primary human foreskin fibroblast hf cells  (ATCC)

Journal: Life Science Alliance

Article Title: Mitochondrial double-stranded RNA homeostasis depends on cell-cycle progression

doi: 10.26508/lsa.202402764

Figure Lengend Snippet: (A) Schematic of the retroviral strategy used to transform human dermal fibroblasts (top). Brightfield images of the resulting cell lines are shown (bottom, scale bar: 200 μm). (B) Cell proliferation of the indicated fibroblast cell lines as measured by confluency (N = 3 independent cultures). Error bars indicate the SD. (C) Colony formation assay with the indicated fibroblast cell line. Images are representative of two technical repeats. (D) Oxygen consumption rates (left) and basal extracellular acidification rate (ECAR) of WT and hTERT-LT-RAS fibroblasts. Oligomycin (Olg), carbonyl cyanide-p-trifluoromethoxyphenylhydrazone (FCCP), and rotenone + antimycin A (Rot + AA) were injected at the time points indicated by the arrows. Mean data values are plotted, and the error bars indicate the SD (N = 4 independent cultures). (E) Immunoblot analysis of mitochondrial and MRG-associated proteins in whole-cell lysates obtained from each fibroblast cell line. Samples were blotted on different membranes which are indicated by the dotted lines. (F) mtDNA levels in transformed fibroblast cell lines relative to WT fibroblasts as measured by qPCR of the indicated mtDNA regions, MT-RNR1 and MT-CYTB. C q values were normalised against nuclear ACTB (N = 3 independent cultures). Bars indicate the mean and the error bars represent the SD. Individual data points are shown as grey circles. (G) Scatter plot of the mitochondrial network area per cell in each fibroblast cell line. Area was measured from the immunofluorescence staining of anti-TOMM20 antibodies and imaged by epifluorescence microscopy. The one-way ANOVA test was used to determine P -values compared with WT. P -value (WT versus hTERT) = 0.0029, P -value (WT versus hTERT-LT) < 0.0001, P -value (WT versus hTERT-LT-Ras) < 0.0001 (number of cells for each cell line = 30, from two independent cultures). Horizontal lines indicate the mean value and error bars represent the SD. Source data are available for this figure.

Article Snippet: Human primary foreskin dermal fibroblast cells, CRL-2097 (ATCC) were transduced with retrovirus particles generated with plasmids encoding pBABE-hTERT-puromycin, pBABE-SV40LT-neomycin, and pMSCV-HRasGV12-blasticidine.

Techniques: Retroviral, Colony Assay, Injection, Western Blot, Transformation Assay, Immunofluorescence, Staining, Epifluorescence Microscopy

Journal: Life Science Alliance

Article Title: Mitochondrial double-stranded RNA homeostasis depends on cell-cycle progression

doi: 10.26508/lsa.202402764

Figure Lengend Snippet: (A) Immunofluorescence of dsRNA, BrU, and DNA in the indicated fibroblast cell lines treated with BrU for 1 h and imaged by confocal microscopy. DAPI staining shown in blue (scale bar: 20 μm). (B) Number of mitochondrial dsRNA foci, BrU foci, and nucleoids per μm 2 of mitochondria determined by immunofluorescence and confocal microscopy. The mitochondrial network was imaged using anti-TOMM20 antibody. Box and whiskers plot represent the number of foci per μm 2 mitochondria for each cell. Whiskers represent minimum and maximum values. Boxes extend from the 25 th to the 75 th percentile with the median plotted in the middle. “+” indicates the mean value (N = 30 cells from two independent cultures). (C) Northern blot of mRNA transcripts of the coding and mirror regions of CYTB and ND5 in the fibroblast cell lines (top). Nuclear 7SL RNA was used as a loading control. Schematic representing the coding and mirror regions that were probed for CYTB and ND5 (bottom). Coding genes: green arrows. Coding regions for tRNAs: pink arrows. (D) Immunofluorescence of dsRNA in WT fibroblasts treated with the indicated siRNA for 48 h and imaged by confocal microscopy. The mitochondrial network was immunostained with anti-TOMM20. DAPI staining shown in blue (scale bar: 20 μm). (E) Immunofluorescence of dsRNA in WT, NME6 KO, and NME6 KO HeLa cells expressing NME6-MycFlag or NME6 H137N -MycFlag and imaged by confocal microscopy. DAPI staining shown in blue (scale bar: 20 μm). (E, F) Scatter plot of mean mt-dsRNA intensity per cell quantified from confocal images as shown in (E) (N = 100–120 cells from two independent cultures). The one-way ANOVA test was used to determine P -values compared with WT. P -value (WT versus NME6 KO) < 0.0001, P -value (WT versus NME6 KO + WT) = 0.0013, P -value (WT versus NME6 KO + H137N) < 0.0001. Horizontal lines indicate the mean value and error bars indicate the SD. (G) Immunofluorescence of dsRNA in WT and NME6 KO HeLa cells incubated with 100 μM nucleosides for 5 d and imaged by confocal microscopy. DAPI staining shown in blue (scale bar: 20 μm). (G, H) Scatter plot of mean mt-dsRNA intensity per cell quantified from confocal images as shown in (G) (N = 100–120 cells from two independent cultures). The Mann-Whitney t test was used to determine the P -value between NME6 KO versus NME6 KO + nuc. P -value < 0.0001. Horizontal lines indicate the mean value and error bars indicate the SD. Source data are available for this figure.

Article Snippet: Human primary foreskin dermal fibroblast cells, CRL-2097 (ATCC) were transduced with retrovirus particles generated with plasmids encoding pBABE-hTERT-puromycin, pBABE-SV40LT-neomycin, and pMSCV-HRasGV12-blasticidine.

Techniques: Immunofluorescence, Confocal Microscopy, Staining, Northern Blot, Control, Expressing, Incubation, MANN-WHITNEY

Journal: Life Science Alliance

Article Title: Mitochondrial double-stranded RNA homeostasis depends on cell-cycle progression

doi: 10.26508/lsa.202402764

Figure Lengend Snippet: (A) Immunofluorescence of FASTKD2 and GRSF1 in the indicated fibroblast cell lines imaged by confocal microscopy. DAPI staining shown in blue (scale bar: 20 μm). (B) Scatter plot of mean mitochondrial J2 intensity per cell quantified from confocal images as shown in . The one-way ANOVA test was used to determine P -values compared with siLuc. P -value (siLuc versus siSUV3) < 0.0001, P -value (siLuc versus siPNPase) < 0.0001 (N = 100 cells from two independent cultures). (C) Immunofluorescence of NME6 and dsRNA in HeLa cells (top) and U2OS cells (bottom) treated with the indicated siRNA for 48 h, imaged by confocal microscopy. DAPI staining is shown in blue (scale bar: 20 μm). (D) Immunofluorescence of dsRNA in WT fibroblasts incubated with 100 μM nucleosides for 5 d and imaged by confocal microscopy. The mitochondrial network was labelled with MitoTracker (MitoT) Deep Red. DAPI staining shown in blue (scale bar: 20 μm).

Article Snippet: Human primary foreskin dermal fibroblast cells, CRL-2097 (ATCC) were transduced with retrovirus particles generated with plasmids encoding pBABE-hTERT-puromycin, pBABE-SV40LT-neomycin, and pMSCV-HRasGV12-blasticidine.

Techniques: Immunofluorescence, Confocal Microscopy, Staining, Incubation

Journal: Life Science Alliance

Article Title: Mitochondrial double-stranded RNA homeostasis depends on cell-cycle progression

doi: 10.26508/lsa.202402764

Figure Lengend Snippet: (A) hTERT fibroblasts were treated with basic FGF (10 ng/ml), aphidicolin (Aph; 6 μM), or a combination of both. Cell proliferation was measured by confluency (N = 3 independent cultures; points indicate mean and error bars represent SD). (B) Immunofluorescence of dsRNA in hTERT fibroblasts treated with FGF/Aph for 48 h and BrU for 1 h and imaged by confocal microscopy. The mitochondrial network was labelled with MitoTracker DeepRed (MitoT; magenta). DAPI staining shown in blue (scale bar: 20 μm). (C) Scatter plot of mean BrU intensity per cell quantified from confocal images as shown in (N = 92–111 cells from two independent cultures). A one-way ANOVA test was used to determine P -values compared with DMSO. P -value (DMSO versus Aph) = 0.0130, P -value (DMSO versus FGF) < 0.0001, P -value (DMSO versus FGF + Aph) < 0.0001. Horizontal lines indicate the mean value and error bars indicate the SD. (D) Scatter plot of mean mt-dsRNA intensity per cell quantified from confocal images as shown in (B) (N = 89–111 cells from two independent cultures). A one-way ANOVA test was used to determine P-values compared to DMSO. P -value (DMSO versus Aph) = 0.2653, P -value (DMSO versus FGF) < 0.0001, P -value (DMSO versus FGF + Aph) = 0.0735. Horizontal lines indicate the mean value and error bars indicate the SD. (E) Heatmap showing relative RNA expression of heavy and light mitochondrial transcripts from hTERT fibroblasts treated with FGF/Aph for 48 h as measured by strand-specific RT-qPCR. Fold changes versus mean DMSO values for each transcript are shown. C q values were normalised against GAPDH (N = 3 independent cultures). One-way ANOVA analysis was performed for each transcript among the four conditions and the resulting P -value is indicated below. (F) mtDNA levels of hTERT fibroblasts treated with FGF/Aph for 48 h as measured by qPCR of the indicated mtDNA regions, MT-RNR1 and MT-CYTB . C q values were normalised against nuclear ACTB (N = 3 independent cultures). Bars indicate the mean and the error bars represent the SD. Individual data points are shown as grey circles. (G) Immunoblot analysis of mitochondrial and MRG-associated proteins in whole-cell lysates obtained from hTERT fibroblasts treated with FGF/Aph for 48 h. Samples were blotted on different membranes which are indicated by the dotted lines. Source data are available for this figure.

Article Snippet: Human primary foreskin dermal fibroblast cells, CRL-2097 (ATCC) were transduced with retrovirus particles generated with plasmids encoding pBABE-hTERT-puromycin, pBABE-SV40LT-neomycin, and pMSCV-HRasGV12-blasticidine.

Techniques: Immunofluorescence, Confocal Microscopy, Staining, RNA Expression, Quantitative RT-PCR, Western Blot

Journal: Life Science Alliance

Article Title: Mitochondrial double-stranded RNA homeostasis depends on cell-cycle progression

doi: 10.26508/lsa.202402764

Figure Lengend Snippet: (A) Immunofluorescence of BrU in hTERT fibroblasts treated with FGF/Aph for 48 h and BrU for 1 h and imaged by confocal microscopy. The mitochondrial network was labelled with MitoTracker DeepRed. DAPI staining is shown in blue (scale bar: 20 μm). (B) Northern blot of mRNA transcripts of the coding and mirror regions of CYTB in hTERT fibroblasts treated with FGF/Aph for 48 h. Nuclear 7SL RNA was used as a loading control. (B, C) Scatter plot of the total mitochondrial network area per cell from confocal images as shown in (B). The one-way ANOVA test was used to determine P-values compared with DMSO. P -value (DMSO versus Aph) < 0.0001, P -value (DMSO versus FGF) > 0.9999, P -value (DMSO versus FGF + Aph) < 0.0001 (Number of cells for each cell line = 108, from two independent cultures). Horizontal lines indicate the mean value and error bars indicate the SD. Source data are available for this figure.

Article Snippet: Human primary foreskin dermal fibroblast cells, CRL-2097 (ATCC) were transduced with retrovirus particles generated with plasmids encoding pBABE-hTERT-puromycin, pBABE-SV40LT-neomycin, and pMSCV-HRasGV12-blasticidine.

Techniques: Immunofluorescence, Confocal Microscopy, Staining, Northern Blot, Control