BUSCADOR RECOLECTA

(A, B) Parental (Par), and Ndufs4−/− RAW 264.7 cells were left untreated or treated with LPS (100 ng/ml). Supernatants were collected at 8 hours for measurement of cytokine concentrations by ELISA (A). Cells were collected at 4 hours for quantification of cytokine transcripts using real-time PCR (expressed as fold increases versus untreated parental cells) (B). (C) Representative flow cytometry plots (left) showing phagocytosis of FITC labeled heat killed E. coli (HKEC) and bar graph representing % of fluorescent cells and the MFI values (right). Ctrl, control. Ns, not significant; *, P <0.05; **, P <0.01; ***, P<0.005; ****, P<0.001. Each point represents a biological replicate. In all experiments, 4 to 5 different Ndufs4−/− clones were used. Data are shown as the mean ± SEM., Peer reviewed

Native gels were incubated with NADH (as a substrate), and nitro blue tetrazolium (NBT, as the electron acceptor). CI activity is shown in purple., Peer reviewed

1D-BNE showing OXPHOS complexes I to IV (CI-CIV) and SCs. Western blot analysis was performed using antibodies against CI (NDUFA9), CIII (Core 2), CIV (COX5A), and CV (ATP5A). Asterisks (*) indicate lower molecular weight SC. Loading control, CII subunit SDHA., Peer reviewed

(A) Activities of MRC complex (I–IV) were assayed spectrophotometrically, and the results were normalized to citrate synthase (CS) activity in mitochondria isolated from parental (Par) and Ndufs4−/− RAW 264.7 cells. (B) Left panel, a representative experiment showing OCR in RAW 264.7 sublines before and after the sequential addition of oligomycin (2.6 μM), FCCP (1 μM), and a combination of rotenone (Rot) and antimycin A (AA) (1 μM). Right panel, basal respiration, maximal respiration, and ATP production. Ns, not significant; *, P <0.05; **, P <0.01; ***, P<0.005; ****, P<0.001. Each point represents a biological replicate. Data are shown as the mean ± SEM., Peer reviewed

(A) Structural model for ovine (Ovis aries) mitochondrial complex I (CI, Ref: 5LNK) [17] displaying the location of the NDUFS4 subunit (colored in black). UCSF ChimeraX (v1.5) [18] was used for visualization. (B) General Cas9 HITI strategy to ablate Ndufs4. Cas9 produces a double stranded DNA break at specific target sequences within the first exon of Ndufs4. Cas9 also excises the HITI donor by cleaving the same target sequence flanking the blasticidin cassette to be inserted. The excised HITI donor is ligated into the genomic site through the NHEJ pathway. The forward integration depicted in the scheme is “locked” and cannot be processed further. A reverse integration could be corrected by continuous excision/repair cycles in virtue of flanking target sites reconstitution. Grey pentagons represent Cas9/gRNA target sequences. Black lines within pentagons indicate Cas9 cleavage sites. (C) Whole cell lysates from each resultant knockout cell line were analyzed by Western blotting using antibodies against Ndufs4 or β-actin (loading control). Correctly targeted clones were named Ndufs4−/− followed by a serial number; Par, parental cell line., Peer reviewed

Increasing evidence demonstrate that the electron transfer chain plays a critical role in controlling the effector functions of macrophages. In this work, we have generated a Ndufs4−/− murine macrophage cell lines. The Ndufs4 gene, which encodes a supernumerary subunit of complex I, is a mutational hotspot in Leigh syndrome patients. Ndufs4−/− macrophages showed decreased complex I activity, altered complex I assembly, and lower levels of maximal respiration and ATP production. These mitochondrial respiration alterations were associated with a shift towards a pro-inflammatory cytokine profile after lipopolysaccharide challenge and improved ability to phagocytose Gram-negative bacteria., Peer reviewed

Parental and Ndufs4−/− RAW 264.7 cells (40,000) were plated on 6-well plates. The cells were cultured in media containing galactose in the complete absence of glucose. The number of viable cells was determined at the indicated time points. Each point represents a biological replicate. Data are shown as the mean ± SD., Peer reviewed

A representative experiment showing OCR in LPS-pretreated RAW 264.7 sublines before and after the sequential addition of oligomycin (2.6 μM), FCCP (1 μM), and a combination of rotenone (Rot) and antimycin A (AA) (1 μM)., Peer reviewed

The NAD/NADH ratio was measured through colorimetric detection in deproteinized cell extracts from parental (Par) and Ndufs4−/− RAW 264.7 cells. *, P <0.05; **, P <0.01; ***, P<0.005; ****, P<0.001. Each point represents a biological replicate. Data are shown as the mean ± SEM., Peer reviewed

Left panel, Western blot for NDUFV1 (N module) and NDUFA9 (Q module). ATP5B and Coomassie staining were used as loading controls. Right panel, schematic representation of CI. The N module contains an NADH oxidation site, while the Q module contains a ubiquinone reduction site. P module is involved in proton-pumping activity. The positions of NDUFV1 and NDUFA9 are indicated. IMM, inner mitochondrial membrane; IMS, intermembrane space., Peer reviewed