Journal: Cancers

Article Title: Pannexin 2 Localizes at ER-Mitochondria Contact Sites

doi: 10.3390/cancers11030343

Figure Lengend Snippet: Pannexin 2 (Panx2) clusters in endoplasmic reticulum (ER) microdomains. ( A ) Panx2 co-fractions with ER and mitochondrial markers. Post-nuclear supernatant from C6 Panx2-enhanced green fluorescent protein (EGFP) cells was fractionated by ultracentrifugation and twenty-four fractions were collected and immunoprobed for various organelle markers. A substantial amount of Panx2 co-fractionated with mitochondrial (voltage dependent anion channel 1 (VDAC1) and ER (Grp78/BiP, calreticulin) markers. ( B ) Three additional fractionations were performed but fractions were paired to allow the probing of all fractions on a single membrane for quantification. Panx2 distribution positively correlated (Pearson’s correlation) with the mitochondrial marker cytochrome C (r = 0.633, p = 0.0273) and the ER protein Grp78/BiP (r = 0.76, p = 0.0041) but not with the Golgi marker GM130 (r = −0.407, p = 0.1892). ( C ) C6 Panx2-EGFP cells were transfected with an ER-targeted mCherry construct and trajectories of Panx2 puncta were tracked in living cells. A large majority of Panx2 puncta were located on the ER network as indicated by the white arrowheads (inset). Scale bars: 10 µm and 5 µm (inset). ( D ) The probability distribution of Panx2 puncta (orange curve) was compared to the probability distribution calculated after randomizing the trajectories of Panx2 puncta within the cytoplasm (purple curve). The distributions, calculated from 13 cells, show that a larger proportion of Panx2 puncta was localized on the ER network prior to randomization. ( E ) Frame sequence showing that Panx2-ER association was stable for over 55 s despite the high mobility of the ER network during that period. Scale bar, 2 µm. ( F ) Mouse brain sections were stained for Panx2 and the ER marker calnexin. Panx2 formed discrete puncta that primarily clustered in ER microdomains as indicated by the white arrowheads (inset). Scale bars, 10 µm (inset 5 µm).

Article Snippet: The anti-Panx2 mouse monoclonal antibody (cat# 75-212, clone N121A/1) was from UC Davis/NIH NeuroMab Facility (Davis, CA, USA) and was used at 2 μg/mL for immunofluorescence (IF), 5 μg/mL for immunogold and 4 μg/mL for Western blot (WB).

Techniques: Membrane, Marker, Transfection, Construct, Sequencing, Staining

Journal: Cancers

Article Title: Pannexin 2 Localizes at ER-Mitochondria Contact Sites

doi: 10.3390/cancers11030343

Figure Lengend Snippet: Panx2 puncta associate with mitochondria. ( A ) The dynamic of Panx2 puncta was analyzed in C6 Panx2-EGFP cells transfected with mitochondria-targeted mCherry. Many Panx2 puncta were closely associated with the mitochondrial network as indicated by the white arrowheads (inset). Scale bar, 10 µm. ( B ) The probability distribution of Panx2 puncta was calculated before and after randomizing the trajectories of Panx2 puncta within the cytoplasm (orange and purple curves respectively). The distributions, computed from 17 cells, show that Panx2 puncta were not randomly distributed within the cytoplasm; more Panx2 foci localized in the proximity of or in direct apposition with mitochondria that can be explained by chance. ( C ) Frame sequence showing that Panx2 association with mitochondria is stable even though the mitochondrial network is dynamic. A Panx2 puncta approaching a mitochondrion in frame a (white arrowhead) established contact with the organelle in frames b and c. The association was briefly lost (frame d) but stably re-established for over 18 s from frame e onward. Scale bar, 2 µm. ( D ) C6 Panx2-EGFP cells were stained with MitoTracker CMXRos and either untreated (control) or treated with antimycin A (40 µM) or the mitochondrial uncouplers valinomycin and carbonyl cyanide m-chlorophenyl hydrazone (CCCP) (100 nM and 10 µM, respectively) for 2 h prior fixing. Several Panx2 puncta (green) remained associated with mitochondria (magenta) following the fragmentation of the mitochondrial network upon treatment with antimycin A, valinomycin, and CCCP as indicated by the arrowheads. Scale bars: 10 µm (top panels) and 2.5 µm (insets). ( E ) Mouse brain sections were stained for Panx2 and ATP synthase α, a subunit of mitochondrial complex V. A large proportion of Panx2 staining overlapped with brain mitochondria as indicated by the white arrowheads (inset). Scale bars, 10 µm (inset 5 µm).

Article Snippet: The anti-Panx2 mouse monoclonal antibody (cat# 75-212, clone N121A/1) was from UC Davis/NIH NeuroMab Facility (Davis, CA, USA) and was used at 2 μg/mL for immunofluorescence (IF), 5 μg/mL for immunogold and 4 μg/mL for Western blot (WB).

Techniques: Transfection, Sequencing, Stable Transfection, Staining, Control

Journal: Cancers

Article Title: Pannexin 2 Localizes at ER-Mitochondria Contact Sites

doi: 10.3390/cancers11030343

Figure Lengend Snippet: Panx2 localizes at ER-mitochondria contact sites in vivo. ( A ) C6 Panx2-EGFP cells were co-transfected with mito-Turquoise and ER-mCherry. Many Panx2 puncta simultaneously contact ER and mitochondrial networks as indicated by the white arrowheads. Scale bars: 10 µm (inset 2.5 µm). ( B ) Mouse cortical brain sections were co-stained for Panx2, the ER marker calnexin and the mitochondrial protein ATP synthase. Panx2 is found on ER microdomains that are closely associated with mitochondria in vivo as indicated by the white arrowheads. Scale bars: 10 µm (inset 2.5 µm). ( C ) Transmission electron micrograph of a mouse cortical brain showing that the gap between ER and mitochondria is often imperceptible at ER-mitochondria contact site. ( D ) Representative image of a mouse brain cortex showing the localization of Panx2 by immunogold. Most gold clusters are found in close association with mitochondria (open arrowheads). Gold clusters not located near mitochondria (solid arrowheads) are located on ER membranes as seen in F. ( E ) Representative example of an immunogold staining from mouse brain cortex showing the presence of Panx2 at ER-mitochondria contact sites (solid arrowhead). Gold particles are in the narrow gap which defines the cytoplasmic region between ER (open arrowheads) and mitochondrial membranes. ( F ) Representative image of a mouse brain cortex showing the localization of Panx2 (solid arrowhead) on ER membranes by immunogold staining. The gold particles are in the cytoplasm and not inside the ER lumen which is consistent with the localization of Panx2 C-terminal tail. ( G , H ) Representative electron micrographs from mouse brain cortex illustrating the absence of gold particles when the anti-Panx2 primary antibody was replaced by an antibody from a non-immunized mouse. The localization of the ER membranes at Mitochondria-associated ER membranes (MAMs) is indicated by open arrowheads. The ER lumen was pseudocolored in cyan ( C , F ) and mitochondria in purple ( C , D , F , G ). Scale bars: 500 nm ( C ), 1 µm ( D , G ), 100 nm ( E , H ) and 200 nm ( F ). ( I ) Brain sections immunoprobed for Panx2 had a much higher density of gold clusters than control sections immunoprobed with an antibody from a non-immunized mouse (respectively 0.796 ± 0.055 vs. 0.097 ± 0.031 cluster per µm 2 ). ( J ) In cortical brain sections immunoprobed for Panx2, 220 out of 363 gold clusters (60.61%) were directly associated with mitochondria. In contrast, only 4 gold clusters were found associated with mitochondria in cortical sections from control brains. Two different brains and a total of forty-one images per group were used for quantification in I and J. Both brains were used in each group.

Article Snippet: The anti-Panx2 mouse monoclonal antibody (cat# 75-212, clone N121A/1) was from UC Davis/NIH NeuroMab Facility (Davis, CA, USA) and was used at 2 μg/mL for immunofluorescence (IF), 5 μg/mL for immunogold and 4 μg/mL for Western blot (WB).

Techniques: In Vivo, Transfection, Staining, Marker, Transmission Assay, Control

Journal: Cancers

Article Title: Pannexin 2 Localizes at ER-Mitochondria Contact Sites

doi: 10.3390/cancers11030343

Figure Lengend Snippet: Panx2 is enriched in purified MAMs but is absent from pure mitochondria. ( A ) MAMs and pure mitochondria were isolated from mouse liver and immunoprobed with different markers. Two distinct bands, labeled fraction #1 (MAMs) and fraction #2, were isolated above the mitochondrial fraction by ultracentrifugation on a Percoll gradient. Panx2 is found in the crude mitochondrial and MAM fractions but not the pure mitochondrial fraction. Purified MAMs were also enriched in Grp78/BiP and ACSL4. ( B ) Isolated MAMs are enriched in Panx2 but are devoid of inner mitochondrial membrane contaminant as shown by the absence of ATP synthase.

Article Snippet: The anti-Panx2 mouse monoclonal antibody (cat# 75-212, clone N121A/1) was from UC Davis/NIH NeuroMab Facility (Davis, CA, USA) and was used at 2 μg/mL for immunofluorescence (IF), 5 μg/mL for immunogold and 4 μg/mL for Western blot (WB).

Techniques: Purification, Isolation, Labeling, Membrane

Journal: Cancers

Article Title: Pannexin 2 Localizes at ER-Mitochondria Contact Sites

doi: 10.3390/cancers11030343

Figure Lengend Snippet: Panx2 sensitizes glioma cells to apoptosis. ( A ) C6 wildtype (WT), C6-Panx1-EGFP and C6 Panx2-EGFP glioma cells were treated for 2 h to 24 h with 1 µM staurosporine (STS) prior to assessing apoptosis by DNA gel electrophoresis. C6 cells expressing Panx2 underwent apoptosis as soon as 4 h after STS application as showed by evidence of DNA fragmentation. In contrast, C6WT and C6-Panx1-EGFP cells did not show appreciable sign of DNA fragmentation within that period. ( B ) C6 WT, C6-Panx1-EGFP and C6 Panx2-EGFP cells were treated with 1 µM STS for 2–12 h prior to immunoblotting for cleaved caspase-3. C6 Panx2-EGFP, but not C6WT and C6-Panx1EGFP, showed strong activation of caspase-3 starting 4 h after STS treatment. ( C ) The normalized intensity of cleaved caspase-3 staining following 1 µM STS treatment for 6 h was analyzed by a two-way mixed factorial ANOVA, with cell type and treatment as independent variables (n = 3). A logarithmic transformation was used to ensure normal distribution of the residuals, homoscedasticity was confirmed using the Levene’s test, and statistical significance was further examined using a post hoc Tukey test. A significant effect, indicated by *, was found for the cell type F 0.05 (1,8) = 12.07 p -value < 0.0084, STS treatment F 0.05 (1,8) = 67.11 p -value < 3.7 × 10 −5 , and the interaction F 0.05 (1,8) = 32.42 p -value < 0.0005. This indicates that STS treatment induced a significant increase in caspase-3 activation in C6 Panx2-EGFP but not C6WT cells.

Article Snippet: The anti-Panx2 mouse monoclonal antibody (cat# 75-212, clone N121A/1) was from UC Davis/NIH NeuroMab Facility (Davis, CA, USA) and was used at 2 μg/mL for immunofluorescence (IF), 5 μg/mL for immunogold and 4 μg/mL for Western blot (WB).

Techniques: DNA Gel Electrophoresis, Expressing, Western Blot, Activation Assay, Staining, Transformation Assay

Journal: Chromosoma

Article Title: FancJ (Brip1) loss-of-function allele results in spermatogonial cell depletion during embryogenesis and altered processing of crossover sites during meiotic prophase I in mice.

doi: 10.1007/s00412-015-0549-2

Figure Lengend Snippet: Fig. 7 Western blot analysis of whole testis lysates from Fancj+/+ and FancjGT/GT males reveals specific alterations in DNA MMR proteins and BLM. a–g Quantitation of protein levels for BRCA1, BLM, MLH1, MLH3, MSH4, MSH5, and MUS81 normalized to β-tubulin, showing significant increases in protein levels in FancjGT/GT (gray bars) compared to Fancj+/+ (black bars) testis extracts for all except BRCA1 and MUS81

Article Snippet: Primary-antibody incubation was performed for 12 h at 4 °C at 1:1000 dilution (antibodies used are: MUS81 (NBP1-32054 from Novus Biochemicals), MSH4 (ab95096 from Abcam), MSH5 (LS-C101911 from LsBio), MLH1 (550838 from BD pharmingen), β-Tubulin (T8328 from Sigma),β-Actin (A2228 from Sigma), BLM (a gift from Dr. Raimundo Freire), BRIP1/FANCJ (Novus, NBP1-31883), and BRCA1 (a gift from Dr. Satoshi Namekawa).

Techniques: Western Blot, Quantitation Assay

Journal: Toxics

Article Title: Effects of Short-Term Exposure to Polystyrene Nanoplastics on the Nervous System: Calcium Homeostasis, BDNF and Synaptic Plasticity

doi: 10.3390/toxics14020178

Figure Lengend Snippet: Role of BDNF in the mechanism of synaptic plasticity impairment. All indicators were detected in neurons from the control and PS-NPs intervention groups. ( A ) Western blot analysis of CREB, BDNF, Cav3.1, and CaMKIIα in neurons, with GAPDH as the loading control. ( B ) Protein expression levels of CREB in the neurons. n = 3. ( C ) Protein expression levels of BDNF in the neurons. n = 3. ( D ) Calcium ion content in the neurons. n = 6. ( E ) Protein expression levels of the calcium channel Cav3.1 in neurons. ( n = 3). ( F ) Protein expression levels of CaMKIIα in neurons. n = 3. (○) represents the individual samples within each group. Quantitative results were normalized, and the data in the bar charts are presented as mean ± SD. ** p < 0.01 or *** p < 0.001, **** p < 0.0001.

Article Snippet: The membranes were blocked for 1 h at room temperature with 5% skim milk (Beyotime, Shanghai, China, P0216-1500 g) and incubated overnight at 4 °C with specific primary antibodies, including Cav3.1 (1:2000, NBP2-59322, Novus, Lone Tree, CO, USA), CaMKIIα (1:2000, 3357S, CST, Danvers, MA, USA), CREB (1:2000, 12208-1-AP, Proteintech, Rosemont, IL, USA), BDNF (1:1000, ab226843, abcam, Cambridge, UK), PSD95 (1:1000, 2507S, CST), SYN (1:2000, 17785-1-AP, Proteintech), ProBDNF (1:1000, 28205-1-AP, Proteintech), and GAPDH (1:2000, UM4002, YouKang, Tianjin, China).

Techniques: Control, Western Blot, Expressing