BUSCADOR RECOLECTA

(A) Fish standard length (SL) plotted against fish age. (B, C) Fish weight (B) and liver weight (C) increases with SL represented in a semi-log plot. (D) Liver-to-body weight ratio during postembryonic growth is constant in adult fish. (N > 10, n ≥ 300 fish). Gender of the corresponding samples is colour coded: male (blue), female (pink), and ND (green). The numerical values that were used to generate the graphs can be found in S1 Data., Peer reviewed

(A) Adult liver displaying clones along the central vein; recombination was induced in hepatoblasts at 26 hpf (n = 9 livers). (B, C) Adult livers exhibiting giant clusters in the ventral lobe (n = 3 livers). For (A-C) total numbers: N = 9, n = 79 livers. (D) Adult liver with a cluster along a central vein (n = 5 livers) and (E) clusters oriented in lateral stripes (n = 10 livers) upon recombination induced in hepatocytes. For (D, E) total numbers N = 4, n = 31 livers). (F) Giant clusters in the ventral lobe are also apparent in juvenile livers when labelling was induced at 4 dpf in hepatocytes (n = 1; total N = 5, n = 42 livers). (G) Confocal section showing the kdrl:GFP+ sinusoidal architecture in the adult liver counterstained with DAPI (N = 1, n = 2 livers). (H-J) No recombined cells were detected in 84% noninduced control livers of long-term lineage tracing experiments showed no recombined cells (H; N = 15, n = 84 livers), and the majority of recombined samples (N = 15, n = 100) show only one recombined clone of a few labelled cells (I, J; N = 15, n = 16 livers)., Peer reviewed

(A-C) Mathematical models simulating hepatoblast differentiation, based on heterogeneous hepatoblast potentials (A, B) or differential proliferation times (C; n = 10). (D, F) Presentation of 10 μm sections from juvenile (D) and adult (F) livers stained for fabp10a:GFP (hepatocytes), tp1:H2B-mCherry (BECs), and DAPI (nuclei). (E) Relative distribution of BECs and hepatocytes in juvenile liver (N = 4, n = 4 livers and 18 ROIs). (G) Relative distribution of BECs and hepatocytes at the organ centre (N = 1, n = 1 liver, 4 sections) or periphery in adult livers (N = 1, n = 1 whole-mount liver). The numerical values that were used to generate the graphs in (A-C, E, G) can be found in S1 Data., Peer reviewed

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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

(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

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