Fig 2: MFSD1 and MFSD3 are expressed specifically in neurons. To determine cell type expression, fluorescent immunostaining on coronal mouse brain sections, with nuclear DAPI staining were run. This revealed co-staining between MFSD1 (a) and MFSD3 (b), with the neuronal marker NeuN as seen in the merged images. No overlap could be seen with either the astrocytic marker GFAP (c, d) or the dendritic marker MAP2 (e, f). Micrographs were acquired using a fluorescence Olympus microscope BX53 with an Olympus DP73 camera

Fig 3: Uptake and distribution of Cy3 labelled SDNP siRNA by microfilariae at 24 hpi. a Fluorescent image of microfilariae from siRNA non-injected mosquitoes (position indicated by a red square). b Bright field image of microfilariae from siRNA non-injected mosquitoes. c Fluorescent image of microfilariae from Cy3 labelled SDNP siRNA-injected mosquitoes (arrow indicates the dispersion of Cy3 labelled siRNA). d Bright field image of microfilariae from Cy3 labelled SDNP siRNA-injected mosquitoes. Fluorescence was visualised using Olympus BX53 using Cy3 filter. Magnification 400×. Scale-bars: a-d, 20 µm

Fig 4: Effects of recombinant KL, Rap, and 3-MA on endogenous renal KL expression in mice with CLP-induced AKI.Notes: (A) Western blot for endogenous renal KL expression. *P<0.05 vs CLP+media. (B) Images of IHC for KL. (C) Quantification of IHC staining. *P<0.05 vs CLP+media. Mice received recombinant KL (0.01 mg/kg) 1 hour after CLP. Rap (1 mg/kg) or 3-MA (30 mg/kg) was administered by intraperitoneal injection 2 hours before CLP surgery. Mouse renal tissues were collected 24 hours after CLP. Sections were stained with anti-KL (1:100, Abcam, Cambirdge, UK) at 4°C overnight. Images at 200× magnification were acquired using a biological imaging microscope (BX53; Olympus Corporation, Tokyo, Japan).Abbreviations: Rap, rapamycin; 3-MA, 3-methyladenine; CLP, cecal ligation and puncture; AKI, acute kidney injury; CMC, carboxymethyl cellulose; IHC, immunohistochemistry.

Fig 5: SCD5 knockdown promotes necrotic cell death of MCF-7 cellsApoptosis/necrosis analysis was performed by an “Apoptosis/Necrosis Detection Kit” (ENZO Life Sciences) on MCF-7 cells that underwent a SCD5 knockdown by a 72-h transient transfection with 60 pmol of SCD5 siRNA oligos. Cells were washed with PBS and incubated with apoptosis (annexin V-EnzoGold)/necrosis (membrane impermeable DNA intercalating dye-7-AAD) dual detection reagent, according to the manufacturer's protocol and observed under an Olympus BX53 fluorescent microscope. The concurrent cell staining with annexin V (yellow) and 7-AAD (red) identifies cells in late apoptosis (yellow arrows in the central right panel, orange arrows in the bottom right panel). Staining with the only necrosis red dye (red in the central panels and pink in the bottom merged images, white arrows) detects necrotic cells. Nuclear counterstaining was performed with the blue fluorescent dye DAPI (turquoise arrow). Control siRNA (MCF-7 transfected with non-targeting siRNA oligos). Negative controls (Ct-) were obtained by omitting incubation with dual detection reagent. MCF-7 cells treated with Staurosporine (STS, 2 µM) were used as positive control (Ct+) for necrosis [37]. Images (magnification, 200x) were captured with ISCapture software (Tucsen Photonics) and are representative of three independent experiments. Data are shown as mean + SD of three independent experiments. *p<0.001 vs control siRNA, Student's t test.