lipofectaminetm rnaimax transfection reagent thermo fisher scientific cat  (Thermo Fisher)

Journal: bioRxiv

Article Title: Rab12 is a regulator of mitophagy and mitochondrial homeostasis

doi: 10.64898/2026.03.29.715103

Figure Lengend Snippet: mt-mKeima/YFP-Parkin HeLa cells were reverse transfected with siRNAs against the notated Rab GTPase family members for 24 h. Following transfection, cells were treated with vehicle or 30 μM FCCP for 4h to induce mitophagy. Cells were immediately live-cell imaged on a confocal microscope for mt-mKeima-based mitophagy analysis to analyze impact of Rab target knockdown on mitophagic flux. Results are graphed as average mt-mKeima (pH 4-5) positive foci per cell, normalized to respective plate scramble (Scr) siRNA controls. All plates also included an siRNA against PINK1 as a positive control for mitophagy impairment. Candidates with a ≥50% increase are presented in blue, and those with a ≥50% decrease in mitophagy readout are presented in red. n = 3 replicates (except Rab2B and Rab15: n = 2 replicates). Data are presented as mean ± SEM.

Article Snippet: A working mix of Lipofectamine RNAiMAX transfection reagent (Fisher Scientific 13-778-150) and Opti-MEM reduced serum medium (Thermo Scientific 31985070) was prepared by diluting 300 μL RNAiMAX in 9.7 mL OptiMEM.

Techniques: Transfection, Microscopy, Knockdown, Positive Control

Journal: bioRxiv

Article Title: Rab12 is a regulator of mitophagy and mitochondrial homeostasis

doi: 10.64898/2026.03.29.715103

Figure Lengend Snippet: ( A ) RNA was collected for qRT-PCR analysis of Rab12 mRNA expression in mt-mKeima/YFP-parkin HeLa cells following 72 h transfection with siRNA against Rab12 (siRab12) or the scramble control (siScr). Internal control for normalization was GAPDH. **** p < 0.0001 determined by unpaired t-test. n = 3 biological replicates with three technical replicates each. ( B ) Representative western blot of lysates collected from mt-mKeima/YFP-parkin HeLa cells transfected with siScr or siRab12 assessed for Rab12 levels and β-actin as a loading control. ( C ) Quantification demonstrates Rab12 protein knockdown with Rab12 siRNA by the same protocol. **** p < 0.0001, as determined by unpaired t-test. n = 3 biological replicates. ( D ) Representative 60X confocal images of YFP-Parkin (green), mt-mKeima at neutral pH (cyan), and mt-mKeima at lysosomal pH (magenta) and ( E ) quantification of mt-mKeima mitophagy analysis demonstrated increased levels of mitophagy induced by treatment with 30 μM FCCP with Rab12 knockdown. ** p < 0.01, **** p < 0.0001, as determined by two-way ANOVA with Bonferroni’s multiple comparisons test. n = 3 biological replicates. Data are presented as mean ± SEM.

Article Snippet: A working mix of Lipofectamine RNAiMAX transfection reagent (Fisher Scientific 13-778-150) and Opti-MEM reduced serum medium (Thermo Scientific 31985070) was prepared by diluting 300 μL RNAiMAX in 9.7 mL OptiMEM.

Techniques: Quantitative RT-PCR, Expressing, Transfection, Control, Western Blot, Knockdown

Journal: bioRxiv

Article Title: Rab12 is a regulator of mitophagy and mitochondrial homeostasis

doi: 10.64898/2026.03.29.715103

Figure Lengend Snippet: ( A ) mt-mKeima/YFP-Parkin HeLa were transfected with siRNA against Rab12 (siRab12) or a scramble control siRNA (siScr) for 72 h. Mito DNA DX analysis of mtDNA lesion frequency showed that Rab12 knockdown caused a significant decrease in mtDNA lesions relative to siScr transfection. n = 3 biological replicates. * p < 0.01, as determined by unpaired t-test. ( B ) No differences in steady state of mtDNA copy number was observed between conditions. n = 3 biological replicates. Data are presented as mean ± SEM.

Article Snippet: A working mix of Lipofectamine RNAiMAX transfection reagent (Fisher Scientific 13-778-150) and Opti-MEM reduced serum medium (Thermo Scientific 31985070) was prepared by diluting 300 μL RNAiMAX in 9.7 mL OptiMEM.

Techniques: Transfection, Control, Knockdown

Journal: EMBO Molecular Medicine

Article Title: Cis -aconitate therapy protects against influenza mortality by dual targeting of viral polymerase and ERK/AKT/NF-κB signaling

doi: 10.1038/s44321-026-00379-8

Figure Lengend Snippet: BEAS-2B cells were infected with influenza A/Scotland/20/74 (H3N2) virus at a MOI = 5 ( A ) or MOI = 1 ( B – F ) for 4 h, then washed and treated with 3.4 mM of cis -aco (CA) or left untreated (Medium). ( A ) Representative images from transmission electron microscopy (upper panel) and scanning electron microscopy (lower panel) show IAV particles budding at 20 h p.i., indicated by arrows (scale bar: 1 µm). ( B – E ) At 8 h p.i., viral protein (green) expression and trafficking were analyzed by ( B , C ) confocal microscopy (scale bar: 20 µm) and ( D , E ) Western blotting to detect viral NP, NS1, and PA proteins. ( C ) Raw integrated density (RawIntDen), calculated as the sum of all pixel values in the region of interest, was measured and normalized to the mean of the IAV condition for each experiment. ( E ) Relative protein levels were normalized to the mean value of “IAV condition” samples, with β-actin as a loading control. ( F ) At 6 h p.i., IAV transcription was quantified by RT-qPCR, measuring M1 viral mRNA levels. ( G ) A minigenome assay was performed in HEK-293T cells to test the effect of cis -aco on viral polymerase activity. Cells were transfected with plasmids encoding PA, PB1, PB2, NP, and the reporter plasmid pPolI-WSN-NA-firefly luciferase. At 20 h post-transfection, cells were treated with 0 or 3.4 mM cis -aco (CA) and luciferase activity was measured at 48 h post-transfection. Results are presented as the mean ± SEM from 3 ( A – C ), 4 ( D , E , G ), or 5 ( F ) independent experiments. The number of data points shown in each bar plot corresponds to the number of independent experiments performed for that condition. Statistical analyses were performed using the Kruskal–Wallis test with Dunn’s multiple comparison test ( G ), the Mann–Whitney test ( C ), or the Wilcoxon matched-pairs rank test ( E , F ). Statistical significance: * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001. .

Article Snippet: InvitrogenTM LipofectamineTM RNAiMAX Transfection Reagent , Fischer scientific , 13-778-150.

Techniques: Infection, Virus, Transmission Assay, Electron Microscopy, Expressing, Confocal Microscopy, Western Blot, Control, Quantitative RT-PCR, Activity Assay, Transfection, Plasmid Preparation, Luciferase, Comparison, MANN-WHITNEY

Journal: EMBO Molecular Medicine

Article Title: Cis -aconitate therapy protects against influenza mortality by dual targeting of viral polymerase and ERK/AKT/NF-κB signaling

doi: 10.1038/s44321-026-00379-8

Figure Lengend Snippet: ( A – E ) BEAS-2B cells were transfected with either cis -aconitate decarboxylase (CAD) or control (scramble) siRNA. At 48 h post-transfection, cells were infected or not with A/Scotland/20/74 (H3N2) virus (IAV) at an MOI = 1 for 4 h, and subsequently treated or not with 3.4 mM cis -aco (CA) for 16 h. ( A ) CAD catalyzes the decarboxylation of cis -aco to produce itaconate. ( B ) Gene knockdown was confirmed by RT-qPCR. ( C , D ) IAV particles production was measured by a plaque-forming units (pfu) assay ( C ) and a neuraminidase activity assay ( D ). ( E ) IL-6 levels in cell supernatants were quantified by ELISA. ( F ) CAD-deficient and wild-type (WT) mice were infected intranasally with 100 pfu of A/Scotland/20/74 (H3N2) virus (IAV) and treated intranasally or not with 30 mg/kg of cis -aco (CA) 20 min p.i. Animal survival was monitored daily. Data are presented as the mean ± SEM and are cumulative from a single experiment ( F ), or 3 ( B ), or 4 ( C – E ) independent experiments. The number of data points shown in each bar plot corresponds to the number of independent experiments performed for that condition. Statistical analyses were performed using the Mixed-effects model test ( B , E ) or the Kruskal–Wallis test with Dunn’s multiple comparison test ( C – E ), or the Log-rank (Mantel–Cox) test ( F ). Statistical significance: * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001. .

Article Snippet: InvitrogenTM LipofectamineTM RNAiMAX Transfection Reagent , Fischer scientific , 13-778-150.

Techniques: Transfection, Control, Infection, Virus, Knockdown, Quantitative RT-PCR, Activity Assay, Enzyme-linked Immunosorbent Assay, Comparison