Journal: Nucleic Acids Research

Article Title: Differential uptake, kinetics and mechanisms of intracellular trafficking of next-generation antisense oligonucleotides across human cancer cell lines

doi: 10.1093/nar/gkz214

Figure Lengend Snippet: RAB5C regulates ASO productive uptake in NSCLC. ( A ) Data showing IC 50 dose–response curves for KRAS mRNA knockdown in H2030 cells transfected with siRNA targeting RNASEH1, RAB5C or negative controls for 72 h and then treated with AZD4785 for 48 h. Data were normalized to housekeeping gene 18S rRNA and PBS control. Results were expressed from the mean of biological duplicates from each experimental run and then averaged across two independent experiments. Error bars represent SEM. ( B ) Western blot analysis of KRas and vinculin levels in lysates from H2030 cells transfected for 72 h with siRNA On-Target SMARTpools for RAB5C, RNASEH1 or non-targeting control (siNTC) before treating with AZD4785 for 48 h. Red box highlights differences in KRas proteins levels between control and siRAB5C transfected cells. ( C ) RT-qPCR was carried out on untreated cells to determine expression levels of RAB5C across the cell panel. Data were normalized to 18S rRNA control and shown relative to H2030 cells. Data shown represent the mean and SEM from a minimum of three experiments. ( D ) RT-qPCR was carried out on untreated cells to determine expression levels of RAB5 isoforms across the cell panel. Data were normalized to 18S rRNA control and shown relative to RAB5C . Data shown represent the mean and SD from a minimum of two experiments. ( E ) Data showing IC 50 dose–response curves for KRAS mRNA knockdown in A549 cells transfected with siRNA targeting RNASEH1, RAB5C or siNTC for 72 h and then treated with AZD4785 for 48 h. Data were normalized to housekeeping gene 18S rRNA and PBS control. Results were expressed from the mean of biological duplicates from an experimental run, error bars represent the range.

Article Snippet: FAM MGB Assay Probes were obtained from Thermo Fisher Applied Biosystems: KRAS (Hs00364284_g1), FOSL1 (Hs00759776_S1), 18S rRNA (4352655), RAB5C (Hs00904926_g1), RNASEH1 (Hs 007702360_s1), KIF1A (Hs00987720_m1), KIF5C (Hs00189672_m1), RAB5A (Hs00702360_s1) and RAB5B (Hs00951468_M1).

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

Journal: Nucleic Acids Research

Article Title: Differential uptake, kinetics and mechanisms of intracellular trafficking of next-generation antisense oligonucleotides across human cancer cell lines

doi: 10.1093/nar/gkz214

Figure Lengend Snippet: Schematic to show the proposed mechanism of ASO productive and non-productive trafficking in the cell. ASO is trafficked to the early endosome where it may be recycled back to the membrane or undergo endosomal escape mediated by Rab5C. The early endosome can fuse with the late endosome or multivesicular body and transport cargo, likely through motor proteins kinesin-1 and-3, either to the lysosome for degradation or to the cell periphery where it fuses with the plasma membrane and is secreted out of the cell via extracellular vesicles.

Article Snippet: FAM MGB Assay Probes were obtained from Thermo Fisher Applied Biosystems: KRAS (Hs00364284_g1), FOSL1 (Hs00759776_S1), 18S rRNA (4352655), RAB5C (Hs00904926_g1), RNASEH1 (Hs 007702360_s1), KIF1A (Hs00987720_m1), KIF5C (Hs00189672_m1), RAB5A (Hs00702360_s1) and RAB5B (Hs00951468_M1).

Techniques: Membrane, Clinical Proteomics

Journal: eLife

Article Title: Early myelination involves the dynamic and repetitive ensheathment of axons which resolves through a low and consistent stabilization rate

doi: 10.7554/eLife.82111

Figure Lengend Snippet: ( A ) The endocytic recycling pathway during sheath initiation and loss. ( B ) Lateral images of oligodendrocytes in the early stages of the ensheathment process in the spinal cord of living larvae at 2.5 dpf labeled with sox10:mScarlet-CAAX (magenta) and expressing either myrf:eGFP-RAB5C , myrf:eGFP-RAB7A , or myrf:eGFP-RAB11A (green). White boxes outline immature sheaths with Rab+ endosomal puncta for each fusion protein. (Scale bar = 5 μm). ( C ) Top panels are grey inset images from the outlined regions in B. The bottom panels are 3D reconstructions of these insets (Membrane in magenta, endosomes in green). ( D ) Quantification of Rab+ endosomal puncta in immature sheaths. Number of puncta in each sheath was normalized by the length of the sheath. Rab5 n = 36 sheaths (9 ventral cells/2 dorsal cells/11 larvae), Rab11 n = 34 sheaths (8 ventral cells/2 dorsal cells/10 larvae), Rab7 n = 33 sheaths (9 ventral cells/2 dorsal cells/11 larvae). Dashed lines represent average values and error bars are SD. (See associated source data and supplementary video files). Figure 7—source data 1. Excel spreadsheet with the Rab5, -7, -11 localization data.

Article Snippet: Plasmids encoding the RAB5C , RAB7A , and RAB11A zebrafish coding sequences (Addgene, RAB5C =80518, RAB7A =80522, and RAB11A =80529) were sequenced, and the RAB7A and RAB11A sequences each had a point mutation ( D63G and Q166R, respectively) relative to the NCBI protein sequences ( RAB7A =NM_200928.1, RAB11A =NM_001007359.1) and relative to other previous work in zebrafish ( ).

Techniques: Labeling, Expressing, Membrane

Journal: eLife

Article Title: Early myelination involves the dynamic and repetitive ensheathment of axons which resolves through a low and consistent stabilization rate

doi: 10.7554/eLife.82111

Figure Lengend Snippet: ( A ) Representative lateral images of ventral oligodendrocytes in the spinal cord of living larvae at 4 days post fertilization (dpf) labeled by sox10:eGFP-CAAX (green) and one of the following: myrf:tagRFP , myrf:tagRFP-RAB5C , myrf:tagRFP-rab5C S36N , myrf:tagRFP-RAB7A , myrf:tagRFP-rab7A T22N , and myrf:tagRFP-RAB11A , myrf:tagRFP-rab11A S25N (all in magenta). The image and data for the control is the same as for the ventral group in (scale bar = 5 μm). ( B ) Sheath number per cell. ( C ) Average sheath length per cell. ( D ) Total sheath length per cell ( myrf:tagRFP n=26 cells/26 larvae, myrf:tagRFP-RAB5C n=28 cells/28 larvae, myrf:tagRFP-rab5C S36N n=27 cells/27 larvae , myrf:tagRFP-RAB7A n=29 cells/29 larvae, myrf:tagRFP-rab7A T22N n=32 cells/32 larvae, and myrf:tagRFP-RAB11A n=30 cells/30 larvae, myrf:tagRFP-rab11A S25N n=27 cells/27 larvae). The dashed lines in each plot represent average values with all data points shown. Error bars are standard deviation. Global significance was determined using a Kruskal-Wallis test for B–D. This global p-value is shown for C since it was not significant. Post hoc multiple comparison tests were not performed for this analysis. Post hoc Dunn’s multiple comparison tests were performed to compare groups in B and D. We compared everything with the control group and compared each wild-type and associated mutant with each other. The different Rab groups were not compared with each other (see associated source data). Figure 8—source data 1. Excel spreadsheet with the sheath analysis data for the Rab5, -7, -11 dominant-negative mutants.

Article Snippet: Plasmids encoding the RAB5C , RAB7A , and RAB11A zebrafish coding sequences (Addgene, RAB5C =80518, RAB7A =80522, and RAB11A =80529) were sequenced, and the RAB7A and RAB11A sequences each had a point mutation ( D63G and Q166R, respectively) relative to the NCBI protein sequences ( RAB7A =NM_200928.1, RAB11A =NM_001007359.1) and relative to other previous work in zebrafish ( ).

Techniques: Labeling, Control, Standard Deviation, Comparison, Mutagenesis, Dominant Negative Mutation

Journal: eLife

Article Title: Early myelination involves the dynamic and repetitive ensheathment of axons which resolves through a low and consistent stabilization rate

doi: 10.7554/eLife.82111

Figure Lengend Snippet: ( A ) Lateral images from the ventral spinal cord of living larvae labeled with sox10 : eGFP-CAAX (in green) and one of the following: myrf:tagRFP , myrf:tagRFP - RAB5C , myrf:tagRFP-rab5C S36N (all in magenta); and time-lapsed for 15 hours from 2.5-3dpf. The first panel is an image taken immediately before starting the time-lapse experiment. The subsequent panels are the same cells at the peak of sheath accumulation, at 3dpf, and at 4dpf. The images and data for the control are the same as for the ventral group in . (Scale bar = 5 μm). ( B ) Total ensheathment attempts per cell. ( C ) Peak sheath number per cell. ( D ) Sheath number at 3dpf per cell. ( E ) Final sheath number per cell at 4dpf. ( F ) Net sheaths lost from the peak to 4dpf. ( G ) Percent of sheaths stabilized during the accumulation phase (peak sheath number/total ensheathment attempts). (H) Percent of sheaths stabilized during the stabilization phase (final sheath number/peak sheath number). ( I ) Percent of total sheaths stabilized across both the accumulation and stabilization phases (final sheath number/total ensheathment attempts). ( J ) Simple linear regression comparing the total number of ensheathment attempts to the final sheath number at 4dpf for each cell. (control n=18 cells/18 larvae, wild-type Rab5 n=18 cells/18 larvae, Rab5DN n=18 cells/17 larvae). The dashed lines in each plot represent average values with all data points shown. The error bars are standard deviation. Significance was determined using global Kruskal-Wallis tests. These p-values are shown for B-D and G since they were not significant. Post hoc multiple comparisons tests were not performed for these analyses. Post hoc Dunn’s multiple comparisons tests were done for E, F, H, and I and the individual p-values are shown. ( J’ ) The slopes of the Rab5WT and Rab5DN regression lines from J were compared to the control in Graphpad by (two-tailed) testing the null hypothesis that the slopes are identical (the lines are parallel). P-values are shown in the table. (See associated source data and supplementary video files). Figure 9—source data 1. Excel spreadsheet with the summary data for the Rab5 dominant-negative mutant oligodendrocyte ensheathment dynamics experiment.

Article Snippet: Plasmids encoding the RAB5C , RAB7A , and RAB11A zebrafish coding sequences (Addgene, RAB5C =80518, RAB7A =80522, and RAB11A =80529) were sequenced, and the RAB7A and RAB11A sequences each had a point mutation ( D63G and Q166R, respectively) relative to the NCBI protein sequences ( RAB7A =NM_200928.1, RAB11A =NM_001007359.1) and relative to other previous work in zebrafish ( ).

Techniques: Labeling, Control, Standard Deviation, Two Tailed Test, Dominant Negative Mutation

Journal: eLife

Article Title: Early myelination involves the dynamic and repetitive ensheathment of axons which resolves through a low and consistent stabilization rate

doi: 10.7554/eLife.82111

Figure Lengend Snippet:

Article Snippet: Plasmids encoding the RAB5C , RAB7A , and RAB11A zebrafish coding sequences (Addgene, RAB5C =80518, RAB7A =80522, and RAB11A =80529) were sequenced, and the RAB7A and RAB11A sequences each had a point mutation ( D63G and Q166R, respectively) relative to the NCBI protein sequences ( RAB7A =NM_200928.1, RAB11A =NM_001007359.1) and relative to other previous work in zebrafish ( ).

Techniques: Plasmid Preparation, Membrane, Mutagenesis, Transgenic Assay, Expressing

Journal: bioRxiv

Article Title: Genetic analysis of rab7 mutants in zebrafish

doi: 10.1101/2023.03.09.531857

Figure Lengend Snippet: A Confocal images of a tip cell from a 32hpf transgenic Tg(kdrl:mCherry-CAAX) S916 embryo injected with the plasmid fli:EGFP-Rab5c (marker of early endosome). The Rab5c signal (EGFP) does not co-localise with CAAX at dots, but Rab5c dots are found close to the apical surface of the expanding lumen. A’ Inverted contrast of eGFP-Rab5c from A shows Rab5c dots (pink circles). Note that these regions do overlap with the CAAX signal (A’’). A’’ Inverted contrast of mCherry-CAAX from A. B Confocal images of a tip cell from a 32hpf transgenic Tg(kdrl:mCherry-CAAX) S916 embryo injected with the plasmid fli:EGFP-Rab11a (marker of recycling endosome). The Rab11a signal (EGFP) does not co-localise with CAAX at dots, but Rab11a dots are found in proximity to the apical surface and is also found along the apical membrane of the expanding lumen. A’ Inverted contrast of EGFP-Rab11a from B shows Rab11a dots (pink circles). Note that these regions overlap with CAAX signal (B’’). B’’ Inverted contrast of mCherry-CAAX from B.

Article Snippet: For generation of the fli1: EGFP-rab5c vector, a pDestTol2CG2-heart-gfp with the independent marker cmlc2:EGFP, a fli1 P-5’entry clone (Addgene, Lawson Lab), an EGFP p-middle entry (Addgene, Kwan, Chien lab) and rab5c p-3’entry clone ( ) were used.

Techniques: Transgenic Assay, Injection, Plasmid Preparation, Marker