grna sequence of anxa5  (GenScript corporation)

Journal: The EMBO Journal

Article Title: Annexin A5 controls VDAC1-dependent mitochondrial Ca 2+ homeostasis and determines cellular susceptibility to apoptosis

doi: 10.1038/s44318-025-00454-9

Figure Lengend Snippet: ( A ) Immunoblots show the AnxA5 expression in WT and AnxA5-KO in HeLa and ( B ) EA.hy926 cells (Experiments are performed in Clone 21 indicated as a red rectangle). Uncropped blots are provided in the Source data. Panel ( A ) shows a redisplay of content from Fig. EV2J. ( C ) Graphical representation of genetically encoded FRET-based mitochondrial matrix targeted Ca 2+ sensor (4mtD3cpv). ( D ) Representative image of HeLa cells transfected with (4mtD3cpv). The cells have been pseudocolored to represent mitochondrial Ca 2+ levels as a ratio under basal (left panel) conditions or upon histamine stimulation (left panel) (Scale bar = 5 μm). ( E ) Average time courses of the 100 μM histamine-induced [Ca 2+ ] Matrix responses in WT (black) and AnxA5-KO (red) in EA.hy926 cells measured in Ca 2+ -free buffer (containing 100 μM EGTA). ( F ) Bar graphs show the basal [Ca 2+ ] Matrix and ( G ) histamine-induced maximum [Ca 2+ ] Matrix levels in WT (black) and AnxA5-KO (red). Data points represent the mean ± SEM (n WT = 9/6; n AnxA5-KO = 12/6). The p -value for ( G ) is p = 0.0011 (** p < 0.01). ( H ) Mean time courses of the histamine-induced [Ca 2+ ] Cyto responses in WT (black) and AnxA5-KO (red) in EA.hy926 cells measured in Ca 2+ -free buffer (containing 100 μM EGTA). ( I ) Bar graphs show the basal [Ca 2+ ] Cyto and ( J ) histamine-induced maximum [Ca 2+ ] Cyto levels in WT (black) and AnxA5-KO (red). Data points represent the mean ± SEM (n WT = 101/6; n AnxA5-KO = 88/6). The p -value for ( J ) is p = 0.0094 (** p < 0.01). ( K ) Representative Immunoblot shows the expression level of AnxA5 transfected either with shControl or shAnxA5. Uncropped blots are provided in the Source Data. ( L ) Bar graph represents immunoblot analysis of AnxA5 expression as mean ± SEM (n shControl = 3; n shAnxA5 = 3). ( M ) Average time courses of the 100 μM histamine-induced [Ca 2+ ] Matrix responses in the presence (dashed lines) and absence (solid lines) of CGP37157 in shControl (black) and shAnxA5 (red) in HeLa cells. Data points represent the mean ± SEM (n shControl = 29/3; n shAnxA5 = 28/4; n shControl-CGP37157 = 56/3; n shAnxA5-CGP37157 = 43/4). Significant differences were assessed with the two-tailed unpaired Student’s t-test (** p < 0.01 and ns: not significant). .

Article Snippet: The gRNA sequence of AnxA5 (5′-3′) GATGCAGAAACTCTTCGGA , Genscript , This study.

Techniques: Western Blot, Expressing, Transfection, Two Tailed Test

Journal: The EMBO Journal

Article Title: Annexin A5 controls VDAC1-dependent mitochondrial Ca 2+ homeostasis and determines cellular susceptibility to apoptosis

doi: 10.1038/s44318-025-00454-9

Figure Lengend Snippet: ( A ) Representative time courses of the 100 μM ATP-induced [Ca 2+ ] Matrix responses in WT (black), AnxA5-KO (red), and rescue (blue) in HeLa cells measured in Ca 2+ (2 mM) containing buffer. ( B ) Bar graphs show the basal [Ca 2+ ] Matrix and ( C ) ATP-induced maximum [Ca 2+ ] Matrix level in WT (black), AnxA5-KO (red), and Rescue (blue). Data points represent the mean ± SEM (n WT = 19/5; n AnxA5-KO = 22/5; n Rescue = 36/9). The p -values were calculated using one-way ANOVA with Tukey’s multiple comparison test. From left to right: p = 0.0034 (** p < 0.01) and p = 0.0404 (* p < 0.05). ( D ) Mean time courses of the ATP-induced [Ca 2+ ] Cyto responses in WT (black) and AnxA5-KO (red) in HeLa cells measured in Ca 2+ (2 mM) containing buffer. ( E ) Bar graphs show the basal [Ca 2+ ] Cyto and ( F ) ATP-induced maximum [Ca 2+ ] Cyto levels in WT (black) and AnxA5-KO (red). Data points represent the mean ± SEM (n WT = 103/6; n AnxA5-KO = 107/6). ( G ) Average time courses of the 100 μM histamine and 15 μM BHQ induced [Ca 2+ ] ER responses in WT (black) and, AnxA5-KO (red) Hela cells. ( H ) Bar graphs show the 100 μM Histamine-induced [Ca 2+ ] ER release and ( I ) 15 μM BHQ-induced [Ca 2+ ] ER drop in WT (black) and AnxA5-KO (red) cells. Data points represent the mean ± SEM (n WT = 22/4; n AnxA5-KO = 27/5). ( J ) Mean time courses of the 100 μM ATP-induced [Ca 2+ ] Matrix responses in WT (black) and AnxA5-KO (red) perivascular cells measured in Ca 2+ -free buffer (containing 100 μM of EGTA). ( K ) Bar graphs show the basal [Ca 2+ ] Matrix and ( L ) ATP-induced maximum [Ca 2+ ] Matrix levels in WT (black) and AnxA5-KO (red) cells. Data points represent the mean ± SEM (n WT = 29/6; n AnxA5-KO = 40/6). The p -value was calculated using a two-tailed unpaired Student’s t-test, p = 0.0062 (** p < 0.01). ( M ) Mean time courses of the 100 μM ATP-induced [Ca 2+ ] Cyto responses in WT (black) and AnxA5-KO (red) in perivascular cells measured in Ca 2+ -free buffer (containing 100 μM of EGTA). ( N ) Bar graphs show the basal [Ca 2+ ] Cyto and ( O ) ATP-induced maximum [Ca 2+ ] Cyto levels in WT (black) and AnxA5-KO (red). Data points represent the mean ± SEM (n WT = 46/6; n AnxA5-KO = 33/6). n represents the number of cells/biological replicates (minimum of 3 independent experiments). Significant differences were assessed using either one-way ANOVA with Tukey’s multiple comparison tests or Kruskal–Wallis test (* p < 0.05, ** p < 0.01 and ns: not significant) and with the unpaired Student’s t-test or Kolmogorov–Smirnov test (** p < 0.001 and ns: not significant). .

Article Snippet: The gRNA sequence of AnxA5 (5′-3′) GATGCAGAAACTCTTCGGA , Genscript , This study.

Techniques: Comparison, Two Tailed Test

Journal: The EMBO Journal

Article Title: Annexin A5 controls VDAC1-dependent mitochondrial Ca 2+ homeostasis and determines cellular susceptibility to apoptosis

doi: 10.1038/s44318-025-00454-9

Figure Lengend Snippet: ( A ) Representative time courses of Ψmito before and after 1 μM FCCP treatment in WT (black) and AnxA5-KO (red) HeLa cells stained with TMRM. ( B ) Bar graph shows the relative mitochondrial membrane potential in WT (black) and AnxA5-KO (red) HeLa cells. Data points represent the mean ± SEM (n WT = 87/9; n AnxA5-KO = 106/10). ( C ) Immunoblots show the expression level of AnxA5, VDAC1, MICU1, MICU2, UCP2, MCU, and EMRE in WT and AnxA5-KO HeLa cell lysates. Uncropped blots are provided in the Source data. ( D ) Bar graph shows the expression level analysis of the respective proteins. Data points represent the mean ± SEM (n WT = 3; n AnxA5-KO = 3). ( E ) Representative confocal images of WT and AnxA5-KO HeLa cells were captured, stained with MTR-CMX (magenta), and expressed ERAT4.03 NA (green). Multiplying the fluorescence signals of mitochondria and ER at the pixel level, and subsequently amplifying the resulting signal, led to the visualization of MAM. Images provide an overview of the cells (scale bar = 10 μm), and magnified views of selected regions indicated by dashed squares (scale bar = 1 μm). ( F ) Bar graphs show the ER-mitochondrial co-localization represented by Pearson’s R-value, ( G ) mitochondrial volume, and ( H ) mitochondrial branching (a lower value indicates more branched mitochondria). Data points represent the mean ± SEM (n WT = 91/11; n AnxA5-KO = 88/11). The p -values were calculated using a two-tailed unpaired Student’s t-test for ( F ), p = 0.8780 (ns); for ( G ), p = 0.0092 (** p < 0.01); and for ( H ), p = 0.0036 (** p < 0.01). ( I ) Representative TEM images of mitochondria in WT and AnxA5-KO HeLa cells, where mitochondria are highlighted with magenta (scale bar = 500 nm). ( J ) Bar graphs show the cristae membrane-amount, measured by calculating the perimeter of cristae and normalizing it to the corresponding perimeter of mitochondria, and ( K ) cristae density was calculated by normalizing the perimeter of cristae to the area of mitochondria. Data points represent the mean ± SEM (n WT = 47/2; n AnxA5-KO = 45/2), n represents the number of mitochondria/biological replicates). ( L ) Schematic illustration depicts the spatial cristae membrane density (ρCM) measurement within segmented mitochondria. Iterative measurements of ρCM density were conducted in gradually downsized circular segments. ( M ) shows the spatial ρCM distribution in WT and AnxA5-KO cells by using the methods depicted in ( L ). The x-axis scale ranges from 0 to 100, with 0 indicating the outermost shell of the mitochondrion and 100 representing the mitochondrial center. Data points represent the mean ± SEM (n WT = 44/3; n AnxA5-KO = 47/3). n represents the number of cells/biological replicates (minimum of 3 independent experiments). Significant differences were assessed with the unpaired Student’s t-test or Kolmogorov–Smirnov test (* p < 0.05, ** p < 0.01, and ns: not significant). .

Article Snippet: The gRNA sequence of AnxA5 (5′-3′) GATGCAGAAACTCTTCGGA , Genscript , This study.

Techniques: Staining, Membrane, Western Blot, Expressing, Fluorescence, Two Tailed Test

Journal: The EMBO Journal

Article Title: Annexin A5 controls VDAC1-dependent mitochondrial Ca 2+ homeostasis and determines cellular susceptibility to apoptosis

doi: 10.1038/s44318-025-00454-9

Figure Lengend Snippet: ( A ) Bar graphs show the calculated percentage localization of AnxA5 in cytosol versus pure mitochondria, ( B ) pure mitochondria versus pure mitochondria + proteinase K (PK) treatment in mitochondria isolated from HeLa cells. Data points represent the mean ± SEM in HeLa cells ( n = 3). ( C ) Bar graphs show the distribution of gold particles in cytosol, mitochondria, and the nucleus using AnxA5 antibody or rabbit antibody as a negative control. Data points represent the mean ± SEM in HeLa (n AnxA5 antibody = 13/3; n Rabbit antibody = 15/3). The p -value is p = 0.0337 (* p < 0.05). ( D ) Bar graphs show the basal [Ca 2+ ] IMS levels in WT (black), AnxA5-KO (red), and Rescue (blue) in HeLa and ( E ) EA.hy926 cells. Data points represent the mean ± SEM in HeLa (n WT = 23/7; n AnxA5-KO = 34/8; n Rescue = 30/9) and in EA.hy926 cells (n WT = 14/6; n AnxA5-KO = 12/6). ( F ) Bar graphs show a histamine-induced maximum [Ca 2+ ] IMS and ( G ) [Ca 2+ ] cyto elevation in WT (black) and AnxA5-KO (red) HeLa cells measured in Ca 2+ -free buffer. The p -values for ( F ), from left to right, are: p = 0.0037 (** p < 0.01), p = 0.0002 (*** p < 0.001), p < 0.0001 (**** p < 0.0001), p = 0.0014 (** p < 0.01), and p = 0.1758 (ns). ( H ) Concentration-response curve of histamine (1, 3, 10, 30, 100 μM) shows maximum [Ca 2+ ] IMS and ( I ) [Ca 2+ ] Cyto rise in WT (black) and AnxA5-KO (red) cells measured in Ca 2+ -free buffer. The values were calculated from panels ( F ) and ( G ). Data points represent the mean ± SEM in IMS (n WT-100 μM-Hist = 19/4; n AnxA5-KO-100 μM-Hist = 14/5; n WT-30μM-Hist = 19/5; n AnxA5-KO-30 μM-Hist = 20/5; n WT-10 μM-Hist = 26/6; n AnxA5-KO-10 μM-Hist = 23/6; n WT-3μM-Hist = 19/6; n AnxA5-KO-3 μM-Hist = 11/6; n WT-1 μM-Hist = 14/3; n AnxA5-KO-1 μM-Hist = 11/3) and in the cytosol (n WT-100 μM-Hist = 50/5; n AnxA5-KO-100 μM-Hist = 33/4; n WT-30 μM-Hist = 50/5; n AnxA5-KO-30 μM-Hist = 40/5; n WT-10 μM-Hist = 46/4; n AnxA5-KO-10 μM-Hist = 46/4; n WT-3 μM-Hist = 39/4; n AnxA5-KO-3 μM-Hist = 25/4; n WT-1 μM-Hist = 30/5; n AnxA5-KO-1 μM-Hist = 28/5). ( J ) Representative immunoblots show the expression of AnxA5 in WT and AnxA5-KO cells transfected either with an empty plasmid (in WT and AnxA5-KO cells) or with AnxA5, AnxA5-2mt, AnxA5-3mt, and AnxA5-5mt (in AnxA5-KO cells). Uncropped blots are provided in the Source Data. Significant differences were assessed using either one-way ANOVA with Tukey’s multiple comparison tests or Kruskal–Wallis test (ns: not significant) and with the two-tailed unpaired Student’s t-test or Kolmogorov–Smirnov (* p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001, and ns: not significant). .

Article Snippet: The gRNA sequence of AnxA5 (5′-3′) GATGCAGAAACTCTTCGGA , Genscript , This study.

Techniques: Isolation, Negative Control, Concentration Assay, Western Blot, Expressing, Transfection, Plasmid Preparation, Comparison, Two Tailed Test

Journal: The EMBO Journal

Article Title: Annexin A5 controls VDAC1-dependent mitochondrial Ca 2+ homeostasis and determines cellular susceptibility to apoptosis

doi: 10.1038/s44318-025-00454-9

Figure Lengend Snippet: ( A ) Representative immunoblots show protein components of subcellular fractions including Homogenate (Homog), cytosol (Cyto), crude mitochondria (C. Mito), and pure mitochondria (P. Mito) obtained from HeLa cells. Marker proteins indicate OMM (VDAC1), the cytosolic leaflet of the OMM (TOM20), IMS (cytochrome-C, Cyt-C), and cytosol (tubulin). To digest proteins localized on the cytosolic leaflet of the OMM, the pure mitochondrial fraction was incubated with 50 µg/ml Proteinase K (PK) for 15 min ( n = 3). Uncropped blots are provided in the Source Data. ( B ) Representative Immuno-electron micrographs of mitochondria from HeLa cells. In the upper panel, individual gold particles indicate the localization of the AnxA5, while the lower panel serves as a negative control where the primary antibody was omitted. Mitochondria were highlighted with magenta and individual gold particles were marked with green arrows (scale bar = 250 nm). ( C ) Representative time courses of the 100 μM histamine-induced [Ca 2+ ] IMS responses in WT (black) and AnxA5-KO (red) in HeLa cells measured in Ca 2+ (2 mM) containing buffer. ( D ) Bar graphs show the histamine-induced maximum [Ca 2+ ] IMS levels in WT (black) and AnxA5-KO (red). Data points represent the mean ± SEM (n WT = 23/7; n AnxA5-KO = 34/8). The p -value was calculated using the two-tailed Kolmogorov–Smirnov test, yielding p < 0.0001 (**** p < 0.0001). ( E ) Mean time courses of the 100 μM histamine-induced [Ca 2+ ] IMS responses of WT (black) and AnxA5-KO (red) EA.hy926 cells measured in Ca 2+ -free buffer (containing 100 μM of EGTA). ( F ) Bar graph shows the histamine-induced maximum [Ca 2+ ] IMS levels in WT (black) and AnxA5-KO (red) cells. Data points represent the mean ± SEM (n WT = 14/6; n AnxA5-KO = 12/6). The p -value was calculated using a two-tailed unpaired Student’s t-test, p = 0.0034 (** p < 0.01). ( G ) Mean time courses of 15 μM BHQ and 100 μM histamine-induced [Ca 2+ ] IMS responses in the absence of extracellular Ca 2+ and upon subsequent store-operated Ca 2+ entry in WT (black) and AnxA5-KO (red) HeLa cells. ( H ) Bar graphs show the BHQ-, ( I ) histamine-, and ( J ) SOCE-induced maximum [Ca 2+ ] IMS responses in WT (black) and AnxA5-KO (red) cells. Data points represent the mean ± SEM (n WT = 23/5; n AnxA5-KO = 16/4). The p -value for ( I ) was calculated using a two-tailed unpaired t-test yielding p = 0.001 (** p < 0.01). ( K ) Table displaying the list of AnxA5 mutation sites and the characteristics of the mutations. L Average time courses of 100 μM ATP-induced [Ca 2+ ] IMS responses were measured in HeLa cells under Ca 2+ -free conditions (containing 100 μM of EGTA). Studied cell groups: WT, AnxA5-KO, AnxA5-KO + AnxA5 (WT AnxA5), AnxA5-KO + AnxA5-2Mt (mutations in the Ca 2+ -binding domain: D144N, E228Q), AnxA5-KO + AnxA5-3Mt (mutations in the Ca 2+ -binding domain: D144N, E228Q, D303N), AnxA5-KO + AnxA5-5Mt (mutations in the self-assembly domain: R18E, R25E, K29E, K58E, K193). Cells were transfected with either an empty plasmid in WT and AnxA5-KO cells or the respective WT or mutant version of AnxA5 in AnxA5-KO cells. ( M ) Bar graphs show the ATP-induced maximum [Ca 2+ ] IMS levels in WT (black), AnxA5-KO (red), AnxA5-KO + AnxA5 (blue), AnxA5-KO + AnxA5-2Mt (orange), AnxA5-KO + AnxA5-3Mt (magenta), AnxA5-KO + AnxA5-5Mt (green). Data points represent the mean ± SEM (n WT = 53/10; n AnxA5-KO = 67/12; n Rescue = 64/11; n AnxA5-KO+2Mt = 55/11; n AnxA5-KO+3Mt = 58/11; n AnxA5-KO+5Mt = 60/12). n represents the number of cells/biological replicates (minimum of 3 independent experiments). The p -values were calculated using a Kruskal–Wallis test, from left to right: p = 0.0011 (** p < 0.01), p < 0.0001 (**** p < 0.0001), p = 0.1488 (ns), p = 0.9038 (ns), p < 0.0001 (**** p < 0.0001). Significant differences were assessed using either one-way ANOVA with Kruskal–Wallis test (** p < 0.01, **** p < 0.0001 and ns: not significant) and with the unpaired Student’s t-test or Kolmogorov–Smirnov test (** p < 0.01, **** p < 0.0001 and ns: not significant). .

Article Snippet: The gRNA sequence of AnxA5 (5′-3′) GATGCAGAAACTCTTCGGA , Genscript , This study.

Techniques: Western Blot, Marker, Incubation, Negative Control, Two Tailed Test, Mutagenesis, Binding Assay, Transfection, Plasmid Preparation

Journal: The EMBO Journal

Article Title: Annexin A5 controls VDAC1-dependent mitochondrial Ca 2+ homeostasis and determines cellular susceptibility to apoptosis

doi: 10.1038/s44318-025-00454-9

Figure Lengend Snippet: ( A ) The schematic illustration depicts the histamine-induced rearrangement of MICU1-CFP and MICU1-YFP FRET, which serve as [Ca 2+ ] IMS sensors. ( B ) Mean time course of 100 µM histamine-induced MICU1 FRET ratio in WT (black) and AnxA5-KO (red) HeLa cells measured in Ca 2+ (2 mM) containing buffer. ( C ) Bar graphs show the histamine-induced change in the MICU1 FRET ratio in WT (black) and AnxA5-KO (red) cells. Data points represent the mean ± SEM (n WT = 19/6; n AnxA5-KO = 24/6). The p -value for ( C ) was calculated using a two-tailed unpaired Student’s t-test, p < 0.0001 (**** p < 0.0001). ( D ) The schematic illustration of cristae membrane dynamics under basal (low) and upon IP 3 -induced [Ca 2+ ] ER release (high Ca 2+ ). ( E ) Bar graph shows cristae membrane movements per frame in MERCs under basal and upon IP 3 -induced [Ca 2+ ] ER release in WT (black), AnxA5-KO (red), and Rescue (blue) HeLa cells. Data points represent the mean ± SEM (n WT = 26/6; n AnxA5-KO = 25/6; n Rescue = 24/6). The p -values were calculated using a two-tailed unpaired Student’s t-test, from left to right: p = 0.0124 (* p < 0.05), p = 0.5845 (ns), and p = 0.0360 (* p < 0.05). ( F ) Representative time course of 100 µM histamine-induced [Ca 2+ ] cristae levels in WT (black) and AnxA5-KO (red) HeLa cells measured in Ca 2+ (2 mM) containing buffer. ( G ) Bar graph shows the histamine-induced maximum [Ca 2+ ] cristae levels in WT (black) and AnxA5-KO (red) cells. Data points represent the mean ± SEM (n WT = 35/6; n AnxA5-KO = 44/6). The p -value was calculated using a two-tailed unpaired Student’s t-test, p < 0.0001 (**** p < 0.0001). ( H ) Representative SIM images of HeLa cells depict the expression of MICU1-YFP (green) and MCU-mcherry (magenta) in WT (left panel) and AnxA5-KO (right panel) before and 90 s after histamine stimulation. ( I ) Bar graph shows the IBM association index of MCU in WT and AnxA5-KO before and 90 s after [Ca 2+ ] ER release. Higher IBM association index values correspond to an increased translocation of MCU from cristae to IBM, as illustrated in the schematic. Data points represent the mean ± SEM (n WT = 84/9; n AnxA5-KO = 88/9). n represents the number of cells/biological replicates (minimum of 3 independent experiments). The p -values were calculated using a one-way ANOVA with Tukey’s multiple comparison test, from left to right: p < 0.0001 (**** p < 0.0001) and p = 0.9586 (ns). Significant differences were assessed using either one-way ANOVA with Tukey’s multiple comparison tests (**** p < 0.0001 and ns: not significant) or with the unpaired Student’s t-test (* p < 0.05, **** p < 0.0001 and ns: not significant). .

Article Snippet: The gRNA sequence of AnxA5 (5′-3′) GATGCAGAAACTCTTCGGA , Genscript , This study.

Techniques: Two Tailed Test, Membrane, Expressing, Translocation Assay, Comparison

Journal: The EMBO Journal

Article Title: Annexin A5 controls VDAC1-dependent mitochondrial Ca 2+ homeostasis and determines cellular susceptibility to apoptosis

doi: 10.1038/s44318-025-00454-9

Figure Lengend Snippet: ( A ) Bar graph shows cristae membrane movements per frame in the whole mitochondria under basal and upon IP 3 -induced [Ca 2+ ] ER release in WT (black), AnxA5-KO (red), and Rescue (blue) in HeLa cells. Data points represent the mean ± SEM (n WT = 26/6; n AnxA5-KO = 25/6; n Rescue = 24/6). ( B ) Bar graph shows the basal [Ca 2+ ] cristae levels in WT (black) and AnxA5-KO (red) cells. Data points represent the mean ± SEM (n WT = 35/6; n AnxA5-KO = 44/6). ( C ) Bar graphs show the mitochondrial area and ( D ) aspect ratio in WT and AnxA5-KO before and 90 s after [Ca 2+ ] ER release. Data points represent the mean ± SEM (n WT = 84/9; n AnxA5-KO = 88/9). The p -values, from left to right, are for ( C ): p = 0.0109 (* p < 0.05), p = 0.0015 (** p < 0.01) and p = 0.5559 (ns); and for ( D ): p < 0.0001 (**** p < 0.0001), p < 0.0001 (**** p < 0.0001), and p = 0.0066 (** p < 0.01). Significant differences were assessed using either one-way ANOVA with Tukey’s multiple comparison tests or the Kruskal–Wallis test and with the two-tailed unpaired Student’s t-test (* p < 0.05, ** p < 0.01, **** p < 0.0001, and ns: not significant). .

Article Snippet: The gRNA sequence of AnxA5 (5′-3′) GATGCAGAAACTCTTCGGA , Genscript , This study.

Techniques: Membrane, Comparison, Two Tailed Test

Journal: The EMBO Journal

Article Title: Annexin A5 controls VDAC1-dependent mitochondrial Ca 2+ homeostasis and determines cellular susceptibility to apoptosis

doi: 10.1038/s44318-025-00454-9

Figure Lengend Snippet: ( A ) Representative image of PLA assay indicating the protein-protein proximity between AnxA5 and VDAC1 in siNeg- and siVDAC1-treated WT, and in AnxA5-KO HeLa cells (scale bar = 20 μm). ( B ) The bar graph shows the ratio of total PLA signals to the number of cells. Data points represent the mean ± SEM (n siNeg = 4; n siVDAC1 = 3; n AnxA5-KO = 4). The p -values were calculated using a Kruskal–Wallis test, from left to right: p = 0.0025 (** p < 0.01), p < 0.0001 (**** p < 0.0001), and p = 0.2271 (ns). ( C ) Representative time courses of 100 μM histamine-induced [Ca 2+ ] Matrix responses in WT_siNeg, AnxA5-KO_siNeg, WT_siVDAC1, AnxA5-KO_siVDAC1, WT_siVDAC1 + VDAC2-FLAG, AnxA5-KO_siVDAC1 + VDAC2-FLAG, WT_siVDAC1 + VDAC3-FLAG, and AnxA5-KO_siVDAC1 + VDAC3-FLAG were measured in a Ca 2+ (2 mM)-containing buffer. ( D ) Bar graphs show the histamine-induced maximum [Ca 2+ ] Matrix levels in WT_siNeg (black), AnxA5-KO_siNeg (red), WT_siVDAC1 (dark yellow), AnxA5-KO_siVDAC1 (orange), WT_siVDAC1 + VDAC2-FLAG (green), AnxA5-KO_siVDAC1 + VDAC2-FLAG (blue), WT_siVDAC1 + VDAC3-FLAG (gray), and AnxA5-KO_siVDAC1 + VDAC3-FLAG (brown). Data points represent the mean ± SEM (n WT_siNeg = 40/9; n AnxA5-KO_siNeg = 47/9; n WT_siVDAC1 = 44/9; n AnxA5-KO_siVDAC1 = 43/7; n WT_siVDAC1+VDAC2-FLAG = 42/6; n AnxA5-KO_siVDAC1+VDAC2-FLAG = 43/8; n WT_siVDAC1+VDAC3-FLAG = 50/7; n AnxA5-KO_siVDAC1+VDAC3-FLAG = 48/8). The p-values were calculated using a Kruskal–Wallis test, from left to right (bottom to top): p = 0.0014 (** p < 0.01), p < 0.0001 (**** p < 0.0001), p = 0.1353 (ns), p = 0.0401, * p < 0.05), p > 0.9999 (ns) and p > 0.9999 (ns). ( E ) The distribution of AnxA5-labeled gold particles within the whole cell was analyzed, showing their localization on mitochondria and cytosol under basal conditions (green) and 20 s after histamine stimulation (magenta). Positive values indicate the distance of the gold particles from the OMM to the mitochondrial matrix side, while negative values represent the distance from the OMM to the cytosol. Data points represent the subcellular localization of the gold particles (n WT-Basal = 10/3; n WT-Histamine = 10/3). ( F ) The relative occurrence of the gold particles was calculated based on the data presented in Fig. 5 ( E ). n represents the number of cells/biological replicates (minimum of 3 independent experiments). ( G ) The schematic illustration depicts the basal localization and histamine-induced translocation of AnxA5 to OMM. Significant differences were assessed using one-way ANOVA with Kruskal–Wallis test (* p < 0.05, ** p < 0.01, **** p < 0.0001 and ns: not significant). .

Article Snippet: The gRNA sequence of AnxA5 (5′-3′) GATGCAGAAACTCTTCGGA , Genscript , This study.

Techniques: Labeling, Translocation Assay

Journal: The EMBO Journal

Article Title: Annexin A5 controls VDAC1-dependent mitochondrial Ca 2+ homeostasis and determines cellular susceptibility to apoptosis

doi: 10.1038/s44318-025-00454-9

Figure Lengend Snippet: ( A ) Representative single-channel traces showing the 35 pS channel in intact mitochondria isolated from the WT, AnxA5KO, AnxA5-KO mitochondria supplemented with recombinant AnxA5 (200 ng/ml added to the patch pipette), WT_siNeg, WT_siVDAC1, and AnxA5KO_siVDAC1. ( B ) Bar graphs show channel occurrence, calculated as the percentage of patches showing single-channel activity relative to the total number of patches on a given experimental day in WT (black), AnxA5-KO (red), AnxA5-KO mitochondria supplemented with recombinant AnxA5 (blue), WT_siNeg (gray), WT_siVDAC1 (dark yellow) and AnxA5KO_siVDAC1 (orange). Data points represent the mean ± SEM (n WT = 14; n AnxA5-KO = 12; n AnxA5-KO + Recombinant AnxA5 = 8; n WT_siNeg = 6; n WT_siVDAC1 = 7; n AnxA5-KO_siVDAC1 = 7). The p -values, from left to right: p = 0.0009 (*** p < 0.001), p < 0.0001 (**** p < 0.0001), p = 0.4726 (ns), p = 0.4094 (ns) and p = 0.066 (ns). ( C ) Bar graphs show NPo at −80 mV in WT (black), AnxA5-KO (red), AnxA5-KO mitochondria supplemented with recombinant AnxA5 (blue), WT_siNeg (gray), WT_siVDAC1 (dark yellow), and AnxA5KO_siVDAC1 (orange). Data points represent the mean ± SEM (n WT = 7; n AnxA5-KO = 13; n AnxA5-KO + Recombinant AnxA5 = 6; n WT_siNeg = 22; n WT_siVDAC1 = 15; n AnxA5-KO_siVDAC1 = 14). The p -values, from left to right: p = 0.0327 (* p < 0.05), p = 0.0002 (*** p < 0.001), p = 0.6267 (ns) and p = 0.2877 (ns). n represents the number of cells/biological replicates (minimum of 3 independent experiments). Significant differences were assessed using one-way ANOVA with Tukey’s multiple comparison tests (* p < 0.05, *** p < 0.001, **** p < 0.0001 and ns: not significant). .

Article Snippet: The gRNA sequence of AnxA5 (5′-3′) GATGCAGAAACTCTTCGGA , Genscript , This study.

Techniques: Isolation, Recombinant, Transferring, Activity Assay, Comparison

Journal: The EMBO Journal

Article Title: Annexin A5 controls VDAC1-dependent mitochondrial Ca 2+ homeostasis and determines cellular susceptibility to apoptosis

doi: 10.1038/s44318-025-00454-9

Figure Lengend Snippet: ( A ) Bar graphs show the basal [Ca 2+ ] Matrix , ( B ) [Ca 2+ ] IMS, and ( C ) [Ca 2+ ] Cyto level in WT (black) and AnxA5-KO (red) cells upon 12 h DMSO, 5 μM and 10 μM cisplatin treatment. Data points represent the mean ± SEM for [Ca 2+ ] Matrix (n WT-DMSO = 63/6; n AnxA5-KO-DMSO = 75/6, n WT-5μM-cisplatin = 67/6; n AnxA5-KO-5μM-cisplatin = 71/6, n WT-10μM-cisplatin = 53/6; n AnxA5-KO-10μM-cisplatin = 54/6), and for [Ca 2+ ] IMS (n WT-DMSO = 64/6; n AnxA5-KO-DMSO = 66/6, n WT-5μM-cisplatin = 60/6; n AnxA5-KO-5μM-cisplatin = 74/6, n WT-10μM-cisplatin = 64/6; n AnxA5-KO-10μM-cisplatin = 63/6), and [Ca 2+ ] Cyto (n WT-DMSO = 64/6; n AnxA5-KO-DMSO = 68/6, n WT-5μM-cisplatin = 74/6; n AnxA5-KO-5μM-cisplatin = 84/6, n WT-10μM-cisplatin = 55/6; n AnxA5-KO-10μM-cisplatin = 83/6). The p -values for ( A ), from left to right, are: p = 0.3780 (ns), p = 0.0320 (* p < 0.05), and p = 0.0022 (** p < 0.01); for ( B ): p = 0.9716 (ns), p = 0.0640 (ns), and p = 0.0039 (** p < 0.01). ( D ) Bar graphs show the percentage of living cells and ( E ) late apoptosis, assessed by Annexin5-FITC/PI staining and FACS analysis in WT (black) and AnxA5-KO (red) cells upon 24 h DMSO, 5 μM and 10 μM cisplatin treatment. ( F ) Bar graphs show the percentage of living cells and ( G ) late apoptosis in WT (black) and AnxA5-KO (red) cells upon 24 h DMSO, 20 μM VBIT-4, 10 μM cisplatin, and cisplatin+VBIT-4 treatment. Data points represent the mean ± SEM (n WT-DMSO = 5; n AnxA5-KO-DMSO = 6, n WT-20μM-VBIT-4 = 6; n AnxA5-KO-20μM-VBIT-4 = 6, n WT-10μM-cisplatin = 6; n AnxA5-KO-10μM-cisplatin = 6, n WT-cisplatin+VBIT-4 = 6; n AnxA5-KO-cisplatin+VBIT-4 = 6). The p -values for ( D ), from left to right, are: p = 0.1775 (ns), p = 0.0043 (** p < 0.01), and p = 0.0095 (** p < 0.01); for ( E ): p = 0.6040 (ns), p = 0.0028 (** p < 0.01), and p < 0.0001 (**** p < 0.0001); for ( F ): p = 0.6787 (ns), p = 0.0582 (ns), p = 0.0037 (** p < 0.01), and p < 0.0001 (**** p < 0.0001); for ( G ): p = 0.8063 (ns), p = 0.1840 (ns), p < 0.0001 (**** p < 0.0001), and p < 0.0001 (**** p < 0.0001). ( H ) Representative immunoblot shows monomeric and dimeric VDAC1 levels. Uncropped blots are provided in the Source Data. ( I ) Bar graph shows the quantification of the immunoblot in WT (black) and AnxA5-KO (red) cells upon 48 h DMSO, 20 μM VBIT-4, 10 μM cisplatin, and cisplatin+VBIT-4 treatment (each color represents the experiments from the same day). Data points represent the mean ± SEM (n WT-all = 4; n AnxA5-KO-all = 4). The p -values, from left to right, are: p = 0.0003 (*** p < 0.001), p = 0.0037 (** p < 0.01), p = 0.0001 (*** p < 0.001), and p = 0.0005 (*** p < 0.001). n represents the number of cells/biological replicates (minimum of 3 independent experiments). Significant differences were assessed using either one-way ANOVA with Tukey’s multiple comparison tests (** p < 0.01, *** p < 0.001 and ns: not significant) and with the two-tailed unpaired Student’s t-test or Kolmogorov–Smirnov test (* p < 0.05, ** p < 0.01, **** p < 0.0001 and ns: not significant). .

Article Snippet: The gRNA sequence of AnxA5 (5′-3′) GATGCAGAAACTCTTCGGA , Genscript , This study.

Techniques: Staining, Western Blot, Comparison, Two Tailed Test

Journal: The EMBO Journal

Article Title: Annexin A5 controls VDAC1-dependent mitochondrial Ca 2+ homeostasis and determines cellular susceptibility to apoptosis

doi: 10.1038/s44318-025-00454-9

Figure Lengend Snippet: ( A ) Representative immunoblot shows monomeric and dimeric VDAC1 levels. Uncropped blots are provided in the Source data. ( B ) Bar graph shows the quantification of the immunoblot in WT (black) and AnxA5-KO (red) cells upon 24 h DMSO, 20 μM VBIT-4, 10 μM cisplatin, and cisplatin+VBIT-4 treatment (each color represents the experiments from the same day). Data points represent the mean ± SEM (n WT-all = 4; n AnxA5-KO-all = 4). ( C ) Representative immunoblot shows monomeric and dimeric VDAC1 levels in response to 48-hour DMSO, 20 μM VBIT-4, 10 μM selenite, and selenite + VBIT-4 treatment. ( D ) Bar graph shows the quantification of the immunoblot in panel ( C ). Data points represent the mean ± SEM (nWT-all = 3; nAnxA5-KO-all = 3). The p -values, from left to right, are: p = 0.0002 (*** p < 0.001), p = 0.0003 (*** p < 0.001), p = 0.0017 (** p < 0.01), and p = 0.0082 (** p < 0.01). ( E ) Bar graphs show the percentage of late apoptosis in WT (black) and AnxA5-KO (red) cells upon 48 h DMSO, 20 μM VBIT-4, 10 μM selenite, and selenite+VBIT-4 treatment. Data points represent the mean ± SEM (nWT-all = 3; nAnxA5-KO-all = 3). The p -values, from left to right, are: p = 0.1688 (ns), p > 0.9999 (ns), p = 0.0448 (* p < 0.05), and p = 0.1932 (ns). ( F ) Representative confocal images of WT and AnxA5-KO HeLa cells, expressing VDAC1-TC (green) and mitoDsRed (red), were captured (Scale bar = 5 μm). ( G ) Bar graph shows the quantification of the obtained confocal images indicating VDAC1 cluster size in μm 2 in WT (black) and AnxA5-KO (red) cells upon 12 h DMSO, 20 μM VBIT-4, 10 μM cisplatin, and cisplatin+VBIT-4 treatment. Data points represent the mean ± SEM (n WT-DMSO = 7; n WT-VBIT-4 = 6; n WT-cisp. = 8; n WT-cisp.+VBIT-4 = 3; n AnxA5-KO-DMSO = 7; n AnxA5-KO-VBIT-4 = 6; n AnxA5-KO-cisp . = 8; n AnxA5-KO-cisp.+VBIT-4 = 3). The p -values, from left to right, are: p = 0.0494 (* p < 0.05), p = 0.2773 (ns), p = 0.0126 (* p < 0.05), and p = 0.0168 (* p < 0.05). Significant differences were assessed using one-way ANOVA with Tukey’s multiple comparison tests or with the unpaired Student’s t-test (* p < 0.05, ** p < 0.01, *** p < 0.005, and ns: not significant). .

Article Snippet: The gRNA sequence of AnxA5 (5′-3′) GATGCAGAAACTCTTCGGA , Genscript , This study.

Techniques: Western Blot, Expressing, Comparison

Journal: The EMBO Journal

Article Title: Annexin A5 controls VDAC1-dependent mitochondrial Ca 2+ homeostasis and determines cellular susceptibility to apoptosis

doi: 10.1038/s44318-025-00454-9

Figure Lengend Snippet: AnxA5 regulates mitochondrial Ca 2+ signaling upon ER Ca 2+ release (upper panel). Apoptotic stimuli induce VDAC1 dimerization and apoptotic cell death (lower panel). Anx5 regulates the dimerization state by localizing in the VDAC1 microenvironment, thus affecting the level of apoptosis (lower left panel). In AnxA5-KO cells, cisplatin/selenite induces enhanced VDAC1 dimerization, leading to elevated apoptosis (lower right panel).

Article Snippet: The gRNA sequence of AnxA5 (5′-3′) GATGCAGAAACTCTTCGGA , Genscript , This study.

Techniques: