Fig 2: Increased levels of HIF-1a in COX4-1-deficient cells. Western blot analysis was performed on cell extracts from both COX4-1-deficient cells (patient and HFF-shCOX 1) and from both corresponding controls. The extracted cells were probed with anti-HIF-1a and anti-actin antibodies as loading control. As a positive control for HIF1-a, untreated (UT) human foreskin fibroblasts (HFF) were preincubated with cobalt chloride to simulate hypoxia (A). The histogram represents the results normalized to actin (B). The figure depicts one representative experiment out of three, showing an increased level of HIF-1a in COX4-1-deficient cells.

Fig 3: Expression of NDRG1-OT1 is induced by HIF-1a, not HIF-2a, under hypoxia.A, B Western blots of HIF-1a in MCF-7 (A) and MDA-MB-453 (B) cells transfected with 12.5 µM siRNA against HIF1A under hypoxia. Top: representative western blots. Bottom: statistical bar chart. Loading control: GAPDH. C, D Western blots of HIF-2a in MCF-7 (C) and MDA-MB-453 (D) cells transfected with 12.5 µM siRNA against HIF2A under hypoxia. Top: representative western blots. Bottom: statistical bar chart. Loading control: GAPDH. E, F Relative expression levels of NDRG1-OT1 in MCF-7 (E) and MDA-MB-453 (F) cells transfected with siRNA against HIF1A, HIF2A, or HIF1A + HIF2A under hypoxia. The relative expression level of each condition was compared with hypoxic group of the negative control (NC), respectively. All data shown are the means ± SDs (n = 3). N normoxia. H hypoxia. *P < 0.05. **P < 0.01.

Fig 4: Neuronal cells undergo a unique metabolic response to SARS-CoV-2 infections.Brain organoids were infected with SARS-CoV-2 and sequenced with 10x single cell sequencing strategies. (A) Projection of cells with SARS-CoV-2 transcripts (in red) onto the UMAP. Percentage of infected cells in each cluster. (B) Change in population representation of example clusters after infection (refer See also Figure S5 for all clusters). (C) Validation of neuronal subtypes using CTIP2, PAX6 and TBR1 antibodies for confocal imaging. (D) Differentially expressed genes (DEGs) from brain organoids infected with ZIKV (Watnabe et al 2017 Cell Reports) were compared with DEGs from SARS-CoV-2 infected organoids. (E) Enriched gene ontology terms (geneontology.com) for upregulated genes from (D). (F) Enriched GO terms in SARS-CoV-2 infected cells (top) and SARS-CoV-2 negative bystander cells from 96 hpi organoids (bottom). (G) Heatmap of genes from metabolic pathways. (H) HIF1A staining of brain organoids that were mock infected versus 96hpi. (I) Quantification of HIF1A positive cells in SARS-CoV-2 infected organoids. Single cell RNA-seq was performed in duplicates with one IPSC line (Y6). HIF1A staining was performed with unique organoid n of 4 per condition, from the same culturing batch, with images from n = 12 cortical regions with two IPSC lines, and student’s t-test was performed (****, P<0.0001).

Fig 5: SCT-1015 triggered the AMPK/HIF1/PDK1 axis. (A) Protein abundance of p-AMPKa, AMPKa, HIF1a, 564pro-OH, 402pro-OH, and PDK1 were analyzed by western blotting after SCT-1015 treatment for 24 h. The groups for western blotting were performed in three independent replicates. (B) PLC5 cells were transfected with the corresponding vector, shAMPKa1 (left), HIF1a overexpression plasmid (middle), or PDK1 overexpression plasmid (right). After 72 h transfection, cells were treated with SCT-1015 (20 µm) for 24 h and subjected to western blotting assay. The groups for western blotting were performed in three independent replicates. (C) PLC5 cells were transfected with the corresponding vector, shAMPKa1 (left), HIF1a overexpression plasmid (middle), or PDK1 overexpression plasmid (right). After 72 h transfection, cells were treated with SCT-1015 (20 µm, 48 h) for viability analysis (bottom, columns, mean; error bars, SD; n = 3) or exposed to SCT-1015 (20 µm, 24 h) for PDH activity analysis (upper, columns, mean; error bars, SD; n = 3).