unc13b  (Santa Cruz Biotechnology)

Journal: Nature

Article Title: TDP-43 loss and ALS-risk SNPs drive mis-splicing and depletion of UNC13A

doi: 10.1038/s41586-022-04436-3

Figure Lengend Snippet: ( a, b ) RNA-seq traces from IGV of representative samples from control (top) and TARDBP KD (bottom) in i 3 Neurons showing intron retention in UNC13A (A) (mean 4.50 ± 1.50 increased IR in KD) and UNC13B (mean 1.86 ± 0.63 increased IR in KD)(B), overlaid with published TDP-43 iCLIP peaks ( c ) Histogram showing number of basescope cryptic foci per nuclei in control (blue) and TDP-43 KD (grey) in WTC11-derived i 3 Neurons, p < 0.0001 unpaired t-test. ( d, e ) RT-qPCR levels of TARDBP and UNC13A with a non-targeting control sgRNA (sgTARDBP −), an intermediate TDP-43 KD (sgTARDBP +) or a higher TDP-43 KD (sgTARDBP ++) in WTC11-derived ( d ) and NCRM-5-derived i 3 Neurons ( e ). n = 4 biological replicates sgTARDBP − ( d ), n = 6 biological replicates sgTARDBP − ( e ), sgTARDBP + ( d, e ) and ++ ( d, e ). plotted as means ± SEM. ( f ) Representative images of UNC13A CE RT-PCR products ( g ) Quantification of the lower gel in ( f ) plotted as means ± SEM, n = 6 biological replicates non-targeting control sgRNA (sgTARDBP −), sgTARDBP +, sgTARDBP ++. Upper gel is quantified in Fig. . One-way ANOVA with multiple comparisons. ( h–k ) Expression of TDP-43 regulated splicing in UNC13A ( h, i ) and UNC13B ( j, k ) across neuronal datasets , in control (blue) and TDP-43 KD (yellow). Intron retention (IR)( i, k ) and CE and fsE PSI ( h, j ) significantly increase after TDP-43 depletion in most experiments, Wilcoxon test ( l ) Relative gene expression levels for TARDBP across neuronal datasets , . Normalized RNA counts are shown as relative to control mean. Numbers show log 2 fold change calculated by DESeq2. Significance shown as adjusted p-values from DESeq2. For (h–l) biological replicates are: iPSC MN Ctrl KD n = 12, TDP-43 KD n = 6; i 3 N Ctrl KD n = 4, TDP-43 KD n = 3; SH-SY5Y, SK-N-DZ a , and SK-N-DZ b Ctrl KD n = 3, TDP-43 KD n = 3, Significance levels reported as * (p < 0.05) ** (p < 0.01) *** (p < 0.001) **** (p < 0.0001).

Article Snippet: Gene expression analysis was performed by qPCR using Taqman Multiplex Universal Master Mix (Thermo 4461882) or Taqman Universal PCR Master Mix (Thermo 4304437) and TaqMan assays (UNC13A-Fam Hs00392638_m1, UNC13B-Fam Hs01066405_m1, TDP-43-Vic Hs00606522_m1, GAPDH-Jun assay 4485713, TDP-43-FAM Hs00606522_m1, UPF1-FAM Hs00161289_m1, HPRT1-FAM Hs02800695_m1) on a QuantStudio 5 or a QuantStudio 6 Flex Real-Time PCR system (Applied Biosystems) and quantified using the ΔΔ C t method .

Techniques: RNA Sequencing, Control, Derivative Assay, Quantitative RT-PCR, Reverse Transcription Polymerase Chain Reaction, Expressing, Gene Expression

Journal: Nature

Article Title: TDP-43 loss and ALS-risk SNPs drive mis-splicing and depletion of UNC13A

doi: 10.1038/s41586-022-04436-3

Figure Lengend Snippet: a , Differential splicing analysis by MAJIQ in control ( n = 4) and CRISPRi TDP-43 depleted (KD) ( n = 3) iPS cell-derived cortical-like i 3 Neurons. Each point denotes a splice junction. b , Representative sashimi plots showing cryptic exon (CE) inclusion between exons 20 and 21 of UNC13A upon TDP-43 knockdown. c , f , Schematics showing intron retention (IR) (orange; bottom), TDP-43 binding region (green), and two ALS- and FTLD-associated SNPs (red) in UNC13A ( c ) and UNC13B ( f ). d , LocusZoom plot of the UNC13A locus in the most recent ALS GWAS ; the dashed line indicates the risk threshold used in that study. Lead SNP rs12973192 is plotted as a purple diamond, other SNPs are coloured by linkage disequilibrium (LD) with rs12973192 in European individuals from 1000 Genomes. Ref. var., reference variant. e , Representative sashimi plot of UNC13B showing inclusion of the FSE upon TDP-43 knockdown. g , BaseScope detection of UNC13A CE (white puncta) in control (top) and TDP-43-knockdown (bottom) i 3 Neurons co-stained for TDP-43 (green), neuronal processes (stained for TUBB3, pink) and nuclei (blue). Scale bar, 5 μm. h , Quantification of RT–PCR products using iPS cell-derived neurons made from an independent iPS cell line, NCRM5, with a non-targeting control short guide RNA (sgRNA) (sgTARDBP−), an intermediate TDP-43 knockdown (sgTARDBP+) or stronger TDP-43 knockdown (sgTARDBP++). Data are mean ± s.e.m. sgControl, n = 6; sgTARDBP+, n = 5; sgTARDBP++, n = 6; one-way ANOVA with multiple comparisons. * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001. i , Schematic of nanopore long reads quantified in j , Extended Data Figs. . j , Percentage of targeted UNC13A long reads with TDP-43-regulated splice events that contain CE, intron retention or both in TDP-43-knockdown SH-SY5Y cells.

Article Snippet: Gene expression analysis was performed by qPCR using Taqman Multiplex Universal Master Mix (Thermo 4461882) or Taqman Universal PCR Master Mix (Thermo 4304437) and TaqMan assays (UNC13A-Fam Hs00392638_m1, UNC13B-Fam Hs01066405_m1, TDP-43-Vic Hs00606522_m1, GAPDH-Jun assay 4485713, TDP-43-FAM Hs00606522_m1, UPF1-FAM Hs00161289_m1, HPRT1-FAM Hs02800695_m1) on a QuantStudio 5 or a QuantStudio 6 Flex Real-Time PCR system (Applied Biosystems) and quantified using the ΔΔ C t method .

Techniques: Control, Derivative Assay, Knockdown, Binding Assay, Variant Assay, Staining, Reverse Transcription Polymerase Chain Reaction

Journal: Nature

Article Title: TDP-43 loss and ALS-risk SNPs drive mis-splicing and depletion of UNC13A

doi: 10.1038/s41586-022-04436-3

Figure Lengend Snippet: Targeted nanopore sequencing reveals UNC13A CE and IR events occur largely independently in-vitro. ( a ) Sanger sequencing of cryptic bands in both SH-SY5Y and SK-N-DZ cells confirm the CE splice junctions. ( b, c ) Crosslink density across UNC13A (chr19) (b) and UNC13B (chr9) ( c ) genomic loci from novel iCLIP on endogenous TDP-43 in SH-SHY5Y cells. Crosslink densities for both genes show peaks at the CE/fsE (red) and retained introns (blue). Coordinates shown in hg38. ( d ) Percentage of all targeted UNC13A long reads in SH-SY5Y cells containing either neither CE nor IR, both, or either CE or IR. Most reads in both control and TDP-43 KD contain neither event, and while IR event is present in controls, CE is only detected in TDP-43 KD. ( e ) Representative trace in TDP-43 KD of UNC13A targeted long reads showing transcript containing either the CE or IR, and transcripts with neither.

Article Snippet: Gene expression analysis was performed by qPCR using Taqman Multiplex Universal Master Mix (Thermo 4461882) or Taqman Universal PCR Master Mix (Thermo 4304437) and TaqMan assays (UNC13A-Fam Hs00392638_m1, UNC13B-Fam Hs01066405_m1, TDP-43-Vic Hs00606522_m1, GAPDH-Jun assay 4485713, TDP-43-FAM Hs00606522_m1, UPF1-FAM Hs00161289_m1, HPRT1-FAM Hs02800695_m1) on a QuantStudio 5 or a QuantStudio 6 Flex Real-Time PCR system (Applied Biosystems) and quantified using the ΔΔ C t method .

Techniques: Nanopore Sequencing, In Vitro, Sequencing, Control

Journal: Nature

Article Title: TDP-43 loss and ALS-risk SNPs drive mis-splicing and depletion of UNC13A

doi: 10.1038/s41586-022-04436-3

Figure Lengend Snippet: Relative gene expression levels for UNC13A ( a ) and UNC13B ( b ) after TDP-43 knockdown across neuronal cell lines , . Normalized RNA counts are shown as relative to control mean. Numbers show log fold change calculated by DESeq2. Significance shown as adjusted p-values from DESeq2. Number of replicates as in Extended data Fig. ( c, d ) RT-qPCR analysis shows TDP-43, UNC13A and UNC13B gene expression is reduced by TARDBP shRNA knockdown in both SH-SY5Y and SK-N-DZ human cell lines. Graphs represent the means ± SEM, n = 6 biological replicates, one sample t-test. ( e ) The 5’ ends of 29 nt reads relative to the annotated start codon from a representative ribosome profiling dataset (TDP-43 KD replicate B). As expected, we detected strong three-nucleotide periodicity, and a strong enrichment of reads across the annotated coding sequence relative to the upstream untranslated region. ( f ) UNC13A, UNC13B, and TDP-43 protein levels, measured by Western Blot, with varying levels of DOX-inducible TDP-43 knockdown in SH-SY5Y cells. Tubulin is used as endogenous control, n = 3. For gel source data, see Supplementary Figure 1. ( g ) Quantification of RT-PCR products from the transcripts containing UNC13A CE, UNC13A intron retention, UNC13B fsE, and UNC13B intron retention, with varying levels of DOX-inducible TDP-43 knockdown in SH-SY5Y cells. Graphs represent the means ± SEM n = 3 biological replicates. ( h ) UPF1 siRNA knock-down led to the rescue of hnRNPL (positive control), UNC13A , and UNC13B transcripts, but not STMN2 . Graphs represent the means ± SEM, n = 4 biological replicates, one-sample t-test. ( l ) UNC13A CE containing-transcript PSI is increased after UPF1 knockdown in i 3 Neurons. Graphs represent the means ± SEM, n = 6 biological replicates. ( j ) RT-PCR products from UNC13A in the setting of mild TDP-43 knockdown (“+”, as for Figure and S4G) with the addition of either DMSO (control) or CHX (NMD inhibition). ( k ) Quantification of ( j ) Graphs represent the means ± SEM, n = 4 biological replicates. Significance levels reported as * (p < 0.05) ** (p < 0.01) *** (p < 0.001) **** (p < 0.0001).

Article Snippet: Gene expression analysis was performed by qPCR using Taqman Multiplex Universal Master Mix (Thermo 4461882) or Taqman Universal PCR Master Mix (Thermo 4304437) and TaqMan assays (UNC13A-Fam Hs00392638_m1, UNC13B-Fam Hs01066405_m1, TDP-43-Vic Hs00606522_m1, GAPDH-Jun assay 4485713, TDP-43-FAM Hs00606522_m1, UPF1-FAM Hs00161289_m1, HPRT1-FAM Hs02800695_m1) on a QuantStudio 5 or a QuantStudio 6 Flex Real-Time PCR system (Applied Biosystems) and quantified using the ΔΔ C t method .

Techniques: Gene Expression, Knockdown, Control, Quantitative RT-PCR, shRNA, Sequencing, Western Blot, Reverse Transcription Polymerase Chain Reaction, Positive Control, Inhibition

Journal: Nature

Article Title: TDP-43 loss and ALS-risk SNPs drive mis-splicing and depletion of UNC13A

doi: 10.1038/s41586-022-04436-3

Figure Lengend Snippet: a , Ribosome profiling of TDP-43-knockdown i 3 Neurons shows a reduction in ribosome occupancy of STMN2 , UNC13A and UNC13B transcripts. b , Mass spectrometry-based proteomic analysis shows dose-dependent reduction in protein abundance of UNC13A and TDP-43 upon TDP-43 knockdown in i 3 Neurons. n = 6 biological replicates. Two-sample t -test. c , Protein and RNA quantification of TDP-43, UNC13A and UNC13B in SH-SY5Y with varying levels of doxycycline-inducible TDP-43 knockdown. n = 3 biological replicates. d , Transcript expression upon treatment with CHX suggests that UNC13A and UNC13B , but not STMN2 , are sensitive to NMD. HNRNPL is used as a positive control. n = 7 biological replicates ( UNC13A , HNRNPL and STMN2 ) and 8 biological replicates ( UNC13B ). One-sample t -test. Data are mean ± s.e.m. ( b – d ).

Article Snippet: Gene expression analysis was performed by qPCR using Taqman Multiplex Universal Master Mix (Thermo 4461882) or Taqman Universal PCR Master Mix (Thermo 4304437) and TaqMan assays (UNC13A-Fam Hs00392638_m1, UNC13B-Fam Hs01066405_m1, TDP-43-Vic Hs00606522_m1, GAPDH-Jun assay 4485713, TDP-43-FAM Hs00606522_m1, UPF1-FAM Hs00161289_m1, HPRT1-FAM Hs02800695_m1) on a QuantStudio 5 or a QuantStudio 6 Flex Real-Time PCR system (Applied Biosystems) and quantified using the ΔΔ C t method .

Techniques: Knockdown, Mass Spectrometry, Quantitative Proteomics, Expressing, Positive Control

Journal: Nature

Article Title: TDP-43 loss and ALS-risk SNPs drive mis-splicing and depletion of UNC13A

doi: 10.1038/s41586-022-04436-3

Figure Lengend Snippet: ( a ) Expression of splice junction reads supporting the UNC13B fsE across tissues and disease subtypes. Junction counts are normalised by library size in millions (junctions per million). Expression of UNC13B fsE is present across controls and ALS/FTLD-non-TDP tissues. Wilcoxon test, significance levels reported as * (p < 0.05) ** (p < 0.01) *** (p < 0.001) **** (p < 0.0001). ( b ) Diagram showing three of the UNC13B transcripts, including the APPRIS principal isoform UNC13B-207 (blue), the NMD sensitive isoform UNC13B-208 (green), and the shorter isoform UNC13B-210 which shares the fsE (light green highlight) and one of the splicing junctions supporting the fsE as UNC13B-208 . ( c ) Expression of three UNC13B isoforms across NYGC cohort and in the five in vitro TDP-43 knockdowns experiments , . UNC13B-210 is expressed across in vivo human tissues, whereas there is almost no expression of UNC13B-210 in any of the in vitro experiments. Box plots ( a, c ): boundaries 25–75th percentiles; midline, median; whiskers, Tukey style.

Article Snippet: Gene expression analysis was performed by qPCR using Taqman Multiplex Universal Master Mix (Thermo 4461882) or Taqman Universal PCR Master Mix (Thermo 4304437) and TaqMan assays (UNC13A-Fam Hs00392638_m1, UNC13B-Fam Hs01066405_m1, TDP-43-Vic Hs00606522_m1, GAPDH-Jun assay 4485713, TDP-43-FAM Hs00606522_m1, UPF1-FAM Hs00161289_m1, HPRT1-FAM Hs02800695_m1) on a QuantStudio 5 or a QuantStudio 6 Flex Real-Time PCR system (Applied Biosystems) and quantified using the ΔΔ C t method .

Techniques: Expressing, In Vitro, In Vivo

Journal: Nature

Article Title: TDP-43 loss and ALS-risk SNPs drive mis-splicing and depletion of UNC13A

doi: 10.1038/s41586-022-04436-3

Figure Lengend Snippet: STMN2 CE PSI correlates with TDP-43 regulated cryptics across NYGC RNA-seq dataset. IRratio in UNC13A exon 31−32 ( a ) and UNC13B exon 21−22 ( b ) across NYGC tissue samples. UNC13A IR was lower in ALS-TDP cases than in controls in cervical spinal, frontal and motor cortices, and higher in FTLD-TDP cases than controls in frontal and temporal cortices. Possibly this reflects differences in the effects of cell type composition in disease state. Box plots ( a, b ): boundaries 25–75th percentiles; midline, median; whiskers, Tukey style..Wilcoxon test, significance levels reported as * (p < 0.05) ** (p < 0.01) *** (p < 0.001) **** (p < 0.0001). ( c–e ) Correlation in ALS/FTLD-TDP cortex between RAP1GAP CE ( c ), PFKP CE ( d ), and UNC13A CE ( e ) with STMN2 CE PSI in patients with at least 30 spliced reads across the CE locus. Spearman’s correlation.

Article Snippet: Gene expression analysis was performed by qPCR using Taqman Multiplex Universal Master Mix (Thermo 4461882) or Taqman Universal PCR Master Mix (Thermo 4304437) and TaqMan assays (UNC13A-Fam Hs00392638_m1, UNC13B-Fam Hs01066405_m1, TDP-43-Vic Hs00606522_m1, GAPDH-Jun assay 4485713, TDP-43-FAM Hs00606522_m1, UPF1-FAM Hs00161289_m1, HPRT1-FAM Hs02800695_m1) on a QuantStudio 5 or a QuantStudio 6 Flex Real-Time PCR system (Applied Biosystems) and quantified using the ΔΔ C t method .

Techniques: RNA Sequencing

Journal: Biomedicines

Article Title: Inhibiting UNC13B Suppresses Cell Proliferation by Upregulating the Apoptotic Pathway in Multiple Myeloma

doi: 10.3390/biomedicines13092086

Figure Lengend Snippet: Relative mRNA expression of UNC13B in multiple myeloma cell lines. ( A ) Quantitative real-time PCR (qPCR) analysis of UNC13B mRNA levels in U266, ARD, and RPMI 8226 cell lines. U266 was used as the reference control for relative quantification, and GAPDH served as the internal reference gene. Expression levels were calculated using the 2 -ΔΔCt method and are presented as fold-change relative to U266. The mean ± standard deviation of UNC13B expression was 1.00 ± 0.26 in U266, 3.07 ± 0.44 in ARD, and 1.41 ± 0.26 in RPMI 8226 cells. Overlaid symbols (●, ■, ▲) denote individual independent experiments ( n = 5 per group); different marker shapes are used only for visual distinction and carry no additional meaning. ( B ) Relative UNC13B mRNA expression in ARD cells following different treatments. The Scramble + 25 μM treatment group was used as the reference for comparison. **** indicate p < 0.0001; ns = not significant ( p ≥ 0.05). Data are presented as the mean ± standard deviation (SD, n = 5 independent experiments).

Article Snippet: Primary antibodies included UNC13B (Santa Cruz, sc-136182, 1:1000), cleaved-PARP (CST, #9541, 1:1000), Bax (~21 kDa; CST, #2772, 1:1000), p21 (CST, #2947, 1:1000), PKC-PAN (#9371, 1:1000), PINK1 (#6946, 1:1000), CDK2 (#2546, 1:1000), AKR7A3 (Abcam, ab227231, 1:1000), and Bim (#2933, 1:1000).

Techniques: Expressing, Real-time Polymerase Chain Reaction, Control, Quantitative Proteomics, Gene Expression, Standard Deviation, Marker, Comparison

Journal: Biomedicines

Article Title: Inhibiting UNC13B Suppresses Cell Proliferation by Upregulating the Apoptotic Pathway in Multiple Myeloma

doi: 10.3390/biomedicines13092086

Figure Lengend Snippet: Effect of UNC13B gene ablation on cell proliferation. The proliferation rate of ARD cells in the shUNC13B group was significantly inhibited ( p < 0.0001), suggesting that UNC13B expression correlated with the proliferative capacity of ARD cells. **** indicate p < 0.0001; Data are presented as mean ± SD ( n = 3 independent experiments).

Article Snippet: Primary antibodies included UNC13B (Santa Cruz, sc-136182, 1:1000), cleaved-PARP (CST, #9541, 1:1000), Bax (~21 kDa; CST, #2772, 1:1000), p21 (CST, #2947, 1:1000), PKC-PAN (#9371, 1:1000), PINK1 (#6946, 1:1000), CDK2 (#2546, 1:1000), AKR7A3 (Abcam, ab227231, 1:1000), and Bim (#2933, 1:1000).

Techniques: Expressing

Journal: Biomedicines

Article Title: Inhibiting UNC13B Suppresses Cell Proliferation by Upregulating the Apoptotic Pathway in Multiple Myeloma

doi: 10.3390/biomedicines13092086

Figure Lengend Snippet: Effect of UNC13B gene ablation on the clonogenic ability of cells. ( A ) Representative images of colony formation in soft agar. ARD cells stably transduced with either shUNC13B or scrambled shRNA were seeded into 6-well plates (1000 cells per well) in soft agar consisting of a 0.35% agar upper layer over a 0.6% agar base layer. After 14 days of incubation at 37 °C in 5% CO 2 , visible colonies (>50 µm in diameter) were observed under a microscope. ( B ) Quantification of colony numbers. The total number of colonies per well was recorded.Overlaid symbols (●, ■) denote individual independent experiments; different marker shapes are used only for visual distinction and have no additional meaning. Data are presented as the mean ± SD. Statistical analysis was performed using a two-tailed unpaired t -test. ** indicates p < 0.01. Scale bar: 500 µm.

Article Snippet: Primary antibodies included UNC13B (Santa Cruz, sc-136182, 1:1000), cleaved-PARP (CST, #9541, 1:1000), Bax (~21 kDa; CST, #2772, 1:1000), p21 (CST, #2947, 1:1000), PKC-PAN (#9371, 1:1000), PINK1 (#6946, 1:1000), CDK2 (#2546, 1:1000), AKR7A3 (Abcam, ab227231, 1:1000), and Bim (#2933, 1:1000).

Techniques: Stable Transfection, Transduction, shRNA, Incubation, Microscopy, Marker, Two Tailed Test

Journal: Biomedicines

Article Title: Inhibiting UNC13B Suppresses Cell Proliferation by Upregulating the Apoptotic Pathway in Multiple Myeloma

doi: 10.3390/biomedicines13092086

Figure Lengend Snippet: Effect of UNC13B gene ablation on the cell cycle in ARD cells. Five days after transduction with shRNA lentiviral vectors, the shUNC13B group showed a reduced fraction of ARD cells in G1 ( p < 0.0001) and an increased fraction in S phase ( p < 0.0001) compared with the Scramble control, with no significant change in G2/M (ns, not significant). Histograms show DNA-content modeling: red areas denote fitted G1 and G2/M peaks, the blue hatched area denotes S phase, and the black line is the measured distribution; ▲ indicates software-generated peak positions (reference only, not used for quantification). **** indicate p < 0.0001.

Article Snippet: Primary antibodies included UNC13B (Santa Cruz, sc-136182, 1:1000), cleaved-PARP (CST, #9541, 1:1000), Bax (~21 kDa; CST, #2772, 1:1000), p21 (CST, #2947, 1:1000), PKC-PAN (#9371, 1:1000), PINK1 (#6946, 1:1000), CDK2 (#2546, 1:1000), AKR7A3 (Abcam, ab227231, 1:1000), and Bim (#2933, 1:1000).

Techniques: Transduction, shRNA, Control, Software, Generated

Journal: Biomedicines

Article Title: Inhibiting UNC13B Suppresses Cell Proliferation by Upregulating the Apoptotic Pathway in Multiple Myeloma

doi: 10.3390/biomedicines13092086

Figure Lengend Snippet: Effect of UNC13B gene ablation on apoptosis in ARD cells. ( A ) Flow cytometric analysis of apoptosis using Annexin V-APC staining. The shUNC13B group exhibited a significantly higher percentage of apoptotic ARD cells compared with the scrambled control group ( p < 0.01); Overlaid symbols (●, ■) denote individual independent experiments; different marker shapes are used only for visual distinction and have no additional meaning. ( B ) Western blot analysis of apoptosis- and signaling-related proteins in ARD cells stably transduced with scrambled shRNA or UNC13B shRNA (shUNC13B) for 72 h. Expression levels of PINK1, CDK2, and AKR7A3 were downregulated, while Bim was upregulated, and PKC levels changed only marginally. In the same samples, UNC13B protein expression was markedly reduced, accompanied by substantial increases in cleaved-PARP (~89 kDa), Bax (~21 kDa), and p21 (CDKN1A) levels. GAPDH served as the loading control. ** indicate p < 0.01.

Article Snippet: Primary antibodies included UNC13B (Santa Cruz, sc-136182, 1:1000), cleaved-PARP (CST, #9541, 1:1000), Bax (~21 kDa; CST, #2772, 1:1000), p21 (CST, #2947, 1:1000), PKC-PAN (#9371, 1:1000), PINK1 (#6946, 1:1000), CDK2 (#2546, 1:1000), AKR7A3 (Abcam, ab227231, 1:1000), and Bim (#2933, 1:1000).

Techniques: Staining, Control, Marker, Western Blot, Stable Transfection, Transduction, shRNA, Expressing

Journal: Oncology letters

Article Title: UNC13B regulates the sensitivity of Wilms' tumor cells to doxorubicin by modulating lysosomes.

doi: 10.3892/ol.2024.14579

Figure Lengend Snippet: Figure 1. Elevated expression of UNC13B in Wilms’ tumor cell lines. (A) Western blot analysis of UNC13B expression in WT‑CLS1, 17.94, G401, SK‑NEP‑1 and HK‑2 cells. Each lane was loaded with 20 µg protein and GAPDH was used as a reference. (B) Analysis of UNC13B transcription levels in different cell lines; n=5. (C) Changes in UNC13B transcription levels 48 h post‑shRNA‑mediated UNC13B knockdown in 17.94 cells compared with the scramble control; n=5. (D) UNC13B expression changes 48 h post‑shRNA‑mediated knockdown in 17.94 cells and (E) statistical analysis of the expression level changes. Each experiment was repeated 3 times, with GAPDH used as a reference. (F) Assessment of cell proliferation post‑knockdown using a Cell Counting Kit‑8 assay, measuring OD450 values at different time points; n=3. Cells were also transfected with non‑target scrambled shRNA as a control. ***P<0.001; ****P<0.0001. UNC13B, unc‑13 homolog B; sh, short hairpin; OD, optical density.

Article Snippet: Initially, cell slides were prepared and fixed with 100% methanol at ‐20 ̊C for 10 min. After three PBS washes, a blocking solution of 3% BSA (Sigma‐Aldrich; Merck KGaA; cat. no. V900933) + 0.3% TritonTM X‐100 in PBS was added at room temperature for 1 h. The blocking solu‐ tion was removed, and the slides were incubated with rabbit UNC13B primary antibodies (1:200; cat. no. NBP2‐93337; Novus Biologicals, Ltd.) at 4 ̊C overnight.

Techniques: Expressing, Wilms Tumor Assay, Western Blot, Knockdown, Control, CCK-8 Assay, Transfection, shRNA

Journal: Oncology letters

Article Title: UNC13B regulates the sensitivity of Wilms' tumor cells to doxorubicin by modulating lysosomes.

doi: 10.3892/ol.2024.14579

Figure Lengend Snippet: Figure 2. UNC13B influences Wilms’ tumor sensitivity to chemotherapy drugs independent of the cell cycle. Evaluation of cell proliferation post‑shRNA‑medi ated UNC13B knockdown after treatment with varying concentrations of (A) vincristine, (B) actinomycin‑D and (C) doxorubicin for 48 h in 17.94 cells, and (D) vincristine, (E) actinomycin‑D and (F) doxorubicin in the G401 cell line, assessed using Cell Counting Kit‑8 assays. Changes in drug sensitivity were analyzed, with dashed lines representing fitted curves for half‑maximal inhibitory concentration calculated using GraphPad software, and the cell number ratio indicating the relative number of viable cells compared between initial cell number and different time points. (G) Cell cycle analysis of 1 µM doxorubicin treatment on control and shUNC13B knockdown cells, detected after 48 h post‑drug treatment. (H) Quantification of the G1, S and G2 phases of the scramble and shUNC13B groups. (I) Typical pseudocolor scatter plots of the apoptosis analysis of UNC13B‑knockdown 17.94 cells after 48 h treatment with 0.5 and 2 µM doxorubicin, and (J) statistical results. **P<0.01; ***P<0.001; ****P<0.0001. UNC13B, unc‑13 homolog B; sh, short hairpin.

Article Snippet: Initially, cell slides were prepared and fixed with 100% methanol at ‐20 ̊C for 10 min. After three PBS washes, a blocking solution of 3% BSA (Sigma‐Aldrich; Merck KGaA; cat. no. V900933) + 0.3% TritonTM X‐100 in PBS was added at room temperature for 1 h. The blocking solu‐ tion was removed, and the slides were incubated with rabbit UNC13B primary antibodies (1:200; cat. no. NBP2‐93337; Novus Biologicals, Ltd.) at 4 ̊C overnight.

Techniques: Wilms Tumor Assay, Knockdown, CCK-8 Assay, Concentration Assay, Software, Cell Cycle Assay, Control

Journal: Oncology letters

Article Title: UNC13B regulates the sensitivity of Wilms' tumor cells to doxorubicin by modulating lysosomes.

doi: 10.3892/ol.2024.14579

Figure Lengend Snippet: Figure 3. UNC13B localizes within vesicles and participates in regulating lysosome formation. (A) Indirect immunofluorescence detecting endogenous UNC13B expression in 17.94 cells and revealing UNC13B localization within cellular vesicles. Green fluorescence represents UNC13B and blue fluorescence represents the cell nucleus. (B) Staining of 17.94 cells with Lyso‑Tracker. Cells were cultured in confocal culture dishes. The Mean Gray Value of red fluores cence was calculated in 7 different random fields using ImageJ software. Objective, 20X. (C) Mean Gray Value, calculated by measuring the grayscale values in indicated fluorescence channel regions of interest. ****P<0.0001. UNC13B, unc‑13 homolog B; sh, short hairpin.

Article Snippet: Initially, cell slides were prepared and fixed with 100% methanol at ‐20 ̊C for 10 min. After three PBS washes, a blocking solution of 3% BSA (Sigma‐Aldrich; Merck KGaA; cat. no. V900933) + 0.3% TritonTM X‐100 in PBS was added at room temperature for 1 h. The blocking solu‐ tion was removed, and the slides were incubated with rabbit UNC13B primary antibodies (1:200; cat. no. NBP2‐93337; Novus Biologicals, Ltd.) at 4 ̊C overnight.

Techniques: Immunofluorescence, Expressing, Fluorescence, Staining, Cell Culture, Software

Journal: Oncology letters

Article Title: UNC13B regulates the sensitivity of Wilms' tumor cells to doxorubicin by modulating lysosomes.

doi: 10.3892/ol.2024.14579

Figure Lengend Snippet: Figure 4. UNC13B modulates cell drug sensitivity by affecting lysosome formation. (A) 17.94 cell line was transiently transfected with the UNC13B‑pCDNA3.1 overexpression vector using Lipofectamine 3,000. Reverse transcription‑quantitative PCR validation was performed 24 h post‑transfection. NC was the trans fection with an empty pcDNA3.1 vector; n=3. (B) UNC13B and LAMP1 expression levels in 17.94 NC cells, UNC13B 17.94 OE cells and UNC13B 17.94 KO cells, using GAPDH as a reference, and (C) the associated semi‑quantitative results. (D) Analysis of Mean Gray Value of Lyso‑Tracker in 6 random fields of 17.94 NC, UNC13B 17.94 OE cells and UNC13B 17.94 KO cells, and (E) representative images of 17.94 NC cells, UNC13B 17.94 OE cells and UNC13B 17.94 KO cells. Lyso‑Tracker indicated lysosomes, as the dye is highly selective for acidic environments, with an excitation wavelength of 577 nm and an emission wavelength of 590 nm. (F) Assessment of doxorubicin sensitivity changes in 17.94 NC, UNC13B 17.94 OE cells and UNC13B 17.94 KO cells; n=3. The cell number ratio indicates the relative number of viable cells compared between initial cell number and different time points. *P<0.05; **P<0.01; ***P<0.001; ****P<0.0001. UNC13B, unc‑13 homolog B; NC, negative control; OE, over‑expressed; KO, knock‑out; LAMP1, lysosomal‑associated membrane protein 1.

Article Snippet: Initially, cell slides were prepared and fixed with 100% methanol at ‐20 ̊C for 10 min. After three PBS washes, a blocking solution of 3% BSA (Sigma‐Aldrich; Merck KGaA; cat. no. V900933) + 0.3% TritonTM X‐100 in PBS was added at room temperature for 1 h. The blocking solu‐ tion was removed, and the slides were incubated with rabbit UNC13B primary antibodies (1:200; cat. no. NBP2‐93337; Novus Biologicals, Ltd.) at 4 ̊C overnight.

Techniques: Transfection, Over Expression, Plasmid Preparation, Biomarker Discovery, Expressing, Negative Control, Membrane

Journal: Oncology letters

Article Title: UNC13B regulates the sensitivity of Wilms' tumor cells to doxorubicin by modulating lysosomes.

doi: 10.3892/ol.2024.14579

Figure Lengend Snippet: Figure 1. Elevated expression of UNC13B in Wilms’ tumor cell lines. (A) Western blot analysis of UNC13B expression in WT‑CLS1, 17.94, G401, SK‑NEP‑1 and HK‑2 cells. Each lane was loaded with 20 µg protein and GAPDH was used as a reference. (B) Analysis of UNC13B transcription levels in different cell lines; n=5. (C) Changes in UNC13B transcription levels 48 h post‑shRNA‑mediated UNC13B knockdown in 17.94 cells compared with the scramble control; n=5. (D) UNC13B expression changes 48 h post‑shRNA‑mediated knockdown in 17.94 cells and (E) statistical analysis of the expression level changes. Each experiment was repeated 3 times, with GAPDH used as a reference. (F) Assessment of cell proliferation post‑knockdown using a Cell Counting Kit‑8 assay, measuring OD450 values at different time points; n=3. Cells were also transfected with non‑target scrambled shRNA as a control. ***P<0.001; ****P<0.0001. UNC13B, unc‑13 homolog B; sh, short hairpin; OD, optical density.

Article Snippet: After electrophoresis, proteins were electrotransferred to a polyvinylidene fluoride membrane, which was blocked with 5% BSA (cat. no. V900933; VetecTM; Sigma‐Aldrich; Merck KGaA)/TBST (0.1% Tween 20) at room temperature for 2 h. Rabbit polyclonal UNC13B (1:1,000; cat. no. NBP2‐93337; Novus Biologicals, Ltd.) and mouse monoclonal lyso‐ somal‐associated membrane protein 1 (LAMP1; 1:1,000; cat. no. sc‐20011; Santa Cruz Biotechnology, Inc.) primary antibodies were added at the appropriate dilution and incu‐ bated at 4 ̊C overnight.

Techniques: Expressing, Wilms Tumor Assay, Western Blot, Knockdown, Control, CCK-8 Assay, Transfection, shRNA

Journal: Oncology letters

Article Title: UNC13B regulates the sensitivity of Wilms' tumor cells to doxorubicin by modulating lysosomes.

doi: 10.3892/ol.2024.14579

Figure Lengend Snippet: Figure 2. UNC13B influences Wilms’ tumor sensitivity to chemotherapy drugs independent of the cell cycle. Evaluation of cell proliferation post‑shRNA‑medi ated UNC13B knockdown after treatment with varying concentrations of (A) vincristine, (B) actinomycin‑D and (C) doxorubicin for 48 h in 17.94 cells, and (D) vincristine, (E) actinomycin‑D and (F) doxorubicin in the G401 cell line, assessed using Cell Counting Kit‑8 assays. Changes in drug sensitivity were analyzed, with dashed lines representing fitted curves for half‑maximal inhibitory concentration calculated using GraphPad software, and the cell number ratio indicating the relative number of viable cells compared between initial cell number and different time points. (G) Cell cycle analysis of 1 µM doxorubicin treatment on control and shUNC13B knockdown cells, detected after 48 h post‑drug treatment. (H) Quantification of the G1, S and G2 phases of the scramble and shUNC13B groups. (I) Typical pseudocolor scatter plots of the apoptosis analysis of UNC13B‑knockdown 17.94 cells after 48 h treatment with 0.5 and 2 µM doxorubicin, and (J) statistical results. **P<0.01; ***P<0.001; ****P<0.0001. UNC13B, unc‑13 homolog B; sh, short hairpin.

Article Snippet: After electrophoresis, proteins were electrotransferred to a polyvinylidene fluoride membrane, which was blocked with 5% BSA (cat. no. V900933; VetecTM; Sigma‐Aldrich; Merck KGaA)/TBST (0.1% Tween 20) at room temperature for 2 h. Rabbit polyclonal UNC13B (1:1,000; cat. no. NBP2‐93337; Novus Biologicals, Ltd.) and mouse monoclonal lyso‐ somal‐associated membrane protein 1 (LAMP1; 1:1,000; cat. no. sc‐20011; Santa Cruz Biotechnology, Inc.) primary antibodies were added at the appropriate dilution and incu‐ bated at 4 ̊C overnight.

Techniques: Wilms Tumor Assay, Knockdown, CCK-8 Assay, Concentration Assay, Software, Cell Cycle Assay, Control

Journal: Oncology letters

Article Title: UNC13B regulates the sensitivity of Wilms' tumor cells to doxorubicin by modulating lysosomes.

doi: 10.3892/ol.2024.14579

Figure Lengend Snippet: Figure 3. UNC13B localizes within vesicles and participates in regulating lysosome formation. (A) Indirect immunofluorescence detecting endogenous UNC13B expression in 17.94 cells and revealing UNC13B localization within cellular vesicles. Green fluorescence represents UNC13B and blue fluorescence represents the cell nucleus. (B) Staining of 17.94 cells with Lyso‑Tracker. Cells were cultured in confocal culture dishes. The Mean Gray Value of red fluores cence was calculated in 7 different random fields using ImageJ software. Objective, 20X. (C) Mean Gray Value, calculated by measuring the grayscale values in indicated fluorescence channel regions of interest. ****P<0.0001. UNC13B, unc‑13 homolog B; sh, short hairpin.

Article Snippet: After electrophoresis, proteins were electrotransferred to a polyvinylidene fluoride membrane, which was blocked with 5% BSA (cat. no. V900933; VetecTM; Sigma‐Aldrich; Merck KGaA)/TBST (0.1% Tween 20) at room temperature for 2 h. Rabbit polyclonal UNC13B (1:1,000; cat. no. NBP2‐93337; Novus Biologicals, Ltd.) and mouse monoclonal lyso‐ somal‐associated membrane protein 1 (LAMP1; 1:1,000; cat. no. sc‐20011; Santa Cruz Biotechnology, Inc.) primary antibodies were added at the appropriate dilution and incu‐ bated at 4 ̊C overnight.

Techniques: Immunofluorescence, Expressing, Fluorescence, Staining, Cell Culture, Software

Journal: Oncology letters

Article Title: UNC13B regulates the sensitivity of Wilms' tumor cells to doxorubicin by modulating lysosomes.

doi: 10.3892/ol.2024.14579

Figure Lengend Snippet: Figure 4. UNC13B modulates cell drug sensitivity by affecting lysosome formation. (A) 17.94 cell line was transiently transfected with the UNC13B‑pCDNA3.1 overexpression vector using Lipofectamine 3,000. Reverse transcription‑quantitative PCR validation was performed 24 h post‑transfection. NC was the trans fection with an empty pcDNA3.1 vector; n=3. (B) UNC13B and LAMP1 expression levels in 17.94 NC cells, UNC13B 17.94 OE cells and UNC13B 17.94 KO cells, using GAPDH as a reference, and (C) the associated semi‑quantitative results. (D) Analysis of Mean Gray Value of Lyso‑Tracker in 6 random fields of 17.94 NC, UNC13B 17.94 OE cells and UNC13B 17.94 KO cells, and (E) representative images of 17.94 NC cells, UNC13B 17.94 OE cells and UNC13B 17.94 KO cells. Lyso‑Tracker indicated lysosomes, as the dye is highly selective for acidic environments, with an excitation wavelength of 577 nm and an emission wavelength of 590 nm. (F) Assessment of doxorubicin sensitivity changes in 17.94 NC, UNC13B 17.94 OE cells and UNC13B 17.94 KO cells; n=3. The cell number ratio indicates the relative number of viable cells compared between initial cell number and different time points. *P<0.05; **P<0.01; ***P<0.001; ****P<0.0001. UNC13B, unc‑13 homolog B; NC, negative control; OE, over‑expressed; KO, knock‑out; LAMP1, lysosomal‑associated membrane protein 1.

Article Snippet: After electrophoresis, proteins were electrotransferred to a polyvinylidene fluoride membrane, which was blocked with 5% BSA (cat. no. V900933; VetecTM; Sigma‐Aldrich; Merck KGaA)/TBST (0.1% Tween 20) at room temperature for 2 h. Rabbit polyclonal UNC13B (1:1,000; cat. no. NBP2‐93337; Novus Biologicals, Ltd.) and mouse monoclonal lyso‐ somal‐associated membrane protein 1 (LAMP1; 1:1,000; cat. no. sc‐20011; Santa Cruz Biotechnology, Inc.) primary antibodies were added at the appropriate dilution and incu‐ bated at 4 ̊C overnight.

Techniques: Transfection, Over Expression, Plasmid Preparation, Biomarker Discovery, Expressing, Negative Control, Membrane

Journal: Oncology Letters

Article Title: UNC13B regulates the sensitivity of Wilms' tumor cells to doxorubicin by modulating lysosomes

doi: 10.3892/ol.2024.14579

Figure Lengend Snippet: Elevated expression of UNC13B in Wilms’ tumor cell lines. (A) Western blot analysis of UNC13B expression in WT-CLS1, 17.94, G401, SK-NEP-1 and HK-2 cells. Each lane was loaded with 20 µg protein and GAPDH was used as a reference. (B) Analysis of UNC13B transcription levels in different cell lines; n=5. (C) Changes in UNC13B transcription levels 48 h post-shRNA-mediated UNC13B knockdown in 17.94 cells compared with the scramble control; n=5. (D) UNC13B expression changes 48 h post-shRNA-mediated knockdown in 17.94 cells and (E) statistical analysis of the expression level changes. Each experiment was repeated 3 times, with GAPDH used as a reference. (F) Assessment of cell proliferation post-knockdown using a Cell Counting Kit-8 assay, measuring OD450 values at different time points; n=3. Cells were also transfected with non-target scrambled shRNA as a control. ***P<0.001; ****P<0.0001. UNC13B, unc-13 homolog B; sh, short hairpin; OD, optical density.

Article Snippet: The full-length UNC13B sequence was synthesized by General Biosystems, Inc. according to the GenBank database (ncbi.nlm.nih.gov/nuccore/NM_001371189.2/, ID no. NM_001371189.2) and inserted into the pcDNA3.1 vector using Bam HI and HindIII restriction enzyme sites.

Techniques: Expressing, Wilms Tumor Assay, Western Blot, shRNA, Knockdown, Control, Cell Counting, Transfection

Journal: Oncology Letters

Article Title: UNC13B regulates the sensitivity of Wilms' tumor cells to doxorubicin by modulating lysosomes

doi: 10.3892/ol.2024.14579

Figure Lengend Snippet: UNC13B influences Wilms’ tumor sensitivity to chemotherapy drugs independent of the cell cycle. Evaluation of cell proliferation post-shRNA-mediated UNC13B knockdown after treatment with varying concentrations of (A) vincristine, (B) actinomycin-D and (C) doxorubicin for 48 h in 17.94 cells, and (D) vincristine, (E) actinomycin-D and (F) doxorubicin in the G401 cell line, assessed using Cell Counting Kit-8 assays. Changes in drug sensitivity were analyzed, with dashed lines representing fitted curves for half-maximal inhibitory concentration calculated using GraphPad software, and the cell number ratio indicating the relative number of viable cells compared between initial cell number and different time points. (G) Cell cycle analysis of 1 µM doxorubicin treatment on control and shUNC13B knockdown cells, detected after 48 h post-drug treatment. (H) Quantification of the G1, S and G2 phases of the scramble and shUNC13B groups. (I) Typical pseudocolor scatter plots of the apoptosis analysis of UNC13B-knockdown 17.94 cells after 48 h treatment with 0.5 and 2 µM doxorubicin, and (J) statistical results. **P<0.01; ***P<0.001; ****P<0.0001. UNC13B, unc-13 homolog B; sh, short hairpin.

Article Snippet: The full-length UNC13B sequence was synthesized by General Biosystems, Inc. according to the GenBank database (ncbi.nlm.nih.gov/nuccore/NM_001371189.2/, ID no. NM_001371189.2) and inserted into the pcDNA3.1 vector using Bam HI and HindIII restriction enzyme sites.

Techniques: Wilms Tumor Assay, shRNA, Knockdown, Cell Counting, Concentration Assay, Software, Cell Cycle Assay, Control

Journal: Oncology Letters

Article Title: UNC13B regulates the sensitivity of Wilms' tumor cells to doxorubicin by modulating lysosomes

doi: 10.3892/ol.2024.14579

Figure Lengend Snippet: UNC13B localizes within vesicles and participates in regulating lysosome formation. (A) Indirect immunofluorescence detecting endogenous UNC13B expression in 17.94 cells and revealing UNC13B localization within cellular vesicles. Green fluorescence represents UNC13B and blue fluorescence represents the cell nucleus. (B) Staining of 17.94 cells with Lyso-Tracker. Cells were cultured in confocal culture dishes. The Mean Gray Value of red fluorescence was calculated in 7 different random fields using ImageJ software. Objective, 20X. (C) Mean Gray Value, calculated by measuring the grayscale values in indicated fluorescence channel regions of interest. ****P<0.0001. UNC13B, unc-13 homolog B; sh, short hairpin.

Article Snippet: The full-length UNC13B sequence was synthesized by General Biosystems, Inc. according to the GenBank database (ncbi.nlm.nih.gov/nuccore/NM_001371189.2/, ID no. NM_001371189.2) and inserted into the pcDNA3.1 vector using Bam HI and HindIII restriction enzyme sites.

Techniques: Immunofluorescence, Expressing, Fluorescence, Staining, Cell Culture, Software

Journal: Oncology Letters

Article Title: UNC13B regulates the sensitivity of Wilms' tumor cells to doxorubicin by modulating lysosomes

doi: 10.3892/ol.2024.14579

Figure Lengend Snippet: UNC13B modulates cell drug sensitivity by affecting lysosome formation. (A) 17.94 cell line was transiently transfected with the UNC13B-pCDNA3.1 overexpression vector using Lipofectamine 3,000. Reverse transcription-quantitative PCR validation was performed 24 h post-transfection. NC was the transfection with an empty pcDNA3.1 vector; n=3. (B) UNC13B and LAMP1 expression levels in 17.94 NC cells, UNC13B 17.94 OE cells and UNC13B 17.94 KO cells, using GAPDH as a reference, and (C) the associated semi-quantitative results. (D) Analysis of Mean Gray Value of Lyso-Tracker in 6 random fields of 17.94 NC, UNC13B 17.94 OE cells and UNC13B 17.94 KO cells, and (E) representative images of 17.94 NC cells, UNC13B 17.94 OE cells and UNC13B 17.94 KO cells. Lyso-Tracker indicated lysosomes, as the dye is highly selective for acidic environments, with an excitation wavelength of 577 nm and an emission wavelength of 590 nm. (F) Assessment of doxorubicin sensitivity changes in 17.94 NC, UNC13B 17.94 OE cells and UNC13B 17.94 KO cells; n=3. The cell number ratio indicates the relative number of viable cells compared between initial cell number and different time points. *P<0.05; **P<0.01; ***P<0.001; ****P<0.0001. UNC13B, unc-13 homolog B; NC, negative control; OE, over-expressed; KO, knock-out; LAMP1, lysosomal-associated membrane protein 1.

Article Snippet: The full-length UNC13B sequence was synthesized by General Biosystems, Inc. according to the GenBank database (ncbi.nlm.nih.gov/nuccore/NM_001371189.2/, ID no. NM_001371189.2) and inserted into the pcDNA3.1 vector using Bam HI and HindIII restriction enzyme sites.

Techniques: Transfection, Over Expression, Plasmid Preparation, Reverse Transcription, Real-time Polymerase Chain Reaction, Biomarker Discovery, Expressing, Negative Control, Knock-Out, Membrane

Journal: Oncology Letters

Article Title: UNC13B regulates the sensitivity of Wilms' tumor cells to doxorubicin by modulating lysosomes

doi: 10.3892/ol.2024.14579

Figure Lengend Snippet: Elevated expression of UNC13B in Wilms’ tumor cell lines. (A) Western blot analysis of UNC13B expression in WT-CLS1, 17.94, G401, SK-NEP-1 and HK-2 cells. Each lane was loaded with 20 µg protein and GAPDH was used as a reference. (B) Analysis of UNC13B transcription levels in different cell lines; n=5. (C) Changes in UNC13B transcription levels 48 h post-shRNA-mediated UNC13B knockdown in 17.94 cells compared with the scramble control; n=5. (D) UNC13B expression changes 48 h post-shRNA-mediated knockdown in 17.94 cells and (E) statistical analysis of the expression level changes. Each experiment was repeated 3 times, with GAPDH used as a reference. (F) Assessment of cell proliferation post-knockdown using a Cell Counting Kit-8 assay, measuring OD450 values at different time points; n=3. Cells were also transfected with non-target scrambled shRNA as a control. ***P<0.001; ****P<0.0001. UNC13B, unc-13 homolog B; sh, short hairpin; OD, optical density.

Article Snippet: The negative controls are scramble sequences synthesized by igebio biotech Co., Ltd. UNC13B shRNA lentiviral vector using lentiCRISPR v2. (cat. no. 52961; Addgene, Inc.) was constructed.

Techniques: Expressing, Wilms Tumor Assay, Western Blot, shRNA, Knockdown, Control, Cell Counting, Transfection

Journal: Oncology Letters

Article Title: UNC13B regulates the sensitivity of Wilms' tumor cells to doxorubicin by modulating lysosomes

doi: 10.3892/ol.2024.14579

Figure Lengend Snippet: UNC13B influences Wilms’ tumor sensitivity to chemotherapy drugs independent of the cell cycle. Evaluation of cell proliferation post-shRNA-mediated UNC13B knockdown after treatment with varying concentrations of (A) vincristine, (B) actinomycin-D and (C) doxorubicin for 48 h in 17.94 cells, and (D) vincristine, (E) actinomycin-D and (F) doxorubicin in the G401 cell line, assessed using Cell Counting Kit-8 assays. Changes in drug sensitivity were analyzed, with dashed lines representing fitted curves for half-maximal inhibitory concentration calculated using GraphPad software, and the cell number ratio indicating the relative number of viable cells compared between initial cell number and different time points. (G) Cell cycle analysis of 1 µM doxorubicin treatment on control and shUNC13B knockdown cells, detected after 48 h post-drug treatment. (H) Quantification of the G1, S and G2 phases of the scramble and shUNC13B groups. (I) Typical pseudocolor scatter plots of the apoptosis analysis of UNC13B-knockdown 17.94 cells after 48 h treatment with 0.5 and 2 µM doxorubicin, and (J) statistical results. **P<0.01; ***P<0.001; ****P<0.0001. UNC13B, unc-13 homolog B; sh, short hairpin.

Article Snippet: The negative controls are scramble sequences synthesized by igebio biotech Co., Ltd. UNC13B shRNA lentiviral vector using lentiCRISPR v2. (cat. no. 52961; Addgene, Inc.) was constructed.

Techniques: Wilms Tumor Assay, shRNA, Knockdown, Cell Counting, Concentration Assay, Software, Cell Cycle Assay, Control

Journal: Oncology Letters

Article Title: UNC13B regulates the sensitivity of Wilms' tumor cells to doxorubicin by modulating lysosomes

doi: 10.3892/ol.2024.14579

Figure Lengend Snippet: UNC13B localizes within vesicles and participates in regulating lysosome formation. (A) Indirect immunofluorescence detecting endogenous UNC13B expression in 17.94 cells and revealing UNC13B localization within cellular vesicles. Green fluorescence represents UNC13B and blue fluorescence represents the cell nucleus. (B) Staining of 17.94 cells with Lyso-Tracker. Cells were cultured in confocal culture dishes. The Mean Gray Value of red fluorescence was calculated in 7 different random fields using ImageJ software. Objective, 20X. (C) Mean Gray Value, calculated by measuring the grayscale values in indicated fluorescence channel regions of interest. ****P<0.0001. UNC13B, unc-13 homolog B; sh, short hairpin.

Article Snippet: The negative controls are scramble sequences synthesized by igebio biotech Co., Ltd. UNC13B shRNA lentiviral vector using lentiCRISPR v2. (cat. no. 52961; Addgene, Inc.) was constructed.

Techniques: Immunofluorescence, Expressing, Fluorescence, Staining, Cell Culture, Software

Journal: Oncology Letters

Article Title: UNC13B regulates the sensitivity of Wilms' tumor cells to doxorubicin by modulating lysosomes

doi: 10.3892/ol.2024.14579

Figure Lengend Snippet: UNC13B modulates cell drug sensitivity by affecting lysosome formation. (A) 17.94 cell line was transiently transfected with the UNC13B-pCDNA3.1 overexpression vector using Lipofectamine 3,000. Reverse transcription-quantitative PCR validation was performed 24 h post-transfection. NC was the transfection with an empty pcDNA3.1 vector; n=3. (B) UNC13B and LAMP1 expression levels in 17.94 NC cells, UNC13B 17.94 OE cells and UNC13B 17.94 KO cells, using GAPDH as a reference, and (C) the associated semi-quantitative results. (D) Analysis of Mean Gray Value of Lyso-Tracker in 6 random fields of 17.94 NC, UNC13B 17.94 OE cells and UNC13B 17.94 KO cells, and (E) representative images of 17.94 NC cells, UNC13B 17.94 OE cells and UNC13B 17.94 KO cells. Lyso-Tracker indicated lysosomes, as the dye is highly selective for acidic environments, with an excitation wavelength of 577 nm and an emission wavelength of 590 nm. (F) Assessment of doxorubicin sensitivity changes in 17.94 NC, UNC13B 17.94 OE cells and UNC13B 17.94 KO cells; n=3. The cell number ratio indicates the relative number of viable cells compared between initial cell number and different time points. *P<0.05; **P<0.01; ***P<0.001; ****P<0.0001. UNC13B, unc-13 homolog B; NC, negative control; OE, over-expressed; KO, knock-out; LAMP1, lysosomal-associated membrane protein 1.

Article Snippet: The negative controls are scramble sequences synthesized by igebio biotech Co., Ltd. UNC13B shRNA lentiviral vector using lentiCRISPR v2. (cat. no. 52961; Addgene, Inc.) was constructed.

Techniques: Transfection, Over Expression, Plasmid Preparation, Reverse Transcription, Real-time Polymerase Chain Reaction, Expressing, Negative Control, Knock-Out, Membrane