje cell while recording the whole-cell current of that cell at -70 mV. CF-activation produced characteristic complex spikes, as shown in the inset. (E) Upper traces : GABAergic inhibitory postsynaptic currents recorded from a Purkinje cell at V hold = -48 mV and with 5 mM Cl - in the pipette solution. The positive polarity of IPSCs indicates Cl - influx. Lower traces : postsynaptic currents, recorded immediately after the climbing-fiber stimulation, displayed decreased amplitudes. (F) IPSCs recorded from a Purkinje cell of an Ano2 -/— mouse before (upper traces) and immediately after (lower traces) CF-activation. (G) The number of detectable IPSC signals decreased by ~47% through climbing-fiber stimulation (before CF: 30.7 ± 6.5 min -1 ; after CF: 14.6 ± 3.4 min -1 ; 8 cells; ctrl ). In slices from Ano2 -/- mice, more IPSCs were detected (54.5 ± 18.5 min -1 ; 4 cells), and the activation of climbing fibers had no effect (52.5 ± 16.2 min -1 ; 4 cells).

Article Snippet: Antibodies used for immunoblotting were guinea-pig anti-ANO1 (dilution 1:400; C-terminus encoding amino acids 962–1040; marked "ANO1 in " in the text) [ ], and a rabbit ANO2 antiserum directed against the extracellular loop that connects TMDs 5 and 6 in ANO2 (Alomone Labs, ACL012, dilution 1:1000, marked “ANO2 ex ” in the text).

Techniques: Activation Assay, Produced, Transferring

Journal: PLoS ONE

Article Title: Anoctamin Calcium-Activated Chloride Channels May Modulate Inhibitory Transmission in the Cerebellar Cortex

doi: 10.1371/journal.pone.0142160

Figure Lengend Snippet: (A) Whole-cell recording from an HEK293 cell transfected with mouse cerebellar ANO2 isoform B at -70 mV. Chloride inward current was activated by diffusion of 7.5 μM Ca 2+ from the pipette into the cell immediately after whole-cell breakthrough (arrow) . (B) Results from current recordings without (black bars) and with (hatched bars) T16Ainh-A01 revealed that neither mouse ANO1 ac nor ANO1 abc were significantly inhibited by 5 μM of the compound, while the ANO2 isoform B showed a significantly reduced current density. At 25 μM T16Ainh-A01 , significant inhibition was also observed with ANO1 ac . Results were averaged from 19–30 cells for each condition. (C) When applied at the ANO2-specific concentration of 5 μM, T16Ainh-A01 blocked DDI : The number of detectable IPSCs was 48.9 ± 12.1 min -1 before and 53.4 ± 15.4 min -1 after CF stimulation. ANO2 +/+ control data from are included for comparison. (D) IPSC traces from wildtype mice in the presence of 5 μM T16Ainh-A01 show no difference in IPSC shapes and amplitudes before (left traces) and after (right traces) CF-activation.

Article Snippet: Antibodies used for immunoblotting were guinea-pig anti-ANO1 (dilution 1:400; C-terminus encoding amino acids 962–1040; marked "ANO1 in " in the text) [ ], and a rabbit ANO2 antiserum directed against the extracellular loop that connects TMDs 5 and 6 in ANO2 (Alomone Labs, ACL012, dilution 1:1000, marked “ANO2 ex ” in the text).

Techniques: Transfection, Diffusion-based Assay, Transferring, Inhibition, Concentration Assay, Control, Comparison, Activation Assay

Journal: PLoS ONE

Article Title: Anoctamin Calcium-Activated Chloride Channels May Modulate Inhibitory Transmission in the Cerebellar Cortex

doi: 10.1371/journal.pone.0142160

Figure Lengend Snippet: (A) At a Cl - concentration of 12 mM in the recording pipette, GABAergic postsynaptic currents were negative (Cl - efflux), indicating that postsynaptic E Cl is less negative than V hold . (B) Shortly after applying 5 μM ANO2 inhibitor, positive currents appear (circles) as some synapses experience a decline of postsynaptic [Cl - ] i , while others still have high Cl - (asterisks) . (C) During the continued presence of the ANO2 inhibitor, virtually all postsynaptic currents reverse to positive polarity (Cl - influx) indicating that GABAergic synapses experience an E Cl more negative than V hold . (D) The collected data from 12 Purkinje cells at [Cl - ] i = 12 mM and V hold = -60 mV demonstrate the polarity reversal of postsynaptic currents (PSCs) actuated by the ANO2 inhibitor. (E) Schematic representation of an hypothesis for the 12 mM [Cl - ] i experiment. In the absence of the ANO2 inhibitor (upper scheme) , the basal activity of ANO2 channels (green) provides a Cl - conductance in the dendritic membrane. ANO2 contributes to the Cl — transport machinery, whose various pathways are represented by the K + /Cl — cotransporter KCC2 (blue) . Together the Cl - pathways stabilize a slightly elevated level of [Cl - ] i which results in a negative driving force (V m —E Cl < 0) for Cl - currents through GABA A receptors in GABAergic synapses (red) . In this situation, Cl - currents are outwardly directed and cause negative postsynaptic currents. Application of the ANO2 inhibitor (lower scheme) reduces the Cl - conductance. This causes a polarity reversal of the Cl - driving force, as the balance shifts towards Cl - extrusion, causing local [Cl - ] i to decrease. This hypothesis provides a qualitative concept for the role of ANO2 channels in the inversion of postsynaptic currents that is depicted in panels A to C. The proximity of Cl — transport pathways and GABAergic synapses, as well as the occurrence of local Cl -. gradients within dendritic segments, are inspired by the model for GABA A -receptor-mediated Cl - gradients in extended dendritic trees proposed by Jedlicka et al. (2011) .

Article Snippet: Antibodies used for immunoblotting were guinea-pig anti-ANO1 (dilution 1:400; C-terminus encoding amino acids 962–1040; marked "ANO1 in " in the text) [ ], and a rabbit ANO2 antiserum directed against the extracellular loop that connects TMDs 5 and 6 in ANO2 (Alomone Labs, ACL012, dilution 1:1000, marked “ANO2 ex ” in the text).

Techniques: Concentration Assay, Transferring, Activity Assay, Membrane