scrambled scr sirna fitc  (Santa Cruz Biotechnology)

Journal: Cell Death & Disease

Article Title: Loss of TMEM55B modulates lipid metabolism through dysregulated lipophagy and mitochondrial function

doi: 10.1038/s41419-025-08210-x

Figure Lengend Snippet: A Primary hepatocytes were isolated from one Tmem55b KO and one WT male littermate and treated with 1 mM oleate for 1 hour, stained with LysoTracker, and imaged on a Nikon spinning disk confocal microscope at 100X (Scale bars = 5 µm). For analysis of lysosome volume, lysosomes were subjected to surface reconstruction in Imaris, and automated segmentation by color-coding based on the volume of the connected components was used for 3D surface rendering of lysosomes (bottom panels). Representative images are shown. Six non-overlapping images were acquired/genotype and quantified. B Lysosomal acid lipase activity was measured on the BD LSRFortessa Cell Analyzer in mouse primary hepatocytes from Tmem55b KO and WT male littermates, N = 2/genotype. C Six-week-old male C57BL/6J mice were treated with an ASO against Tmem55b or NTC at a dose of 25 mg/kg body weight/week and fed a Western diet (0.2% cholesterol, 42% fat) for 6 weeks. Free fatty acid levels were measured in the liver tissue lysate, N = 8/ASO treatment. D Six-week-old male WT and Tmem55b KO mice were fed a GAN diet for 12 weeks, and p62 and LC3B-I and II protein levels were detected by immunoblot in mouse liver tissues and quantified with Image J, N = 6/genotype. E HepG2 cells were transfected with TMEM55B or Scr siRNAs. After 48 hr, HepG2 cells were labeled with anti-PLIN2 and LysoTracker and examined by confocal microscopy (63X, scale bars = 10 µm), N = 30 cells Scr siRNA, N = 33 cells TMEM55B siRNA. F Electron micrographs of HepG2 cells showing lysosome-containing lipid droplets (LDs) with TMEM55B knockdown (63X, scale bars=10 µm), N = 5 images Scr siRNA, n = 6 images TMEM55B siRNA. G HepG2 cells were transfected with TMEM55B or Scr siRNAs. After transfection, HepG2 cells were incubated with 1 µm of red BODIPY™ 558/568 C12-FA overnight, stained with anti-LC3B antibody and subsequently Goat anti-rabbit IgG H&L, and examined by confocal microscopy. N = 33 cells Scr siRNA, N = 37 cells TMEM55B siRNA. Representative images shown. Fluorescence was quantified on the BD LSRFortessa Cell Analyzer as the median fluorescence values of 10,000 gated events. Results are presented as mean ± s.e.m. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001 vs. Scr siRNA or WT by Student’s t -test. WT wildtype Tmem55b fl/fl mice, KO Tmem55b whole-body knockout mice, ASO antisense oligonucleotides, NTC non-target control, LC3B Microtubule-Associated Protein 1 Light Chain 3 Beta, Gapdh glyceraldehyde-3-phosphate dehydrogenase, Scr siRNA scramble small interfering RNA, LDs lipid droplets, FA fatty acid.

Article Snippet: E HepG2 cells were transfected with MitoKeima plasmid and siRNAs targeting TMEM55B or scramble siRNA (Scr) using the AMAXA Nucleofector.

Techniques: Isolation, Staining, Microscopy, Activity Assay, Western Blot, Transfection, Labeling, Confocal Microscopy, Knockdown, Incubation, Fluorescence, Knock-Out, Control, Small Interfering RNA

Journal: Cell Death & Disease

Article Title: Loss of TMEM55B modulates lipid metabolism through dysregulated lipophagy and mitochondrial function

doi: 10.1038/s41419-025-08210-x

Figure Lengend Snippet: A Schematic representation of the FA pulse-chase assays. HepG2 cells were transfected with TMEM55B and Scr siRNAs, pulsed with Green C12-FA (2 µM) for 16 hours, washed, and chased with substrate-limited DMEM supplemented with 0.5 mM of glucose, 1 mM of glutamine, 0.5 mM of carnitine, and 1% FBS for 24 hours. Cells were then labeled with LysoTracker Red (shown as Red), and MitoTracker Deep Red (shown as white), and imaged with Zeiss 710 confocal microscope at 63X (Scale bars=10 µm) B Representative images. C Quantification of C12-FA localization to lysosomes before (Hr0) and after (Hr24) the chase. D Quantification of C12-FA localization to mitochondria before (Hr0) and after (Hr24) the chase. N = 67 cells Scr siRNA, N = 69 cells TMEM55B siRNA at Hr0; N = 93 cells Scr siRNA, N = 79 cells TMEM55B siRNA at Hr24. Results are presented as mean ± s.e.m. ** p < 0.01, **** p < 0.0001 vs Hr0; ### p < 0.001, #### p < 0.0001 vs Scr by two-way ANOVA. Scr siRNA scramble small interfering RNA, LDs lipid droplets, FA fatty acid.

Article Snippet: E HepG2 cells were transfected with MitoKeima plasmid and siRNAs targeting TMEM55B or scramble siRNA (Scr) using the AMAXA Nucleofector.

Techniques: Pulse Chase, Transfection, Labeling, Microscopy, Small Interfering RNA

Journal: Cell Death & Disease

Article Title: Loss of TMEM55B modulates lipid metabolism through dysregulated lipophagy and mitochondrial function

doi: 10.1038/s41419-025-08210-x

Figure Lengend Snippet: A Oxygen consumption rate (OCR) of primary hepatocytes from one female Tmem55b KO ( N = 32 technical replicates/genotype) and one female WT mouse ( N = 30 technical replicates). B HepG2 cells were transfected with siRNAs targeting TMEM55B or scrambled siRNA and OCR was measured by Seahorse XF Analyzer with and without 0.125 mM palmitic acid (FA) and/or 40 µM etomoxir (Eto), N = 6 technical replicates/siRNA. C siRNA-transfected HepG2 cells were incubated with 50 nM TMRE for 25 minutes to stain mitochondrial membrane potential, and fluorescence was quantified in 10,000 gated events on the BD LSRFortessa Cell Analyzer. N = 5 technical replicates Scr siRNA, N = 12 technical replicates TMEM55B siRNA. D Primary hepatocytes were isolated from one male WT and one male Tmem55b KO mouse, incubated with MitoTracker Deep Red (shown as red) and LysoTracker Red DND-99 (shown as white), and visualized using live cell imaging on a Nikon spinning disk confocal microscope at 100X (Scale bars=7 µm). In these images, red indicates healthy mitochondria, while unhealthy mitochondria are observed as blue or purple. Images were subjected to Gaussian filtering and background subtraction in Imaris v9.6. Mitochondria were subjected to surface reconstruction in Imaris, and automated segmentation by color-coding based on the volume of the connected components was used for 3D surface rendering of mitochondria. Non-overlapping images from WT ( N = 13) and KO ( N = 19) were quantified. E HepG2 cells were transfected with MitoKeima plasmid and siRNAs targeting TMEM55B or scramble siRNA (Scr) using the AMAXA Nucleofector. After 48 hours, cells were treated with BSA or 1 mM PA for 1 hour and imaged and quantified on a Nikon spinning disk confocal microscope at 60X. N = 5 images/siRNA. F Primary hepatocytes from one female Tmem55b KO and one WT mouse were treated with DMSO (sham) or 20uM Urolithin A (UA) for 16 hours, followed by 1 mM palmitate (PA) for 1 hour. Cells were then incubated with 5 µM MitoSox and imaged at 60X on Nikon spinning disk confocal microscope. N = 5 non-overlapping images/genotype (Scale bars=10 µm). Representative images are shown. Results are presented as mean ± s.e.m. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001 vs. Scr or WT by Student’s t -test or 2-way ANOVA. WT wildtype Tmem55b fl/fl mice, KO Tmem55b whole-body knockout mice, Scr siRNA scramble small interfering RNA, FA fatty acid, ETO etomoxir,TMRE Tetramethylrhodamine ethyl ester, BSA bovine serum albumin, PA palmitate, DMSO dimethyl sulfoxide, UA urolithin a.

Article Snippet: E HepG2 cells were transfected with MitoKeima plasmid and siRNAs targeting TMEM55B or scramble siRNA (Scr) using the AMAXA Nucleofector.

Techniques: Transfection, Incubation, Staining, Membrane, Fluorescence, Isolation, Live Cell Imaging, Microscopy, Plasmid Preparation, Knock-Out, Small Interfering RNA

Journal: Cell Death & Disease

Article Title: Loss of TMEM55B modulates lipid metabolism through dysregulated lipophagy and mitochondrial function

doi: 10.1038/s41419-025-08210-x

Figure Lengend Snippet: A Primary hepatocytes isolated from one male Tmem55b KO and one male WT mouse were incubated with 2 µm BODIPY C12-FA for 30 min, and fluorescence was quantified by the BD LSRFortessa Cell Analyzer in 10,000 gated events, N = 4 technical replicates/genotype. B HepG2 cells were transfected with siRNA targeting TMEM55B or a scrambled control, and after 48 hr treated with 2 µm BODIPY C12-FA for 30 mins. Fluorescence was quantified by flow cytometry as described above, N = 3 technical replicates/treatment. C Transfected HepG2 cells were incubated with 2 µm BODIPY C12-FA for 30 mins before live cell imaging with Nikon spinning disk confocal microscope at 100X (Scale bars=3 µm), N = 10 non-overlapping images Scr siRNA, N = 12 images TMEM55B siRNA. Mitochondria and FAs were subjected to surface reconstruction in Imaris. Volume of FA containing particles is indicated by color. D Huh7 cells were transfected with TMEM55B and Scr siRNAs, incubated with 1 mM oleate for 24 hr, stained with Nile red, and visualized using confocal microscopy (Scale bars=10 µm), N = 10 cells Scr siRNA, N = 15 cells siTMEM55B siRNA. E Control and JIP4 -knockout (KO) HepG2 cells were transfected with siRNAs targeting TMEM55B or scrambled control, incubated with 200 ng/ml BODIPY493/503, and the fluorescence was quantified by the BD LSRFortessa Cell Analyzer in 10,000 gated events, N = 4 technical replicates/siRNA. F HepG2 cells were transfected with siRNAs targeting TMEM55B ( siTMEM55B ) or JIP4 (siJIP4) individually or together, or a scrambled control siRNA. After 48 hours, cells were incubated with 1 mM oleate for 24 hours, stained with LipidTOX Red, and imaged with Zeiss 710 confocal microscope at 63X (Scale bars=10 µm), N = 32 cells Scr siRNA, N = 59 cells TMEM55B siRNA, N = 44 cells JIP4 siRNA, N = 41 cells TMEM55B/JIP4 siRNAs. Representative images are shown. Results are presented as mean ± s.e.m. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001 vs. Scr by Student’s t -test. WT wildtype Tmem55b fl/fl mice, KO Tmem55b whole-body knockout mice, Scr siRNA scramble small interfering RNA, JIP4 C-Jun-amino-terminal kinase-interacting protein 4, also known as SPAG9.

Article Snippet: E HepG2 cells were transfected with MitoKeima plasmid and siRNAs targeting TMEM55B or scramble siRNA (Scr) using the AMAXA Nucleofector.

Techniques: Isolation, Incubation, Fluorescence, Transfection, Control, Flow Cytometry, Live Cell Imaging, Microscopy, Staining, Confocal Microscopy, Knock-Out, Small Interfering RNA

Journal: Communications Biology

Article Title: Primary cilia prevent activation of the cGAS-STING pathway during mouse decidualization

doi: 10.1038/s42003-025-08030-x

Figure Lengend Snippet: A TMEM67 and acetylated tubulin (AcTub) immunofluorescence in mouse uterine stroma on day 4 of pregnancy. Scale bar, 5 μm. Inset scale bars, 3 μm. B TMEM67 and Arl13b immunofluorescence in cultured uterine stromal cells on day 4 of pregnancy. Scale bar, 5 μm. Inset scale bars, 3 μm. C Western blot analysis and quantification of TMEM67 protein in stromal cells under in vitro decidualization (E + P) for 2 and 3 days. D Relative mRNA abundances of Tmem67 , Prl8a2 and Prl3c1 after stromal cells were transfected with scrambled negative control siRNA (siScr) or siRNA targeting Tmem67 (siTMEM67) for 24 h and further induced for in vitro decidualization for 48 h. E , F Arl13b and γ-tubulin immunofluorescence, the percentage of ciliated cells, and cilia length in mouse uterine stromal cells after transfected with siScr or siTMEM67 for 24 h and induced for in vitro decidualization for 48 h. Scale bars, 10 μm. Inset scale bars, 5 μm. All images were representative of three biologically independent experiments. For violin plots, the solid lines indicated the median and the dotted lines indicated the first and third quartiles. Other data were presented as means ± SD from three biologically independent experiments. The two-tailed Student’s t -test was used for comparing two groups. One-way ANOVA test was used for comparing more than two groups. * P < 0.05, ** P < 0.01, *** P < 0.001, and **** P < 0.0001.

Article Snippet: The small interfering RNA (siRNA) and scrambled negative control (Scr) siRNA were designed and synthesized by Ribobio Co, Ltd. (Guangzhou, China) with sequences as follows: siRNA-mouse- Ift88 : 5’-GCTTGGAGCTTATTACATT-3’, siRNA-mouse- Tmem67 : 5’-CTAGAAACCCATCAGACTA-3’, siRNA-mouse- Ccl6 : 5’-GAAGCAAGGCCCAAGATCT-3’, siRNA-mouse- Pcsk9 : 5’-GAACCTACATTGTGGTGCT-3’, and siRNA-mouse- Rbp4 : 5’-CAACATCATCGCTGAGTTT-3’. siRNA was transfected with Lipofectamine 2000 kit (11668019, Invitrogen, Carlsbad, CA).

Techniques: Immunofluorescence, Cell Culture, Western Blot, In Vitro, Transfection, Negative Control, Two Tailed Test