Structured Review

Abcam anti mcherry antibody
OsNF-YC11 and OsNF-YC12 interact with OsNF-YB1 in vivo and mediate the nuclear localization of OsNF-YB1. (A) Observation of OsNF-YB1 subcellular localization showed a dual cytosolic–nuclear localization in root cells and nucleus-specific localization in aleurone layer cells. Root of young seedlings and aleurone layer cells of 10 DAF seeds expressing pUbi:OsNF-YB1-GFP were observed. Bars: 50 µm (upper) or 20 µm (bottom). (B) OsNF-YB1-GFP protein showed a dual cytosolic–nuclear localization in the presence of red fluorescent protein (RFP) or OsNF-YC2-RFP, and was translocated to nucleus of rice protoplast cells in the presence of OsNF-YC11-RFP or OsNF-YC12-RFP. Bars: 5 µm. (C, D) Co-immunoprecipitation analysis revealed the interaction of OsNF-YB1-GFP and <t>OsNF-YC11-mCherry</t> (C), and OsNF-YB1-GFP and OsNF-YC12-mCherry (D) in tobacco cells. Total protein extracts (Input) or immunoprecipitated (IP) fractions using an anti-GFP antibody were analyzed using anti-GFP or anti-mCherry antibodies.
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Images

1) Product Images from "Rice aleurone layer specific OsNF-YB1 regulates grain filling and endosperm development by interacting with an ERF transcription factor"

Article Title: Rice aleurone layer specific OsNF-YB1 regulates grain filling and endosperm development by interacting with an ERF transcription factor

Journal: Journal of Experimental Botany

doi: 10.1093/jxb/erw409

OsNF-YC11 and OsNF-YC12 interact with OsNF-YB1 in vivo and mediate the nuclear localization of OsNF-YB1. (A) Observation of OsNF-YB1 subcellular localization showed a dual cytosolic–nuclear localization in root cells and nucleus-specific localization in aleurone layer cells. Root of young seedlings and aleurone layer cells of 10 DAF seeds expressing pUbi:OsNF-YB1-GFP were observed. Bars: 50 µm (upper) or 20 µm (bottom). (B) OsNF-YB1-GFP protein showed a dual cytosolic–nuclear localization in the presence of red fluorescent protein (RFP) or OsNF-YC2-RFP, and was translocated to nucleus of rice protoplast cells in the presence of OsNF-YC11-RFP or OsNF-YC12-RFP. Bars: 5 µm. (C, D) Co-immunoprecipitation analysis revealed the interaction of OsNF-YB1-GFP and OsNF-YC11-mCherry (C), and OsNF-YB1-GFP and OsNF-YC12-mCherry (D) in tobacco cells. Total protein extracts (Input) or immunoprecipitated (IP) fractions using an anti-GFP antibody were analyzed using anti-GFP or anti-mCherry antibodies.
Figure Legend Snippet: OsNF-YC11 and OsNF-YC12 interact with OsNF-YB1 in vivo and mediate the nuclear localization of OsNF-YB1. (A) Observation of OsNF-YB1 subcellular localization showed a dual cytosolic–nuclear localization in root cells and nucleus-specific localization in aleurone layer cells. Root of young seedlings and aleurone layer cells of 10 DAF seeds expressing pUbi:OsNF-YB1-GFP were observed. Bars: 50 µm (upper) or 20 µm (bottom). (B) OsNF-YB1-GFP protein showed a dual cytosolic–nuclear localization in the presence of red fluorescent protein (RFP) or OsNF-YC2-RFP, and was translocated to nucleus of rice protoplast cells in the presence of OsNF-YC11-RFP or OsNF-YC12-RFP. Bars: 5 µm. (C, D) Co-immunoprecipitation analysis revealed the interaction of OsNF-YB1-GFP and OsNF-YC11-mCherry (C), and OsNF-YB1-GFP and OsNF-YC12-mCherry (D) in tobacco cells. Total protein extracts (Input) or immunoprecipitated (IP) fractions using an anti-GFP antibody were analyzed using anti-GFP or anti-mCherry antibodies.

Techniques Used: In Vivo, Expressing, Immunoprecipitation

2) Product Images from "An improved auxin-inducible degron system preserves native protein levels and enables rapid and specific protein depletion"

Article Title: An improved auxin-inducible degron system preserves native protein levels and enables rapid and specific protein depletion

Journal: Genes & Development

doi: 10.1101/gad.328237.119

The interaction of ARF-PB1 and AID mediates rescue of endogenous protein levels. ( A ). Positions on the X -axis are relative to ARF16 residue numbers. ( B ) The amino acids D994 and D998 of ARF16 and K114 of IAA17 are shown in the left panel. The amino acid side chains corresponding to D183 and D187 of IAA17 and K944 of ARF16 are highlighted in the right ) into each chain of an A. thaliana ). These mutations in ARF16, which disrupt the electrostatic binding interface, fail to rescue chronic ZNF143 ( C ) and TEAD4 ( D ) degradation. ( E ) These mutations disrupt this coimmunoprecipitation of mCherry-AID with eGFP-tagged ARF16-PB1. Consistent with lower stability of the ARF16-MT in C and D , the mutant GFP-ARF16-PB1 plasmid was transfected at a concentration three times higher than the wild type to achieve comparable expression of each protein. ( F ) ARF16-PB1 is detected upon mCherry-AID immunoprecipitation; however, we were unable to detect TIR1 associating with AID.
Figure Legend Snippet: The interaction of ARF-PB1 and AID mediates rescue of endogenous protein levels. ( A ). Positions on the X -axis are relative to ARF16 residue numbers. ( B ) The amino acids D994 and D998 of ARF16 and K114 of IAA17 are shown in the left panel. The amino acid side chains corresponding to D183 and D187 of IAA17 and K944 of ARF16 are highlighted in the right ) into each chain of an A. thaliana ). These mutations in ARF16, which disrupt the electrostatic binding interface, fail to rescue chronic ZNF143 ( C ) and TEAD4 ( D ) degradation. ( E ) These mutations disrupt this coimmunoprecipitation of mCherry-AID with eGFP-tagged ARF16-PB1. Consistent with lower stability of the ARF16-MT in C and D , the mutant GFP-ARF16-PB1 plasmid was transfected at a concentration three times higher than the wild type to achieve comparable expression of each protein. ( F ) ARF16-PB1 is detected upon mCherry-AID immunoprecipitation; however, we were unable to detect TIR1 associating with AID.

Techniques Used: Binding Assay, Mutagenesis, Plasmid Preparation, Transfection, Concentration Assay, Expressing, Immunoprecipitation

3) Product Images from "Ca2+ signals initiate at immobile IP3 receptors adjacent to ER-plasma membrane junctions"

Article Title: Ca2+ signals initiate at immobile IP3 receptors adjacent to ER-plasma membrane junctions

Journal: Nature Communications

doi: 10.1038/s41467-017-01644-8

STIM1 translocates to ER-PM junctions adjacent to immobile IP 3 R puncta. a , b TIRFM images show that thapsigargin (Tg, 1 µM, 5 min) causes formation of STIM1-mCherry puncta at ER-PM junctions (i). Immobile IP 3 Rs (ii, white, identified from overlays of pseudocoloured EGFP-IP 3 R distribution at 30-s intervals) abut STIM1 puncta (iii). Enlarged image of the boxed area shows all immobile EGFP-IP 3 R1 (arrows) and STIM1-mCherry without pseudocolours for clarity (iv). Scale bars = 10 µm (i–iii), 2 µm (iv). c Similar overlay images from three additional Tg-treated cells, with all immobile IP 3 R punta identified with arrows. Scale bars = 2 µm. d Fluorescence intensity profiles for EGFP-IP 3 R1 measured at 30-s intervals (green and magenta) and STIM1-mCherry for transects drawn across Tg-treated cells. Results show that immobile IP 3 Rs (white, where green and magenta coincide) and STIM1 after store depletion are juxtaposed, but not perfectly aligned. Distances (μm) between the peaks of the fluorescence intensity for STIM1 and immobile IP 3 R are shown; 84 ± 9% (mean ± SD, n = 3 cells) of the centroids of immobile EGFP-IP 3 R1 puncta were within twice their radius (2 r = 0.64–0.96 μm) of a STIM1 punctum. e STIM1-mCherry fluorescence was measured before ( F 0 ) and after Tg treatment ( F Tg ) at ROI with twice the radius of underlying mobile or immobile EGFP-IP 3 R1 puncta. Summary results show the change in mCherry fluorescence (Δ F = ( F Tg − F 0 )/ F 0 , mean ± SEM, n = 3 cells, 29–33 puncta). * P
Figure Legend Snippet: STIM1 translocates to ER-PM junctions adjacent to immobile IP 3 R puncta. a , b TIRFM images show that thapsigargin (Tg, 1 µM, 5 min) causes formation of STIM1-mCherry puncta at ER-PM junctions (i). Immobile IP 3 Rs (ii, white, identified from overlays of pseudocoloured EGFP-IP 3 R distribution at 30-s intervals) abut STIM1 puncta (iii). Enlarged image of the boxed area shows all immobile EGFP-IP 3 R1 (arrows) and STIM1-mCherry without pseudocolours for clarity (iv). Scale bars = 10 µm (i–iii), 2 µm (iv). c Similar overlay images from three additional Tg-treated cells, with all immobile IP 3 R punta identified with arrows. Scale bars = 2 µm. d Fluorescence intensity profiles for EGFP-IP 3 R1 measured at 30-s intervals (green and magenta) and STIM1-mCherry for transects drawn across Tg-treated cells. Results show that immobile IP 3 Rs (white, where green and magenta coincide) and STIM1 after store depletion are juxtaposed, but not perfectly aligned. Distances (μm) between the peaks of the fluorescence intensity for STIM1 and immobile IP 3 R are shown; 84 ± 9% (mean ± SD, n = 3 cells) of the centroids of immobile EGFP-IP 3 R1 puncta were within twice their radius (2 r = 0.64–0.96 μm) of a STIM1 punctum. e STIM1-mCherry fluorescence was measured before ( F 0 ) and after Tg treatment ( F Tg ) at ROI with twice the radius of underlying mobile or immobile EGFP-IP 3 R1 puncta. Summary results show the change in mCherry fluorescence (Δ F = ( F Tg − F 0 )/ F 0 , mean ± SEM, n = 3 cells, 29–33 puncta). * P

Techniques Used: Fluorescence

Depletion of ER Ca 2+ stores causes native STIM1 to accumulate at functional ER-PM junctions adjacent to immobile IP 3 R puncta. a , b Representative TIRFM images of EGFP-IP 3 R1 HeLa cells fixed and immunostained for STIM1 before ( a ) or after treatment with thapsigargin (Tg, 1 µM, 15 min) to deplete the ER of Ca 2+ ( b ). Overlaid images of Tg-treated cells show no significant co-localization of STIM1 and IP 3 R puncta (Pearson’s coefficient with Costes’ automatic threshold: 0.331 ± 0.026, n = 7 cells). c Distribution of mobile (green and magenta) and immobile (white) IP 3 R puncta in Tg-treated cell. Scale bars ( a – c ) = 10 µm. d Enlargements of the boxed regions in b show that immobile IP 3 R puncta (identified before fixation ( c ), with all shown by arrowheads) abut STIM1 puncta without coinciding with them. Scale bars = 2 µm. e Fluorescence intensity profiles for EGFP-IP 3 R1 and STIM1 across the lines shown in d . Distances (μm) between the peaks of the fluorescence intensity for STIM1 and immobile IP 3 R are shown. f Co-localization of CFP-STIM1 (pseudocoloured green) and mCherry-Orai1 (red) puncta in a Tg-treated HeLa cell. We used tagged proteins because available antibodies do not reliably detect endogenous Orai1. Scale bar = 10 µm (2 μm in enlargement)
Figure Legend Snippet: Depletion of ER Ca 2+ stores causes native STIM1 to accumulate at functional ER-PM junctions adjacent to immobile IP 3 R puncta. a , b Representative TIRFM images of EGFP-IP 3 R1 HeLa cells fixed and immunostained for STIM1 before ( a ) or after treatment with thapsigargin (Tg, 1 µM, 15 min) to deplete the ER of Ca 2+ ( b ). Overlaid images of Tg-treated cells show no significant co-localization of STIM1 and IP 3 R puncta (Pearson’s coefficient with Costes’ automatic threshold: 0.331 ± 0.026, n = 7 cells). c Distribution of mobile (green and magenta) and immobile (white) IP 3 R puncta in Tg-treated cell. Scale bars ( a – c ) = 10 µm. d Enlargements of the boxed regions in b show that immobile IP 3 R puncta (identified before fixation ( c ), with all shown by arrowheads) abut STIM1 puncta without coinciding with them. Scale bars = 2 µm. e Fluorescence intensity profiles for EGFP-IP 3 R1 and STIM1 across the lines shown in d . Distances (μm) between the peaks of the fluorescence intensity for STIM1 and immobile IP 3 R are shown. f Co-localization of CFP-STIM1 (pseudocoloured green) and mCherry-Orai1 (red) puncta in a Tg-treated HeLa cell. We used tagged proteins because available antibodies do not reliably detect endogenous Orai1. Scale bar = 10 µm (2 μm in enlargement)

Techniques Used: Functional Assay, Fluorescence

Endogenous IP 3 R1s form puncta. a In-gel fluorescence of lysates from EGFP-IP 3 R1 HeLa cells (GR) and control (WT) cells demonstrates that the only fluorescence is associated with EGFP-IP 3 R1 (green arrow). Results typical of four gels. Positions of selected M r markers (kDa) are shown ( a , c , d ). b TIRFM images of EGFP-IP 3 R1 HeLa cells showing a marker for the ER lumen (mCherry-ER). The merged image and an enlargement of the boxed area show co-localization of EGFP-IP 3 R1 with mCherry-ER (Pearson’s coefficient with Costes’ automatic threshold = 0.93 ± 0.02; Costes P value = 1.00, n = 4 cells). Scale bar = 5 µm (2 µm for enlargement). c Western blots (WBs) for IP 3 R1-3 show expression of tagged (green arrow, ~290 kDa) and untagged (black arrow, ~260 kDa) IP 3 R1 in GR and WT cells, respectively. Expression of IP 3 R subtypes in GR cells is shown relative to control (WT) cells (%, mean ± SD, n = 3 for IP 3 R2 and IP 3 R3, n = 4 for IP 3 R1). Comparisons of band intensities using paired Student’s t -tests indicated no significant differences between WT and EGFP-IP 3 R1 cells. d WB (IP 3 R1-3 antibodies) from lysates of EGFP-IP 3 R1 HeLa cells after immunoprecipitation with GFP-Trap. Eluate lanes were loaded with sample equivalent to 1.5 times the amounts loaded in the lysate lanes. Numbers show % of each subtype detected in the pull-down ( n = 2). e Photobleaching of a punctum showing the final bleaching step (bracket) and the initial fluorescence (dashed line) used to calculate the total number of fluorophores ( n ). FU, fluorescence units. f Single-step photobleaching results (284 puncta from five cells, Supplementary Fig. 5 ) were used to calculate the number of tetrameric IP 3 Rs per punctum (8.4 ± 7)
Figure Legend Snippet: Endogenous IP 3 R1s form puncta. a In-gel fluorescence of lysates from EGFP-IP 3 R1 HeLa cells (GR) and control (WT) cells demonstrates that the only fluorescence is associated with EGFP-IP 3 R1 (green arrow). Results typical of four gels. Positions of selected M r markers (kDa) are shown ( a , c , d ). b TIRFM images of EGFP-IP 3 R1 HeLa cells showing a marker for the ER lumen (mCherry-ER). The merged image and an enlargement of the boxed area show co-localization of EGFP-IP 3 R1 with mCherry-ER (Pearson’s coefficient with Costes’ automatic threshold = 0.93 ± 0.02; Costes P value = 1.00, n = 4 cells). Scale bar = 5 µm (2 µm for enlargement). c Western blots (WBs) for IP 3 R1-3 show expression of tagged (green arrow, ~290 kDa) and untagged (black arrow, ~260 kDa) IP 3 R1 in GR and WT cells, respectively. Expression of IP 3 R subtypes in GR cells is shown relative to control (WT) cells (%, mean ± SD, n = 3 for IP 3 R2 and IP 3 R3, n = 4 for IP 3 R1). Comparisons of band intensities using paired Student’s t -tests indicated no significant differences between WT and EGFP-IP 3 R1 cells. d WB (IP 3 R1-3 antibodies) from lysates of EGFP-IP 3 R1 HeLa cells after immunoprecipitation with GFP-Trap. Eluate lanes were loaded with sample equivalent to 1.5 times the amounts loaded in the lysate lanes. Numbers show % of each subtype detected in the pull-down ( n = 2). e Photobleaching of a punctum showing the final bleaching step (bracket) and the initial fluorescence (dashed line) used to calculate the total number of fluorophores ( n ). FU, fluorescence units. f Single-step photobleaching results (284 puncta from five cells, Supplementary Fig. 5 ) were used to calculate the number of tetrameric IP 3 Rs per punctum (8.4 ± 7)

Techniques Used: Fluorescence, Marker, Western Blot, Expressing, Immunoprecipitation

IP 3 Rs form mobile and immobile puncta. a Time-lapse TIRFM images (0.6-s intervals) of EGFP-IP 3 R1 in cells expressing mCherry-ER. Track of a single particle, with the first and last positions shown by white and yellow arrows, respectively. Scale bar = 5 µm. b Representative epifluorescence image of an EGFP-IP 3 R1 HeLa cell with perinuclear (blue) and peripheral (magenta) regions highlighted for FRAP analysis (circular bleached area, radius = 1.84 μm). The boxed area is enlarged to show pre- and post-bleach (after 120 s) images of the peripheral region. Scale bars = 5 µm. c Normalized fluorescence intensities recorded from peripheral or perinuclear regions in a typical FRAP experiment with live and fixed EGFP-IP 3 R1 HeLa cells. d , e Summary results show mobile fractions ( M f , mean ± SEM) ( d ) and diffusion coefficients ( D , mean and all values) ( e ) for perinuclear (25 cells) and peripheral regions (26 cells). **** P
Figure Legend Snippet: IP 3 Rs form mobile and immobile puncta. a Time-lapse TIRFM images (0.6-s intervals) of EGFP-IP 3 R1 in cells expressing mCherry-ER. Track of a single particle, with the first and last positions shown by white and yellow arrows, respectively. Scale bar = 5 µm. b Representative epifluorescence image of an EGFP-IP 3 R1 HeLa cell with perinuclear (blue) and peripheral (magenta) regions highlighted for FRAP analysis (circular bleached area, radius = 1.84 μm). The boxed area is enlarged to show pre- and post-bleach (after 120 s) images of the peripheral region. Scale bars = 5 µm. c Normalized fluorescence intensities recorded from peripheral or perinuclear regions in a typical FRAP experiment with live and fixed EGFP-IP 3 R1 HeLa cells. d , e Summary results show mobile fractions ( M f , mean ± SEM) ( d ) and diffusion coefficients ( D , mean and all values) ( e ) for perinuclear (25 cells) and peripheral regions (26 cells). **** P

Techniques Used: Expressing, Fluorescence, Diffusion-based Assay

4) Product Images from "Fast purification of recombinant monomeric amyloid-β from E. coli and amyloid-β-mCherry aggregates from mammalian cells"

Article Title: Fast purification of recombinant monomeric amyloid-β from E. coli and amyloid-β-mCherry aggregates from mammalian cells

Journal: bioRxiv

doi: 10.1101/2020.05.13.093534

Deconvoluted mass spectrum of AβM(E22G)-mCherry shows many protein species present. The AβM(E22G)-mCherry sample displayed multiple peaks on the chromatograph prior to MS, two fractions were electrosprayed for MS analysis (a. (eluting at 4. 5 mins) and b (eluting at 4.9 mins)). The expected MW of AβM(E22G)-mCherry is ∼32.3 kDa (highlighted in the red box in a.), however this is not the most abundant species isolated from HEK293 cells. A larger species of ∼36.3 kDa (a.) and a degraded product of ∼25.6 kDa (b.) are instead the dominant protein species by MS. Also present are many other species of differing MW.
Figure Legend Snippet: Deconvoluted mass spectrum of AβM(E22G)-mCherry shows many protein species present. The AβM(E22G)-mCherry sample displayed multiple peaks on the chromatograph prior to MS, two fractions were electrosprayed for MS analysis (a. (eluting at 4. 5 mins) and b (eluting at 4.9 mins)). The expected MW of AβM(E22G)-mCherry is ∼32.3 kDa (highlighted in the red box in a.), however this is not the most abundant species isolated from HEK293 cells. A larger species of ∼36.3 kDa (a.) and a degraded product of ∼25.6 kDa (b.) are instead the dominant protein species by MS. Also present are many other species of differing MW.

Techniques Used: Isolation

Large aggregated structures are present in purified AβE22G-mCherry fractions. 10 μL of AβE22G-mCherry sample was incubated on a 400 mesh carbon coated copper grid and negatively stained with 2% uranyl acetate before imaging with TEM. White scale bar = 100 nm, black scale bar = 500 nm.
Figure Legend Snippet: Large aggregated structures are present in purified AβE22G-mCherry fractions. 10 μL of AβE22G-mCherry sample was incubated on a 400 mesh carbon coated copper grid and negatively stained with 2% uranyl acetate before imaging with TEM. White scale bar = 100 nm, black scale bar = 500 nm.

Techniques Used: Purification, Incubation, Staining, Imaging, Transmission Electron Microscopy

Varying aggregate sizes of AβM(E22G)-mCherry isolated by ion exchange chromatography exhibit weak fluorescence. AβM(E22G)-mCherry lysed from HEK293 cells by sonication. (a.) The soluble fraction was applied to the column (shown up to the first dotted line in a. and the unbound protein was washed from the column (shown up to the second dotted line in a. The chromatograph of absorption at 280 nm show protein elution from the HiScreen Capto Q ImpRes column eluted over a gradient of 0-100% of buffer B containing 1 M NaCl over seven column volumes, followed by two column volumes of 100% buffer B (dashed line showing gradient in a. (b) In order to determine when AβM(E22G)-mCherry eluted off the column, the fractions were collected and analysed using SDS-PAGE on a 4-12% bis-tris gel and (b.i) Coomassie blue staining, or transferred to a membrane for Western blot using antibodies against (b.ii.) Aβ and (b.iii.) mCherry. The sample prior to IEX (P) contained many proteins, including aggregated Aβ and mCherry. Protein bands correlating to ∼ 32.3 kDa (shown by the black arrow in b.iii.) show the predicted MW for monomeric AβM(E22G)-mCherry. Fraction 2 contained the highest content of AβM(E22G)-mCherry, although the presence of degraded mCherry (b.iii., grey arrow) and aggregated Aβ (b.ii., star) were also apparent. The morphology of the purified AβM(E22G)-mCherry was determined by TEM and both (c.i.) oligomers and (c.ii., Supplementary Figure 7 ) larger aggregates were present. (c.iii.) A section view of an AβM(E22G)-mCherry aggregate inside a cell prior to purification reveals a similar structure to those identified by TEM after purification. These aggregates were also analysed to determine whether they were fluorescent using widefield imaging with a 561 nm laser, both (d.i.) small oligomers and (d.ii.) large aggregates were weakly fluorescent (also see Supplementary Figure 8 ). Black scale bar = 100 nm, white scale bar = 2 μm.
Figure Legend Snippet: Varying aggregate sizes of AβM(E22G)-mCherry isolated by ion exchange chromatography exhibit weak fluorescence. AβM(E22G)-mCherry lysed from HEK293 cells by sonication. (a.) The soluble fraction was applied to the column (shown up to the first dotted line in a. and the unbound protein was washed from the column (shown up to the second dotted line in a. The chromatograph of absorption at 280 nm show protein elution from the HiScreen Capto Q ImpRes column eluted over a gradient of 0-100% of buffer B containing 1 M NaCl over seven column volumes, followed by two column volumes of 100% buffer B (dashed line showing gradient in a. (b) In order to determine when AβM(E22G)-mCherry eluted off the column, the fractions were collected and analysed using SDS-PAGE on a 4-12% bis-tris gel and (b.i) Coomassie blue staining, or transferred to a membrane for Western blot using antibodies against (b.ii.) Aβ and (b.iii.) mCherry. The sample prior to IEX (P) contained many proteins, including aggregated Aβ and mCherry. Protein bands correlating to ∼ 32.3 kDa (shown by the black arrow in b.iii.) show the predicted MW for monomeric AβM(E22G)-mCherry. Fraction 2 contained the highest content of AβM(E22G)-mCherry, although the presence of degraded mCherry (b.iii., grey arrow) and aggregated Aβ (b.ii., star) were also apparent. The morphology of the purified AβM(E22G)-mCherry was determined by TEM and both (c.i.) oligomers and (c.ii., Supplementary Figure 7 ) larger aggregates were present. (c.iii.) A section view of an AβM(E22G)-mCherry aggregate inside a cell prior to purification reveals a similar structure to those identified by TEM after purification. These aggregates were also analysed to determine whether they were fluorescent using widefield imaging with a 561 nm laser, both (d.i.) small oligomers and (d.ii.) large aggregates were weakly fluorescent (also see Supplementary Figure 8 ). Black scale bar = 100 nm, white scale bar = 2 μm.

Techniques Used: Isolation, Ion Exchange Chromatography, Fluorescence, Sonication, SDS Page, Staining, Western Blot, Purification, Transmission Electron Microscopy, Imaging

Purified AβE22G-mCherry from HEK293 cells remains fluorescent. AβE22G-mCherry isolated from HEK293 cells and purified by ion exchange chromatography was incubated on a glass coverslip and imaged with widefield microscopy. A 561 nm laser was used to excite the sample and (a.) large aggregated structures and (b.) small aggregated structures displayed fluorescence. Scale bar = 8 μm.
Figure Legend Snippet: Purified AβE22G-mCherry from HEK293 cells remains fluorescent. AβE22G-mCherry isolated from HEK293 cells and purified by ion exchange chromatography was incubated on a glass coverslip and imaged with widefield microscopy. A 561 nm laser was used to excite the sample and (a.) large aggregated structures and (b.) small aggregated structures displayed fluorescence. Scale bar = 8 μm.

Techniques Used: Purification, Isolation, Ion Exchange Chromatography, Incubation, Microscopy, Fluorescence

More AβM(E22G)-mCherry is present in the soluble fraction than the insoluble fraction. AβM(E22G)-mCherry expressed in HEK293 cells was lysed from the cell by sonication and centrifuged to separate the soluble and insoluble fractions. Analysis of the two fractions by Western blot using an anti-Aβ antibody (E610) (α-Aβ) and reprobing the same membrane with an anti-mCherry antibody (α –mCherry) shows more AβM(E22G)-mCherry in the soluble fraction.
Figure Legend Snippet: More AβM(E22G)-mCherry is present in the soluble fraction than the insoluble fraction. AβM(E22G)-mCherry expressed in HEK293 cells was lysed from the cell by sonication and centrifuged to separate the soluble and insoluble fractions. Analysis of the two fractions by Western blot using an anti-Aβ antibody (E610) (α-Aβ) and reprobing the same membrane with an anti-mCherry antibody (α –mCherry) shows more AβM(E22G)-mCherry in the soluble fraction.

Techniques Used: Sonication, Western Blot

5) Product Images from "Enhancer identification and activity evaluation in the red flour beetle, Tribolium castaneum"

Article Title: Enhancer identification and activity evaluation in the red flour beetle, Tribolium castaneum

Journal: bioRxiv

doi: 10.1101/199729

hb enhancer analysis in Tribolium . (A) FAIRE profiles at the hb locus. Orange bar: blastoderm enhancer activity when introduced in Drosophila , purple bars: SCRMshaw predictions, red bar: the 1340bp fragment tested in this study ( hb -PE1). (B) hb expression at the early germband stage detected by in situ hybridization for hb transcript. (C) mCherry reporter gene expression of piggyGUM- hb -PE1 detected by in situ hybridization for mCherry transcript. Scale bar: 100 µm (B, C).
Figure Legend Snippet: hb enhancer analysis in Tribolium . (A) FAIRE profiles at the hb locus. Orange bar: blastoderm enhancer activity when introduced in Drosophila , purple bars: SCRMshaw predictions, red bar: the 1340bp fragment tested in this study ( hb -PE1). (B) hb expression at the early germband stage detected by in situ hybridization for hb transcript. (C) mCherry reporter gene expression of piggyGUM- hb -PE1 detected by in situ hybridization for mCherry transcript. Scale bar: 100 µm (B, C).

Techniques Used: Activity Assay, Expressing, In Situ Hybridization

6) Product Images from "Global and transcription-coupled repair of 8-oxoG is initiated by nucleotide excision repair proteins"

Article Title: Global and transcription-coupled repair of 8-oxoG is initiated by nucleotide excision repair proteins

Journal: Nature Communications

doi: 10.1038/s41467-022-28642-9

DDB2 facilitates 8-oxoG repair and is rapidly recruited to sites of 8-oxoG within telomeric DNA. a , b Immunofluorescence and quantification of 8-oxoG in cells transfected with control, DDB2 or OGG1 siRNA. c Schematic of the repair enzyme-based assay for 8-oxoG quantification in DNA. Genomic DNA containing 8-oxoG is treated with FPG to convert 8-oxoG to one nucleotide gaps. Treating with S1 nuclease converts the gaps to double stranded breaks (DSBs). The cleaved DNA is subjected to pulse field gel electrophoresis (PFGE) to track repair, as damaged DNA migrates faster than repaired DNA. d Quantification of 8-oxoG repair in U2OS cells transfected with control or DDB2 siRNA and treated with KBrO3. e Clonogenic cell survival curves in U2OS WT and DDB2 knockout (KO) cells treated with a range of concentrations of KBrO3. f Schematic of dye plus light treatment. Cells stably expressing FAP-TRF1 were treated with dye (100 nM, 15 min) plus light (660 nm, 10 min) to introduce 8-oxoG lesions at telomeres. g (left) Recruitment of DDB2-mCherry to 8-oxoG sites at telomeres in untreated, dye alone, light alone, and dye plus light treated cells. (right) Percentage telomeres colocalized with DDB2-mCherry. h Proximity ligation assay (PLA) for DDB2-mCherry and TRF1 in untreated cells and cells treated with dye (100 nM, 15 min) plus light (660 nm, 10 min). Data ( a , b , d , g , h ) represent mean ± SEM from two to three independent experiments. “ n ” represents the number of cells scored for each condition. Data ( e ) shows one representative experiment (performed in triplicate) from three independent experiments, mean ± SD. One-way ANOVA (Sidak multiple comparison test) ( b , g ), Student’s two-tailed Student’s t -test ( h ) and two-way ANOVA (Sidak multiple comparison test) ( d , e ) were performed for statistical analysis: * p
Figure Legend Snippet: DDB2 facilitates 8-oxoG repair and is rapidly recruited to sites of 8-oxoG within telomeric DNA. a , b Immunofluorescence and quantification of 8-oxoG in cells transfected with control, DDB2 or OGG1 siRNA. c Schematic of the repair enzyme-based assay for 8-oxoG quantification in DNA. Genomic DNA containing 8-oxoG is treated with FPG to convert 8-oxoG to one nucleotide gaps. Treating with S1 nuclease converts the gaps to double stranded breaks (DSBs). The cleaved DNA is subjected to pulse field gel electrophoresis (PFGE) to track repair, as damaged DNA migrates faster than repaired DNA. d Quantification of 8-oxoG repair in U2OS cells transfected with control or DDB2 siRNA and treated with KBrO3. e Clonogenic cell survival curves in U2OS WT and DDB2 knockout (KO) cells treated with a range of concentrations of KBrO3. f Schematic of dye plus light treatment. Cells stably expressing FAP-TRF1 were treated with dye (100 nM, 15 min) plus light (660 nm, 10 min) to introduce 8-oxoG lesions at telomeres. g (left) Recruitment of DDB2-mCherry to 8-oxoG sites at telomeres in untreated, dye alone, light alone, and dye plus light treated cells. (right) Percentage telomeres colocalized with DDB2-mCherry. h Proximity ligation assay (PLA) for DDB2-mCherry and TRF1 in untreated cells and cells treated with dye (100 nM, 15 min) plus light (660 nm, 10 min). Data ( a , b , d , g , h ) represent mean ± SEM from two to three independent experiments. “ n ” represents the number of cells scored for each condition. Data ( e ) shows one representative experiment (performed in triplicate) from three independent experiments, mean ± SD. One-way ANOVA (Sidak multiple comparison test) ( b , g ), Student’s two-tailed Student’s t -test ( h ) and two-way ANOVA (Sidak multiple comparison test) ( d , e ) were performed for statistical analysis: * p

Techniques Used: Immunofluorescence, Transfection, Enzymatic Assay, Nucleic Acid Electrophoresis, Knock-Out, Stable Transfection, Expressing, Introduce, Proximity Ligation Assay, Two Tailed Test

DDB2 is required for efficient OGG1 recruitment to 8-oxoG. a DDB2-mCherry and OGG1-GFP associate at 8-oxoG sites as shown by PLA after dye (100 nM, 15 min) plus light (660 nm, 10 min) treatment, over a period of 3 h. Antibodies against mCherry and GFP were used. b Quantification of PLA. c Accumulation of DDB2-mCherry at telomeric 8-oxoG 30 min post dye plus light treatment in U2OS-FAP-TRF1 cells transfected with control or OGG1 siRNA. d Percent telomeres colocalized with DDB2-mCherry as shown in (c). e Recruitment of OGG1-GFP at damaged telomeres in cells transfected with control or DDB2 siRNA. f Percent telomeres colocalized with OGG1-GFP as shown in ( e ). Data ( a – f ) represents mean ± SEM from two independent experiments. “ n ” represents the number of cells scored for each condition. One-way ANOVA (Sidak multiple comparison test): * p
Figure Legend Snippet: DDB2 is required for efficient OGG1 recruitment to 8-oxoG. a DDB2-mCherry and OGG1-GFP associate at 8-oxoG sites as shown by PLA after dye (100 nM, 15 min) plus light (660 nm, 10 min) treatment, over a period of 3 h. Antibodies against mCherry and GFP were used. b Quantification of PLA. c Accumulation of DDB2-mCherry at telomeric 8-oxoG 30 min post dye plus light treatment in U2OS-FAP-TRF1 cells transfected with control or OGG1 siRNA. d Percent telomeres colocalized with DDB2-mCherry as shown in (c). e Recruitment of OGG1-GFP at damaged telomeres in cells transfected with control or DDB2 siRNA. f Percent telomeres colocalized with OGG1-GFP as shown in ( e ). Data ( a – f ) represents mean ± SEM from two independent experiments. “ n ” represents the number of cells scored for each condition. One-way ANOVA (Sidak multiple comparison test): * p

Techniques Used: Proximity Ligation Assay, Transfection

DDB2 binds sparse telomeric 8-oxoG independently of the DDB1-Cul4A-RBX1 E3 ligase. a Representative images showing recruitment of DDB2-mCherry to telomeric 8-oxoG in cells transfected with control, DDB1 or Cul4A siRNA. b Quantification of a. c , e DDB2-mCherry and GFP-DDB1 ( c ) or DDB2-mCherry and GFP-Cul4A ( e ) accumulation at 8-oxoG sites after dye (100 nM, 15 min) plus light (660 nm, 10 min) treatment. d , f Quantification of c and e respectively. g Western blot for DDB2 in U2OS-FAP-TRF1 cells treated with UVC, potassium bromate (KBrO 3 ) or dye plus light at indicated doses. Independent experiments are represented by black circles. h Colocalization of DDB2-mCherry and GFP-Cul4A at damaged telomeres in U2OS-FAP-TRF1 cells transfected with control or OGG1 siRNA. i Quantification of h. Data ( a – h ) represents mean ± SEM from two independent experiments. ‘n’ represents the number of cells scored for each condition. One-way ANOVA (Sidak multiple comparison test) ( b , i ) was performed for statistical analysis: * p
Figure Legend Snippet: DDB2 binds sparse telomeric 8-oxoG independently of the DDB1-Cul4A-RBX1 E3 ligase. a Representative images showing recruitment of DDB2-mCherry to telomeric 8-oxoG in cells transfected with control, DDB1 or Cul4A siRNA. b Quantification of a. c , e DDB2-mCherry and GFP-DDB1 ( c ) or DDB2-mCherry and GFP-Cul4A ( e ) accumulation at 8-oxoG sites after dye (100 nM, 15 min) plus light (660 nm, 10 min) treatment. d , f Quantification of c and e respectively. g Western blot for DDB2 in U2OS-FAP-TRF1 cells treated with UVC, potassium bromate (KBrO 3 ) or dye plus light at indicated doses. Independent experiments are represented by black circles. h Colocalization of DDB2-mCherry and GFP-Cul4A at damaged telomeres in U2OS-FAP-TRF1 cells transfected with control or OGG1 siRNA. i Quantification of h. Data ( a – h ) represents mean ± SEM from two independent experiments. ‘n’ represents the number of cells scored for each condition. One-way ANOVA (Sidak multiple comparison test) ( b , i ) was performed for statistical analysis: * p

Techniques Used: Transfection, Western Blot

7) Product Images from "Age-related Macular Degeneration patient deep phenotyping and whole genome sequencing analysis identifies coding variants linking small low-luminance visual deficit to fat storage defects"

Article Title: Age-related Macular Degeneration patient deep phenotyping and whole genome sequencing analysis identifies coding variants linking small low-luminance visual deficit to fat storage defects

Journal: bioRxiv

doi: 10.1101/2022.06.22.497149

Lipid droplet (LD) fusion occurs less frequently and more slowly in pre-adipocytes expressing the AMD CIDEC rare variants. (A) Representative time-lapse images over 40 minutes of LDs in cells co-expressing GFP-tagged CIDEC WT (taken from Video 1 ) or each of the rare variants, and mCherry-tagged PLIN1. Scale bar: 2 μm. (B) Percentage of LDs undergoing fusion during the 6-hour analysis. N=3 (mean ± SD, Student’s t test, *p
Figure Legend Snippet: Lipid droplet (LD) fusion occurs less frequently and more slowly in pre-adipocytes expressing the AMD CIDEC rare variants. (A) Representative time-lapse images over 40 minutes of LDs in cells co-expressing GFP-tagged CIDEC WT (taken from Video 1 ) or each of the rare variants, and mCherry-tagged PLIN1. Scale bar: 2 μm. (B) Percentage of LDs undergoing fusion during the 6-hour analysis. N=3 (mean ± SD, Student’s t test, *p

Techniques Used: Expressing

AMD CIDEC variants in the CIDE-N domain decrease dimerization affinity and all four variants decrease binding to effector partners PLIN1, AS160 and RAB8A. (A) 3xflag-tagged CIDEC wild-type (WT) was co-transfected with the indicated GFP-tagged CIDEC variants in HEK 293T cells. GFP alone was used as negative control. 3xflag-tagged CIDEC WT was immuno-precipitated (IP) using anti-Flag and pulled-down proteins were immuno-blotted (IB) with anti-GFP and anti-Flag. Total cell lysate was immunoblotted with anti-GFP to control for CIDEC-GFP expression levels. (B-E) HEK 293T cells were co-transfected with 3xFlag-CIDEC WT, E186X or AMD variants, and either PLIN1-mCherry (B), AS160-GFP (C) or RAB8A-mCherry (D). After immunoprecipitation (IP) of the 3xFlag-CIDEC, pulled-down proteins were probed with anti-mCherry or anti-GFP, and anti-Flag. Co-transfection with mCherry or GFP alone was used as negative controls. Total cell lysates were immunoblotted (IB) with anti-mCherry or anti-GFP to control for PLIN1, AS160 and RAB8A expression levels. (E) Representative fluorescence images of 3T3-L1 pre-adipocytes lipid droplets containing CIDEC-GFP wild-type (WT) or variants and RAB8A-mCherry. Scale bar: 2 μm.
Figure Legend Snippet: AMD CIDEC variants in the CIDE-N domain decrease dimerization affinity and all four variants decrease binding to effector partners PLIN1, AS160 and RAB8A. (A) 3xflag-tagged CIDEC wild-type (WT) was co-transfected with the indicated GFP-tagged CIDEC variants in HEK 293T cells. GFP alone was used as negative control. 3xflag-tagged CIDEC WT was immuno-precipitated (IP) using anti-Flag and pulled-down proteins were immuno-blotted (IB) with anti-GFP and anti-Flag. Total cell lysate was immunoblotted with anti-GFP to control for CIDEC-GFP expression levels. (B-E) HEK 293T cells were co-transfected with 3xFlag-CIDEC WT, E186X or AMD variants, and either PLIN1-mCherry (B), AS160-GFP (C) or RAB8A-mCherry (D). After immunoprecipitation (IP) of the 3xFlag-CIDEC, pulled-down proteins were probed with anti-mCherry or anti-GFP, and anti-Flag. Co-transfection with mCherry or GFP alone was used as negative controls. Total cell lysates were immunoblotted (IB) with anti-mCherry or anti-GFP to control for PLIN1, AS160 and RAB8A expression levels. (E) Representative fluorescence images of 3T3-L1 pre-adipocytes lipid droplets containing CIDEC-GFP wild-type (WT) or variants and RAB8A-mCherry. Scale bar: 2 μm.

Techniques Used: Binding Assay, Transfection, Negative Control, Expressing, Immunoprecipitation, Cotransfection, Fluorescence

8) Product Images from "Defining totipotency using criteria of increasing stringency"

Article Title: Defining totipotency using criteria of increasing stringency

Journal: bioRxiv

doi: 10.1101/2020.03.02.972893

In vitro capacity and potential of candidate totipotent stem cells to give rise to trophoblast stem cells A) Differentiation of L-EPSCs to TSCs. 2iLif ESCs or L-EPSCs were subjected to TSC differentiation conditions. RNA was collected at D0, D3, D6, D9, D12 and qPCR was used to measure the expression of TE marker genes. B) Silencing of extra-embryonic gene expression was maintained in L-EPSCs exposed to TSC differentiation conditions. Expression of TSC markers: Elf5, Eomes, Tfap2c, Cdx2, Ascl2, Esx1 and Nanog as control at D3, D6, D9 and D12 of differentiation by RT-qPCR. C) Experimental design of ESC, L-EPSC or D-EPSC to TSC conversion (−4OH) or reprogramming experiments. ESC, L-EPSC or D-EPSC lines harbored a tamoxifen(4OH)-inducible Cdx2 transgene, were heterozygous for Oct4 ( Pou5f1 ) and contained an Elf5-2a-mCherry reporter. Cells switched to TSC media with or without 4OH were analyzed by flow cytometry on indicated days for TSC markers CD40, ELF5 and PLET1. D) Flow cytometry analysis of ESC, L-EPSC or D-EPSC cells switched to TSC media with or without 4OH on indicated days. Percentages of cells positive of each TSC marker, or cells positive for all three markers are shown. Three biological replicates were performed, error bars indicate standard deviation of mean. E) Flow cytometry analysis of TSCs containing an Elf5-2s-mCherry reporter. Percentages of cells positive of each TSC marker, or cells positive for all three markers are shown. Three biological replicates were performed, error bars indicate standard deviation of mean.
Figure Legend Snippet: In vitro capacity and potential of candidate totipotent stem cells to give rise to trophoblast stem cells A) Differentiation of L-EPSCs to TSCs. 2iLif ESCs or L-EPSCs were subjected to TSC differentiation conditions. RNA was collected at D0, D3, D6, D9, D12 and qPCR was used to measure the expression of TE marker genes. B) Silencing of extra-embryonic gene expression was maintained in L-EPSCs exposed to TSC differentiation conditions. Expression of TSC markers: Elf5, Eomes, Tfap2c, Cdx2, Ascl2, Esx1 and Nanog as control at D3, D6, D9 and D12 of differentiation by RT-qPCR. C) Experimental design of ESC, L-EPSC or D-EPSC to TSC conversion (−4OH) or reprogramming experiments. ESC, L-EPSC or D-EPSC lines harbored a tamoxifen(4OH)-inducible Cdx2 transgene, were heterozygous for Oct4 ( Pou5f1 ) and contained an Elf5-2a-mCherry reporter. Cells switched to TSC media with or without 4OH were analyzed by flow cytometry on indicated days for TSC markers CD40, ELF5 and PLET1. D) Flow cytometry analysis of ESC, L-EPSC or D-EPSC cells switched to TSC media with or without 4OH on indicated days. Percentages of cells positive of each TSC marker, or cells positive for all three markers are shown. Three biological replicates were performed, error bars indicate standard deviation of mean. E) Flow cytometry analysis of TSCs containing an Elf5-2s-mCherry reporter. Percentages of cells positive of each TSC marker, or cells positive for all three markers are shown. Three biological replicates were performed, error bars indicate standard deviation of mean.

Techniques Used: In Vitro, Real-time Polymerase Chain Reaction, Expressing, Marker, Quantitative RT-PCR, Flow Cytometry, Standard Deviation

9) Product Images from "Novel frameshift variant in MYL2 reveals molecular differences between dominant and recessive forms of hypertrophic cardiomyopathy"

Article Title: Novel frameshift variant in MYL2 reveals molecular differences between dominant and recessive forms of hypertrophic cardiomyopathy

Journal: bioRxiv

doi: 10.1101/790196

In vitro analysis of stability and localization of MYL2 variants. (A) Primary structure of MYL2 showing N-terminal C-terminal EF-hand domains, Serine 15 residue that is target of MLCK phosphorylation, three stopgain variants (red octagons), the frameshift variant identified in the proband (red block showing extension of C-terminal end) and missense variant associated with HCM (orange oval) that maps the C-terminal domain. The frameshift mutation changes the canonical MYL2 sequence (‘Ref.’, black text) from residue 144 onwards, modifying the last 20 amino acids of the protein in addition to adding 36 non-canonical amino acids to the C-terminus (‘fs’, red text). (B) Western blot image showing overexpression of EGFP-tagged MYL2 wt and variant cDNAs. The stability of the frameshift variant is significantly affected while other loss-of-function variants did not show a significant reduction in stability. mCherry signal analyzed using western blot does not show any significant changes between MYL2 wt and variant sequences. Loading controls: mCherry and Actin. (C) Immunofluorescence analysis of EGFP tagged MYL2 wt and variants (green) co-overexpressing mCherry from the same transcript (red) in H9c2 cells. EGFP tagged MYL2-wt localizes to the cytoskeleton, while stopgain variants (E22*, K62*, E97*) do not localize to the cytoskeleton and are observed in a diffuse pattern. EGFP signal from the frameshift variant (fs) is significantly reduced. Localization of the missense (G162R) variant is in a pattern similar to the wild type. mCherry signal from the transfected cells does not change between variants. (D) Immunofluorescence images of EGFP tagged MYL2 wt and variants transfected and MG-132 treated H9c2 cells showing rescue of MYL2-fs variant signal following MG-132 treatment. Scale bar indicates 50 uM length. Quantitation of signal from the western and immunofluorescence are provided in Fig S3.
Figure Legend Snippet: In vitro analysis of stability and localization of MYL2 variants. (A) Primary structure of MYL2 showing N-terminal C-terminal EF-hand domains, Serine 15 residue that is target of MLCK phosphorylation, three stopgain variants (red octagons), the frameshift variant identified in the proband (red block showing extension of C-terminal end) and missense variant associated with HCM (orange oval) that maps the C-terminal domain. The frameshift mutation changes the canonical MYL2 sequence (‘Ref.’, black text) from residue 144 onwards, modifying the last 20 amino acids of the protein in addition to adding 36 non-canonical amino acids to the C-terminus (‘fs’, red text). (B) Western blot image showing overexpression of EGFP-tagged MYL2 wt and variant cDNAs. The stability of the frameshift variant is significantly affected while other loss-of-function variants did not show a significant reduction in stability. mCherry signal analyzed using western blot does not show any significant changes between MYL2 wt and variant sequences. Loading controls: mCherry and Actin. (C) Immunofluorescence analysis of EGFP tagged MYL2 wt and variants (green) co-overexpressing mCherry from the same transcript (red) in H9c2 cells. EGFP tagged MYL2-wt localizes to the cytoskeleton, while stopgain variants (E22*, K62*, E97*) do not localize to the cytoskeleton and are observed in a diffuse pattern. EGFP signal from the frameshift variant (fs) is significantly reduced. Localization of the missense (G162R) variant is in a pattern similar to the wild type. mCherry signal from the transfected cells does not change between variants. (D) Immunofluorescence images of EGFP tagged MYL2 wt and variants transfected and MG-132 treated H9c2 cells showing rescue of MYL2-fs variant signal following MG-132 treatment. Scale bar indicates 50 uM length. Quantitation of signal from the western and immunofluorescence are provided in Fig S3.

Techniques Used: In Vitro, Variant Assay, Blocking Assay, Mutagenesis, Sequencing, Western Blot, Over Expression, Immunofluorescence, Transfection, Quantitation Assay

In vivo functional analysis of MYL2 variants using Drosophila Mlc2 substitution assay in the heart. (A) Schematics showing Hand-enhancer driven GAL4 (Hand-GAL4) used to knockdown Mlc2 expression using RNAi in the heart, and to overexpress human MYL2-wt and variant cDNAs for functional substitution. In parallel, the Hand-GAL4 drives the expression of UAS-CD8::mCherry which facilitates visualization of the heart. (B-top) Immunofluorescence image of the Drosophila heart from UAS-CD8::mCherry; Hand-GAL4 animals showing expression of mCherry in the heart chambers and aorta. (B-bottom) Magnified image of the A7-A8 posterior denticle band region showing expression of mCherry in both cardiomyocytes as well as pericardial cells. Merged imaged on the right shows cardiac cells marked by Pericardin expression (green), mCherry driven by Hand-GAL4 (red) and Phalloidin staining of actin (blue) marking both cardiomyocytes and skeletal muscle. No expression of mCherry was detected in the skeletal muscle. (C) Histogram showing developmental lethality due to Mlc2 RNAi knockdown using Hand-GAL4 and rescue by overexpression of human MYL2-wt and variants. Partial rescue of the lethality is observed with wildtype MYL2 cDNA overexpression while no significant difference is observed for the tested variants. Percentage of adult survivors calculated based on the fraction of adults with the obtained desired genotype compared to Mendelian expected ratio for each cross. Significance analyzed using Brown-Forsythe and Welch ANOVA across all samples followed by multiple comparisons using Games-Howell multiple comparison test. Significance indicated using * adj. P-Value
Figure Legend Snippet: In vivo functional analysis of MYL2 variants using Drosophila Mlc2 substitution assay in the heart. (A) Schematics showing Hand-enhancer driven GAL4 (Hand-GAL4) used to knockdown Mlc2 expression using RNAi in the heart, and to overexpress human MYL2-wt and variant cDNAs for functional substitution. In parallel, the Hand-GAL4 drives the expression of UAS-CD8::mCherry which facilitates visualization of the heart. (B-top) Immunofluorescence image of the Drosophila heart from UAS-CD8::mCherry; Hand-GAL4 animals showing expression of mCherry in the heart chambers and aorta. (B-bottom) Magnified image of the A7-A8 posterior denticle band region showing expression of mCherry in both cardiomyocytes as well as pericardial cells. Merged imaged on the right shows cardiac cells marked by Pericardin expression (green), mCherry driven by Hand-GAL4 (red) and Phalloidin staining of actin (blue) marking both cardiomyocytes and skeletal muscle. No expression of mCherry was detected in the skeletal muscle. (C) Histogram showing developmental lethality due to Mlc2 RNAi knockdown using Hand-GAL4 and rescue by overexpression of human MYL2-wt and variants. Partial rescue of the lethality is observed with wildtype MYL2 cDNA overexpression while no significant difference is observed for the tested variants. Percentage of adult survivors calculated based on the fraction of adults with the obtained desired genotype compared to Mendelian expected ratio for each cross. Significance analyzed using Brown-Forsythe and Welch ANOVA across all samples followed by multiple comparisons using Games-Howell multiple comparison test. Significance indicated using * adj. P-Value

Techniques Used: In Vivo, Functional Assay, Expressing, Variant Assay, Immunofluorescence, Staining, Over Expression

10) Product Images from "A dual role for cell plate‐associated PI4Kβ in endocytosis and phragmoplast dynamics during plant somatic cytokinesis"

Article Title: A dual role for cell plate‐associated PI4Kβ in endocytosis and phragmoplast dynamics during plant somatic cytokinesis

Journal: The EMBO Journal

doi: 10.15252/embj.2018100303

Functional association of PI4Kβ1 and MPK4 in a protein complex at the cell plate Experiments were performed to assess possible physical interaction, co‐localization at the cell plate, and functional interplay of PI4Kβ1 and MPK4. PI4Kβ1 interacts with MPK4 in yeast two‐hybrid tests. MPK4 was used as a bait, while PI4Kβ1 was divided into two fragments to be used as prey. The results are representative for three independent experiments. In vivo co‐immunoprecipitation of MPK4 and PI4Kβ1 from 14‐day‐old plants. PI4Kβ1 was specifically co‐precipitated with MPK4‐myc using anti‐myc antibodies. Arrowhead, Migration of PI4Kβ1. The results are representative for three independent experiments. Coordination of MPK4‐YFP and mCherry‐PI4Kβ1 during cytokinesis in 5‐day‐old root meristem cells. MPK4 co‐localized with PI4Kβ1 from early to late cytokinesis and gradually concentrated at the leading edges of cell plates. Images were obtained with an LSM880 in Airyscan super‐resolution (SR) mode. Scale bars, 10 μm. Live‐cell time lapse of MPK4‐YFP and mRFP FAPP1‐PH during cytokinesis, obtained with an LSM880 in Airyscan resolution versus sensitivity (R‐S) mode. The time series is representative for six cells from five roots. Arrowheads indicate the initiation and leading edges of the growing cell plate. Scale bar, 10 μm. Left panels: The distribution of mRFP FAPP1‐PH was observed at the cell plate at the end of cytokinesis in root meristem cells of wild‐type controls (top) and mpk4‐2 mutants (bottom). Intensity profiles were recorded as indicated by the dashed lines. Scale bars, 10 μm. Right top, intensity of mRFP FAPP1‐PH at the cell plate normalized vs. the intensity at the apical plasma membrane (wild type, n = 43 cells, 34 roots; mpk4‐2 , n = 32 cells, 28 roots). Right bottom, intensity of mRFP FAPP1‐PH at the TGN normalized vs. the intensity at the apical plasma membrane (wild type, n = 40 cells, 29 roots; mpk4‐2 , n = 26 cells, 23 roots). *, a significant difference ( P
Figure Legend Snippet: Functional association of PI4Kβ1 and MPK4 in a protein complex at the cell plate Experiments were performed to assess possible physical interaction, co‐localization at the cell plate, and functional interplay of PI4Kβ1 and MPK4. PI4Kβ1 interacts with MPK4 in yeast two‐hybrid tests. MPK4 was used as a bait, while PI4Kβ1 was divided into two fragments to be used as prey. The results are representative for three independent experiments. In vivo co‐immunoprecipitation of MPK4 and PI4Kβ1 from 14‐day‐old plants. PI4Kβ1 was specifically co‐precipitated with MPK4‐myc using anti‐myc antibodies. Arrowhead, Migration of PI4Kβ1. The results are representative for three independent experiments. Coordination of MPK4‐YFP and mCherry‐PI4Kβ1 during cytokinesis in 5‐day‐old root meristem cells. MPK4 co‐localized with PI4Kβ1 from early to late cytokinesis and gradually concentrated at the leading edges of cell plates. Images were obtained with an LSM880 in Airyscan super‐resolution (SR) mode. Scale bars, 10 μm. Live‐cell time lapse of MPK4‐YFP and mRFP FAPP1‐PH during cytokinesis, obtained with an LSM880 in Airyscan resolution versus sensitivity (R‐S) mode. The time series is representative for six cells from five roots. Arrowheads indicate the initiation and leading edges of the growing cell plate. Scale bar, 10 μm. Left panels: The distribution of mRFP FAPP1‐PH was observed at the cell plate at the end of cytokinesis in root meristem cells of wild‐type controls (top) and mpk4‐2 mutants (bottom). Intensity profiles were recorded as indicated by the dashed lines. Scale bars, 10 μm. Right top, intensity of mRFP FAPP1‐PH at the cell plate normalized vs. the intensity at the apical plasma membrane (wild type, n = 43 cells, 34 roots; mpk4‐2 , n = 32 cells, 28 roots). Right bottom, intensity of mRFP FAPP1‐PH at the TGN normalized vs. the intensity at the apical plasma membrane (wild type, n = 40 cells, 29 roots; mpk4‐2 , n = 26 cells, 23 roots). *, a significant difference ( P

Techniques Used: Functional Assay, In Vivo, Immunoprecipitation, Migration

Cytokinetic defects of the Arabidopsis thaliana pi4kβ1 pi4kβ2 double mutant The Arabidopsis pi4kβ1 pi4kβ2 double mutant displays a growth defect and cytokinetic defects. The growth phenotype of the Arabidopsis pi4kβ1 pi4kβ2 double mutant can be complemented by ectopic expression of PI4Kβ1 or PI4Kβ2 or by mCherry‐tagged PI4Kβ1 expressed from a genomic fragment. Plants shown are 1 month old. Scale bar, 10 cm. The irregularities in the pattern of root epidermal cell division orientation of the pi4kβ1 pi4kβ2 double mutant are also complemented by ectopic expression of PI4Kβ variants, as shown by propidium iodide (PI) staining of 5‐day‐old roots. Arrowhead, oblique cell wall. Insets, magnifications of areas showing cell wall stubs. Scale bars, 50 μm. Quantifications of cell wall stubs, oblique cell walls and root meristem size (wild type, n = 15 roots; pi4kβ1 , n = 15 roots; pi4kβ2 , n = 14 roots; pi4kβ1 pi4kβ2 , n = 18 roots; pPI4Kβ1:PI4Kβ1 , n = 14 roots, pPI4Kβ2:PI4Kβ2 , n = 15 roots). n.d., not detected. Data are mean ± SD. Lowercase letters indicate a significant increase of oblique cell walls (Welch ANOVA with Games‐Howell post hoc test; P
Figure Legend Snippet: Cytokinetic defects of the Arabidopsis thaliana pi4kβ1 pi4kβ2 double mutant The Arabidopsis pi4kβ1 pi4kβ2 double mutant displays a growth defect and cytokinetic defects. The growth phenotype of the Arabidopsis pi4kβ1 pi4kβ2 double mutant can be complemented by ectopic expression of PI4Kβ1 or PI4Kβ2 or by mCherry‐tagged PI4Kβ1 expressed from a genomic fragment. Plants shown are 1 month old. Scale bar, 10 cm. The irregularities in the pattern of root epidermal cell division orientation of the pi4kβ1 pi4kβ2 double mutant are also complemented by ectopic expression of PI4Kβ variants, as shown by propidium iodide (PI) staining of 5‐day‐old roots. Arrowhead, oblique cell wall. Insets, magnifications of areas showing cell wall stubs. Scale bars, 50 μm. Quantifications of cell wall stubs, oblique cell walls and root meristem size (wild type, n = 15 roots; pi4kβ1 , n = 15 roots; pi4kβ2 , n = 14 roots; pi4kβ1 pi4kβ2 , n = 18 roots; pPI4Kβ1:PI4Kβ1 , n = 14 roots, pPI4Kβ2:PI4Kβ2 , n = 15 roots). n.d., not detected. Data are mean ± SD. Lowercase letters indicate a significant increase of oblique cell walls (Welch ANOVA with Games‐Howell post hoc test; P

Techniques Used: Mutagenesis, Expressing, Staining

PI4Kβ1 localization at the cell plate and the TGN during cytokinesis in Arabidopsis root cells In vivo localization of mCherry‐PI4Kβ1 expressed from the pPI4Kβ1 promoter in root tips of 5‐day‐old complemented pi4kβ1 pi4kβ2 plants. The mCherry‐PI4Kβ1 distribution was imaged using a Zeiss LSM880 in Airyscan Virtual Pinhole (VP) mode with the pinhole set to 2. Arrowheads, nascent cell plates decorated by mCherry‐PI4Kβ1. Scale bar, 20 μm. Whole‐mount immunostaining of 5‐day‐old seedlings expressing mCherry‐PI4kβ1 in the pi4kβ1 pi4kβ2 double mutant background using anti‐tubulin (red) and anti‐mCherry (green) antibodies, and DAPI (blue). (I), (II), magnifications of regions marked in b, representing early and late cytokinetic stages. Scale bars, 20 μm. .
Figure Legend Snippet: PI4Kβ1 localization at the cell plate and the TGN during cytokinesis in Arabidopsis root cells In vivo localization of mCherry‐PI4Kβ1 expressed from the pPI4Kβ1 promoter in root tips of 5‐day‐old complemented pi4kβ1 pi4kβ2 plants. The mCherry‐PI4Kβ1 distribution was imaged using a Zeiss LSM880 in Airyscan Virtual Pinhole (VP) mode with the pinhole set to 2. Arrowheads, nascent cell plates decorated by mCherry‐PI4Kβ1. Scale bar, 20 μm. Whole‐mount immunostaining of 5‐day‐old seedlings expressing mCherry‐PI4kβ1 in the pi4kβ1 pi4kβ2 double mutant background using anti‐tubulin (red) and anti‐mCherry (green) antibodies, and DAPI (blue). (I), (II), magnifications of regions marked in b, representing early and late cytokinetic stages. Scale bars, 20 μm. .

Techniques Used: In Vivo, Immunostaining, Expressing, Mutagenesis

Disordered phragmoplast dynamics in cytokinetic root cells of the Arabidopsis pi4kβ1 pi4kβ2 double mutant The dynamic transitions of phragmoplast microtubules were recorded by live‐cell time‐lapse imaging. Five‐day‐old seedlings expressing mCherry‐TUA5 were imaged at 1 frame per 2 min. Times are given relative to the instance when the cell plate contacted the peripheral plasma membrane, defined as t0. Cells are outlined by dashed lines in the first and last frames of each series. White arrowhead, ectopic stabilization of microtubules in central phragmoplasts of the pi4kβ1 pi4kβ2 double mutant. Scale bars, 10 μm. Plot profiles obtained from dashed lines marked in (A). Black arrowhead, ectopic stabilization of microtubules in central phragmoplasts of the pi4kβ1 pi4kβ2 double mutant. 3D projections from time points selected from (A), when the transition to a ring phragmoplast has occurred in wild type, but solid phragmoplast persisted in the double mutant. Scale bars, 10 μm. Duration of phragmoplast persistence during cytokinesis, determined from initiation to disbanding. ***, a significant difference ( P
Figure Legend Snippet: Disordered phragmoplast dynamics in cytokinetic root cells of the Arabidopsis pi4kβ1 pi4kβ2 double mutant The dynamic transitions of phragmoplast microtubules were recorded by live‐cell time‐lapse imaging. Five‐day‐old seedlings expressing mCherry‐TUA5 were imaged at 1 frame per 2 min. Times are given relative to the instance when the cell plate contacted the peripheral plasma membrane, defined as t0. Cells are outlined by dashed lines in the first and last frames of each series. White arrowhead, ectopic stabilization of microtubules in central phragmoplasts of the pi4kβ1 pi4kβ2 double mutant. Scale bars, 10 μm. Plot profiles obtained from dashed lines marked in (A). Black arrowhead, ectopic stabilization of microtubules in central phragmoplasts of the pi4kβ1 pi4kβ2 double mutant. 3D projections from time points selected from (A), when the transition to a ring phragmoplast has occurred in wild type, but solid phragmoplast persisted in the double mutant. Scale bars, 10 μm. Duration of phragmoplast persistence during cytokinesis, determined from initiation to disbanding. ***, a significant difference ( P

Techniques Used: Mutagenesis, Imaging, Expressing

11) Product Images from "Regulation of mitochondrial iron homeostasis by sideroflexin 2"

Article Title: Regulation of mitochondrial iron homeostasis by sideroflexin 2

Journal: The Journal of Physiological Sciences

doi: 10.1007/s12576-018-0652-2

Mitochondrial localization of SFXN2. mCherry-SFXN2 ( a ) and SFXN2-mCherry ( b ) colocalized with MitoTracker. Bar = 5 μm. mCherry-SFXN2 ( c ) and SFXN2-mCherry ( d ) colocalized with MitoTracker and endogenous Tomm20. Arrows indicate mCherry-SFXN2 or SFXN2-mCherry, while arrowheads indicate Tomm20. The fluorescence intensities along the dashed lines are shown as line profile graphs. Bars = 1 μm. e Mitochondria were isolated from HEK293 cells transfected with SFXN2-mCherry and then digested with trypsin. SFXN2, Mitofusin1, and Timm50 were detected by Western blotting. The arrow indicates bands corresponding to SFXN2-mCherry. f Quantification of SFXN2-mCherry, Mitofusin1, and Timm50. n = 3–4 each. * p
Figure Legend Snippet: Mitochondrial localization of SFXN2. mCherry-SFXN2 ( a ) and SFXN2-mCherry ( b ) colocalized with MitoTracker. Bar = 5 μm. mCherry-SFXN2 ( c ) and SFXN2-mCherry ( d ) colocalized with MitoTracker and endogenous Tomm20. Arrows indicate mCherry-SFXN2 or SFXN2-mCherry, while arrowheads indicate Tomm20. The fluorescence intensities along the dashed lines are shown as line profile graphs. Bars = 1 μm. e Mitochondria were isolated from HEK293 cells transfected with SFXN2-mCherry and then digested with trypsin. SFXN2, Mitofusin1, and Timm50 were detected by Western blotting. The arrow indicates bands corresponding to SFXN2-mCherry. f Quantification of SFXN2-mCherry, Mitofusin1, and Timm50. n = 3–4 each. * p

Techniques Used: Fluorescence, Isolation, Transfection, Western Blot

Mitochondrial targeting signal in SFXN2. The N-terminus ( a ) or C-terminus ( b ) of SFXN2 was conjugated to mCherry and then expressed in HeLa cells. SFXN2 Nterm -mCherry and mCherry-SFXN2 Cterm were diffusely distributed. c SFXN2 was truncated at the end of the second transmembrane domain and conjugated to mCherry. The fusion protein colocalized with MitoTracker. d A SFXN2 fragment containing the N-terminus and the first transmembrane domain was conjugated to mCherry. The fusion protein colocalized with mitochondria. Bars = 5 μm. e Pearson’s colocalization coefficient was calculated to examine the extent of colocalization between the fluorescence of MitoTracker and the fluorescence of each chimeric protein ( a–d ). n = 5 for SFXN2 Nterm -mCherry (Nterm) and mCherry-SFXN2 Cterm (Cterm), n = 16 for SFXN2 N-TM2 -mCherry (TM2), and n = 14 for SFXN2 N-TM1 -mCherry (TM1). * p
Figure Legend Snippet: Mitochondrial targeting signal in SFXN2. The N-terminus ( a ) or C-terminus ( b ) of SFXN2 was conjugated to mCherry and then expressed in HeLa cells. SFXN2 Nterm -mCherry and mCherry-SFXN2 Cterm were diffusely distributed. c SFXN2 was truncated at the end of the second transmembrane domain and conjugated to mCherry. The fusion protein colocalized with MitoTracker. d A SFXN2 fragment containing the N-terminus and the first transmembrane domain was conjugated to mCherry. The fusion protein colocalized with mitochondria. Bars = 5 μm. e Pearson’s colocalization coefficient was calculated to examine the extent of colocalization between the fluorescence of MitoTracker and the fluorescence of each chimeric protein ( a–d ). n = 5 for SFXN2 Nterm -mCherry (Nterm) and mCherry-SFXN2 Cterm (Cterm), n = 16 for SFXN2 N-TM2 -mCherry (TM2), and n = 14 for SFXN2 N-TM1 -mCherry (TM1). * p

Techniques Used: Fluorescence

12) Product Images from "Sorting nexin-27 regulates AMPA receptor trafficking through the synaptic adhesion protein LRFN2"

Article Title: Sorting nexin-27 regulates AMPA receptor trafficking through the synaptic adhesion protein LRFN2

Journal: bioRxiv

doi: 10.1101/2020.04.27.063248

LRFNs interact with AMPA receptors and regulate their membrane trafficking A , Immunofluorescence staining of endogenous surface GluA1 and GluA2 in DIV20 rat cortical neurons transduced with mCherry-LRFN2 expressing sindbis virus. A mCherry antibody was used to stain for surface LRFN2 expression (shown in red). Scale bars, 20 μm. White boxes indicate the 20 μm section zoomed in. B , Representative fluorescence intensity plots shown of the 20 μm zoomed in sections for surface LRFN2 and GluA1 in (A; region i). C , Representative fluorescence intensity plots shown of the 20 μm zoomed in sections for surface LRFN2 and GluA2 in (A; region iii). D , Fluorescence-based western analysis after GFP-Trap immunoprecipitation of full-length SEP-GluA1 or SEP-GluA2 co-expressed with full length mCherry-LRFN1, mCherry-LRFN2 or mCherry-LRFN4 in HEK293T cells. E , Schematic of LRFN2 constructs mCherry-LRFN2 FL (full-length) and N-terminal mutants. LRR, leucine rich repeat; NT/CT, N/C-terminal domains of LRR; Ig, immunoglobulin domain; FNIII, fibronectin type-III; TM, transmembrane; PDZbm, PDZ binding motif. F , Fluorescence-based western analysis after RFP-Trap immunoprecipitation of mCherry-LRFN2 wild-type (LRFN2 FL) or N-terminal mutants co-expressed with full-length SEP-GluA1 or myc-GluA2 in HEK293T cells.
Figure Legend Snippet: LRFNs interact with AMPA receptors and regulate their membrane trafficking A , Immunofluorescence staining of endogenous surface GluA1 and GluA2 in DIV20 rat cortical neurons transduced with mCherry-LRFN2 expressing sindbis virus. A mCherry antibody was used to stain for surface LRFN2 expression (shown in red). Scale bars, 20 μm. White boxes indicate the 20 μm section zoomed in. B , Representative fluorescence intensity plots shown of the 20 μm zoomed in sections for surface LRFN2 and GluA1 in (A; region i). C , Representative fluorescence intensity plots shown of the 20 μm zoomed in sections for surface LRFN2 and GluA2 in (A; region iii). D , Fluorescence-based western analysis after GFP-Trap immunoprecipitation of full-length SEP-GluA1 or SEP-GluA2 co-expressed with full length mCherry-LRFN1, mCherry-LRFN2 or mCherry-LRFN4 in HEK293T cells. E , Schematic of LRFN2 constructs mCherry-LRFN2 FL (full-length) and N-terminal mutants. LRR, leucine rich repeat; NT/CT, N/C-terminal domains of LRR; Ig, immunoglobulin domain; FNIII, fibronectin type-III; TM, transmembrane; PDZbm, PDZ binding motif. F , Fluorescence-based western analysis after RFP-Trap immunoprecipitation of mCherry-LRFN2 wild-type (LRFN2 FL) or N-terminal mutants co-expressed with full-length SEP-GluA1 or myc-GluA2 in HEK293T cells.

Techniques Used: Immunofluorescence, Staining, Transduction, Expressing, Fluorescence, Western Blot, Immunoprecipitation, Construct, Binding Assay

The membrane trafficking of LRFN2 is dependent on SNX27. A , Immunofluorescence staining of DIV20 rat hippocampal neurons co-transduced with mCherry-LRFN2 and GFP-SNX27 expressing sindbis viruses. Scale bars, 20 μm. White boxes indicate the 20 μm section zoomed in. B , Representative fluorescence intensity plot shown of the 20 μm zoomed in section from (i). C-D , Fluorescence-based western analysis of DIV19 rat cortical neurons transduced with either a control or SNX27 shRNA for: C , endogenous SNX27 D , endogenous LRFN2. Actin was used as a protein load control. Quantification from five independent experiments (N = 5). Data expressed as a percentage of the control shRNA and analysed by an unpaired t-test. E-F , Fluorescence-based western analysis after surface biotinylation and streptavidin agarose capture of membrane proteins of DIV19 rat cortical neurons transduced with either a control or SNX27 shRNA for: E , endogenous surface GluA1 and GluA2. Total levels of endogenous SNX27 are also shown. Quantification from four independent experiments (N = 4). Data expressed as a percentage of the control shRNA and analysed by an unpaired t-test. F , endogenous surface LRFN2. Total levels of endogenous SNX27 are also shown. Quantification from six independent experiments (N = 6). Data expressed as a percentage of the control shRNA and analysed by an unpaired t-test. In all figures error bars represent mean ± SEM. ***, P≤0.001; **, P≤0.01.
Figure Legend Snippet: The membrane trafficking of LRFN2 is dependent on SNX27. A , Immunofluorescence staining of DIV20 rat hippocampal neurons co-transduced with mCherry-LRFN2 and GFP-SNX27 expressing sindbis viruses. Scale bars, 20 μm. White boxes indicate the 20 μm section zoomed in. B , Representative fluorescence intensity plot shown of the 20 μm zoomed in section from (i). C-D , Fluorescence-based western analysis of DIV19 rat cortical neurons transduced with either a control or SNX27 shRNA for: C , endogenous SNX27 D , endogenous LRFN2. Actin was used as a protein load control. Quantification from five independent experiments (N = 5). Data expressed as a percentage of the control shRNA and analysed by an unpaired t-test. E-F , Fluorescence-based western analysis after surface biotinylation and streptavidin agarose capture of membrane proteins of DIV19 rat cortical neurons transduced with either a control or SNX27 shRNA for: E , endogenous surface GluA1 and GluA2. Total levels of endogenous SNX27 are also shown. Quantification from four independent experiments (N = 4). Data expressed as a percentage of the control shRNA and analysed by an unpaired t-test. F , endogenous surface LRFN2. Total levels of endogenous SNX27 are also shown. Quantification from six independent experiments (N = 6). Data expressed as a percentage of the control shRNA and analysed by an unpaired t-test. In all figures error bars represent mean ± SEM. ***, P≤0.001; **, P≤0.01.

Techniques Used: Immunofluorescence, Staining, Transduction, Expressing, Fluorescence, Western Blot, shRNA

13) Product Images from "An Excitatory and Epileptogenic Effect of Dentate Gyrus Mossy Cells in a Mouse Model of Epilepsy"

Article Title: An Excitatory and Epileptogenic Effect of Dentate Gyrus Mossy Cells in a Mouse Model of Epilepsy

Journal: Cell reports

doi: 10.1016/j.celrep.2019.10.100

Inhibiting MCs with iDREADDs Attenuates Pilocarpine-Induced SE (A1) Timeline. Mice underwent surgery for viral injection and implant of an EEG recording assembly. SE was induced 2–4 weeks later with the convulsant pilocarpine (day 1). EEG was conducted 24 h later (day 2; see Figure 3 ), neuronal injury was assessed in a subset of mice on day 3 (see Figure 4 ), and the remaining mice were recorded by vEEG for 2 weeks to quantify chronic seizures (see Figure 5 ). (A2) Expanded day 1 timeline shows the sequence of procedures in more detail. (B) The Cre-dependent viral construct used in iDREADDs experiments was AAV-hSyn-DIO-hM4D(Gi)-mCherry. (C1) Schematic of the hippocampus. (C2) Virus was injected into the dorsal and ventral hippocampus, bilaterally. (D) Subdural screw electrodes were positioned over the left frontal cortex (FC), the left and right dorsal hippocampus (HC), and the right occipital cortex (OC) for vEEG recordings. (E) Representative 6-h EEG recording showing SE onset (the first 30 min of SE). After a ~1-h baseline period, all mice were injected with CNO, followed by pilocarpine 30 min later. Seizures began within the next 60 min and lasted several hours. (F1) There were no group differences in the latency to the first electrographic seizure. (F2) The latency to the first convulsive seizure was significantly delayed in iDREADDs compared to Cre −/− mice. (G1) There were no group differences in baseline EEG power. (G2) MC inhibition selectively increased theta power during the CNO period. (G3) MC inhibition reduced theta, alpha, beta, and gamma power during SE onset. (H1 and H2) Representative 2-h EEG record and corresponding spectrograms showing the pilocarpine injection and SE onset. Note that EEG power was reduced in iDREADDs relative to Cre −/− mice during SE onset. Data are represented as mean ± SEM. *p
Figure Legend Snippet: Inhibiting MCs with iDREADDs Attenuates Pilocarpine-Induced SE (A1) Timeline. Mice underwent surgery for viral injection and implant of an EEG recording assembly. SE was induced 2–4 weeks later with the convulsant pilocarpine (day 1). EEG was conducted 24 h later (day 2; see Figure 3 ), neuronal injury was assessed in a subset of mice on day 3 (see Figure 4 ), and the remaining mice were recorded by vEEG for 2 weeks to quantify chronic seizures (see Figure 5 ). (A2) Expanded day 1 timeline shows the sequence of procedures in more detail. (B) The Cre-dependent viral construct used in iDREADDs experiments was AAV-hSyn-DIO-hM4D(Gi)-mCherry. (C1) Schematic of the hippocampus. (C2) Virus was injected into the dorsal and ventral hippocampus, bilaterally. (D) Subdural screw electrodes were positioned over the left frontal cortex (FC), the left and right dorsal hippocampus (HC), and the right occipital cortex (OC) for vEEG recordings. (E) Representative 6-h EEG recording showing SE onset (the first 30 min of SE). After a ~1-h baseline period, all mice were injected with CNO, followed by pilocarpine 30 min later. Seizures began within the next 60 min and lasted several hours. (F1) There were no group differences in the latency to the first electrographic seizure. (F2) The latency to the first convulsive seizure was significantly delayed in iDREADDs compared to Cre −/− mice. (G1) There were no group differences in baseline EEG power. (G2) MC inhibition selectively increased theta power during the CNO period. (G3) MC inhibition reduced theta, alpha, beta, and gamma power during SE onset. (H1 and H2) Representative 2-h EEG record and corresponding spectrograms showing the pilocarpine injection and SE onset. Note that EEG power was reduced in iDREADDs relative to Cre −/− mice during SE onset. Data are represented as mean ± SEM. *p

Techniques Used: Mouse Assay, Injection, Sequencing, Construct, Inhibition

Inhibiting MCs during SE Reduces Neuronal Injury (A) Timeline of the entire study showing the timing of day 3, when data in the figure were taken. (B) Schematic of the hippocampus and sectioning planes. The dorsal hippocampus was evaluated with coronal sections, whereas the ventral hippocampus was evaluated using horizontal sections. (C and D) Representative dorsal (C) and ventral (D) hM4D(Gi)-mCherry and FluoroJade photomicrographs in Cre −/− and iDREADDs mice 3 days after SE. Note the viral expression in the IML (arrow) and hilus in iDREADDs mice, but not Cre −/− . Viral expression coincided with reduced FluoroJade staining, suggesting that there was less hilar and CA3 neurodegeneration when MCs were inhibited during SE. Scale bars: 200 μm, 500 μm, and 50 μm (insets). (E1a and E2a) Quantification of iDREADDs mouse sections revealed significantly fewer hilar FluoroJade-stained cells compared to Cre −/− . (E1b and E2b) There was a smaller area of the CA3 cell layer stained by FluoroJade (arrows) in iDREADDs compared to Cre −/− mice. (E1c and E2c) FluoroJade staining did not significantly differ between Cre −/− and iDREADDs mice in CA1. d. FluoroJade staining in the GCL was minimal in both groups. Data are represented as mean ± SEM. *p
Figure Legend Snippet: Inhibiting MCs during SE Reduces Neuronal Injury (A) Timeline of the entire study showing the timing of day 3, when data in the figure were taken. (B) Schematic of the hippocampus and sectioning planes. The dorsal hippocampus was evaluated with coronal sections, whereas the ventral hippocampus was evaluated using horizontal sections. (C and D) Representative dorsal (C) and ventral (D) hM4D(Gi)-mCherry and FluoroJade photomicrographs in Cre −/− and iDREADDs mice 3 days after SE. Note the viral expression in the IML (arrow) and hilus in iDREADDs mice, but not Cre −/− . Viral expression coincided with reduced FluoroJade staining, suggesting that there was less hilar and CA3 neurodegeneration when MCs were inhibited during SE. Scale bars: 200 μm, 500 μm, and 50 μm (insets). (E1a and E2a) Quantification of iDREADDs mouse sections revealed significantly fewer hilar FluoroJade-stained cells compared to Cre −/− . (E1b and E2b) There was a smaller area of the CA3 cell layer stained by FluoroJade (arrows) in iDREADDs compared to Cre −/− mice. (E1c and E2c) FluoroJade staining did not significantly differ between Cre −/− and iDREADDs mice in CA1. d. FluoroJade staining in the GCL was minimal in both groups. Data are represented as mean ± SEM. *p

Techniques Used: Mouse Assay, Expressing, Staining

14) Product Images from "The ER cargo receptor SURF4 facilitates efficient erythropoietin secretion"

Article Title: The ER cargo receptor SURF4 facilitates efficient erythropoietin secretion

Journal: bioRxiv

doi: 10.1101/866954

Thrombopoietin secretion does not depend on SURF4. ( A ) A construct that expresses TPO-eGFP and A1AT-mCherry from the same CMV promoter was assembled and used to generate a reporter cell line stably expressing these two fusion proteins. ( B ) Intracellular and extracellular TPO-eGFP and A1AT-mCherry protein abundance was determined by Western blot using anti-eGFP and anti-mCherry antibodies, respectively. ( C ) Flow cytometry histograms showing absence of intracellular accumulation of TPO following SURF4 deletion in HEK293T cells. ( D ) Quantification of cellular mean flourescence intensity of TPO-eGFP and A1AT-mCherry in cells transduced with SURF4 -targeting sgRNAs (n=29). Results were normalized to mean flourescence intensity of cells transduced with non-targeting sgRNAs. As a positive control, the same experiment was performed in parallel in reporter cell lines expressing EPO-eGFP and A1AT-mCherry (n=48). **** p
Figure Legend Snippet: Thrombopoietin secretion does not depend on SURF4. ( A ) A construct that expresses TPO-eGFP and A1AT-mCherry from the same CMV promoter was assembled and used to generate a reporter cell line stably expressing these two fusion proteins. ( B ) Intracellular and extracellular TPO-eGFP and A1AT-mCherry protein abundance was determined by Western blot using anti-eGFP and anti-mCherry antibodies, respectively. ( C ) Flow cytometry histograms showing absence of intracellular accumulation of TPO following SURF4 deletion in HEK293T cells. ( D ) Quantification of cellular mean flourescence intensity of TPO-eGFP and A1AT-mCherry in cells transduced with SURF4 -targeting sgRNAs (n=29). Results were normalized to mean flourescence intensity of cells transduced with non-targeting sgRNAs. As a positive control, the same experiment was performed in parallel in reporter cell lines expressing EPO-eGFP and A1AT-mCherry (n=48). **** p

Techniques Used: Construct, Stable Transfection, Expressing, Western Blot, Flow Cytometry, Transduction, Positive Control

CRISPR/Cas9 loss-of-function screen to identify genes that affect intracellular EPO levels. ( A ) Screen strategy: 24 hours following transduction of the CRISPR library, puromycin selection was applied for 3 days. At day 14, cells with unchanged mCherry but with top or bottom 7% eGFP fluorescence were isolated. sgRNAs abundance was then determined in each cell population. ( B ) Gene level enrichment score was calculated for every gene using MAGeCK (see methods). Each gene is represented by a bubble, the size of which is proportional to number of sgRNAs with significant enrichment in the eGFP high population. SURF4 has the highest MAGeCK enrichment score and is the only gene for which the false discovery rate (FDR) is
Figure Legend Snippet: CRISPR/Cas9 loss-of-function screen to identify genes that affect intracellular EPO levels. ( A ) Screen strategy: 24 hours following transduction of the CRISPR library, puromycin selection was applied for 3 days. At day 14, cells with unchanged mCherry but with top or bottom 7% eGFP fluorescence were isolated. sgRNAs abundance was then determined in each cell population. ( B ) Gene level enrichment score was calculated for every gene using MAGeCK (see methods). Each gene is represented by a bubble, the size of which is proportional to number of sgRNAs with significant enrichment in the eGFP high population. SURF4 has the highest MAGeCK enrichment score and is the only gene for which the false discovery rate (FDR) is

Techniques Used: CRISPR, Transduction, Selection, Fluorescence, Isolation

A reporter HEK293T cell line stably expressing EPO-eGFP and A1AT-mCherry. ( A ) A construct that expresses EPO-eGFP and A1AT-mCherry from the same CMV promoter was assembled and used to generate the reporter cell line. A P2A sequence separates EPO-eGFP from A1AT-mCherry. ( B ) Intracellular and extracellular EPO-eGFP and A1AT-mCherry protein abundance was determined by Western blot using anti-eGFP and anti-mCherry antibodies, respectively. E: ER form of EPO; F: fully-glycosylated EPO. ( C ) Protein abundance was quantified using Image J with GAPDH as control. ( D ) Inhibiting ER to Golgi transport with Brefeldin A (BFA) leads to intracellular accumulation of EPO-eGFP and A1AT-mCherry, as measured by fluorescence intensity ( E ) LMAN1 deletion results in intracellular accumulation of A1AT with no effect on EPO.
Figure Legend Snippet: A reporter HEK293T cell line stably expressing EPO-eGFP and A1AT-mCherry. ( A ) A construct that expresses EPO-eGFP and A1AT-mCherry from the same CMV promoter was assembled and used to generate the reporter cell line. A P2A sequence separates EPO-eGFP from A1AT-mCherry. ( B ) Intracellular and extracellular EPO-eGFP and A1AT-mCherry protein abundance was determined by Western blot using anti-eGFP and anti-mCherry antibodies, respectively. E: ER form of EPO; F: fully-glycosylated EPO. ( C ) Protein abundance was quantified using Image J with GAPDH as control. ( D ) Inhibiting ER to Golgi transport with Brefeldin A (BFA) leads to intracellular accumulation of EPO-eGFP and A1AT-mCherry, as measured by fluorescence intensity ( E ) LMAN1 deletion results in intracellular accumulation of A1AT with no effect on EPO.

Techniques Used: Stable Transfection, Expressing, Construct, Sequencing, Western Blot, Fluorescence

15) Product Images from "Whole-brain mapping of monosynaptic inputs to midbrain cholinergic neurons"

Article Title: Whole-brain mapping of monosynaptic inputs to midbrain cholinergic neurons

Journal: bioRxiv

doi: 10.1101/2020.08.14.251546

Transsynaptic retrograde tracing of midbrain cholinergic neurons A , Schematic of the experimental procedure. AAV5-FLEX-TVA-mCherry and AAV8-FLEX-RG helper viruses were injected into the PPN or LDT of rats that expressed Cre in cholinergic neurons. Two weeks after these injections, a modified rabies virus SADΔG-eGFP (EnvA) was injected into the same area and the brain was processed after 7 days. B , Location of the site of injections in the PPN and LDT. C-E , Injections were confined to the borders of the PPN ( C-C’ ) and LDT ( D-D’ ), as determined by ChAT immunolabeling ( C” and D” ). E , Starter neurons were identified by the expression of the TVA helper reporter and SADΔ-eGFP. Scale bars C D: 1 mm. E-F: 100 µm.
Figure Legend Snippet: Transsynaptic retrograde tracing of midbrain cholinergic neurons A , Schematic of the experimental procedure. AAV5-FLEX-TVA-mCherry and AAV8-FLEX-RG helper viruses were injected into the PPN or LDT of rats that expressed Cre in cholinergic neurons. Two weeks after these injections, a modified rabies virus SADΔG-eGFP (EnvA) was injected into the same area and the brain was processed after 7 days. B , Location of the site of injections in the PPN and LDT. C-E , Injections were confined to the borders of the PPN ( C-C’ ) and LDT ( D-D’ ), as determined by ChAT immunolabeling ( C” and D” ). E , Starter neurons were identified by the expression of the TVA helper reporter and SADΔ-eGFP. Scale bars C D: 1 mm. E-F: 100 µm.

Techniques Used: Retrograde Tracing, Injection, Modification, Immunolabeling, Expressing

16) Product Images from "Interferon-responsive genes are targeted during the establishment of human cytomegalovirus latency"

Article Title: Interferon-responsive genes are targeted during the establishment of human cytomegalovirus latency

Journal: bioRxiv

doi: 10.1101/791673

IFI16 downregulation is maintained during long term latency of undifferentiated monocytes and CD34 + progenitor cells. A) US28 expressing and empty vector THP-1 cells were either left untreated or treated with PMA for 48 hours before cell lysates were harvested. These lysates were then subject to western blotting for IFI16 and actin as a loading control, with molecular weight markers annotated. B) At 48 h.p.i, either undifferentiated CD14 + monocytes, or monocytes pre-differentiated for 7 days with GM-CSF/IL-4/LPS, were fixed and stained for IFI16 and imaged as before at 40X magnification. White arrows indicate corresponding infected cells. C) CD14+ monocytes were infected with HCMV GATA2mCherry, or left uninfected. At the indicated times, cells were fixed and stained for IFI16 or MNDA and imaged as before. D) Primary CD34 + hematopoietic progenitor cells from two donors, or Kasumi-3 cells, were infected infected with HCMV GATA2mCherry, or left uninfected. At the indicated times, cells were fixed and stained for IFI16 and imaged as before. E) Quantification of at least 3 fields of view from D), presented as the proportion of cells with low IFI16 expression in the infected, mCherry positive and uninfected, mCherry negative populations. UI – uninfected. Statistical analysis was performed by Fischer’s Exact test on the total number of cells in each category. **** indicates P
Figure Legend Snippet: IFI16 downregulation is maintained during long term latency of undifferentiated monocytes and CD34 + progenitor cells. A) US28 expressing and empty vector THP-1 cells were either left untreated or treated with PMA for 48 hours before cell lysates were harvested. These lysates were then subject to western blotting for IFI16 and actin as a loading control, with molecular weight markers annotated. B) At 48 h.p.i, either undifferentiated CD14 + monocytes, or monocytes pre-differentiated for 7 days with GM-CSF/IL-4/LPS, were fixed and stained for IFI16 and imaged as before at 40X magnification. White arrows indicate corresponding infected cells. C) CD14+ monocytes were infected with HCMV GATA2mCherry, or left uninfected. At the indicated times, cells were fixed and stained for IFI16 or MNDA and imaged as before. D) Primary CD34 + hematopoietic progenitor cells from two donors, or Kasumi-3 cells, were infected infected with HCMV GATA2mCherry, or left uninfected. At the indicated times, cells were fixed and stained for IFI16 and imaged as before. E) Quantification of at least 3 fields of view from D), presented as the proportion of cells with low IFI16 expression in the infected, mCherry positive and uninfected, mCherry negative populations. UI – uninfected. Statistical analysis was performed by Fischer’s Exact test on the total number of cells in each category. **** indicates P

Techniques Used: Expressing, Plasmid Preparation, Western Blot, Molecular Weight, Staining, Infection

IFI16, MNDA, and HLA-DR are downregulated in latently infected CD14 + monocytes. Primary CD14 + monocytes were isolated from peripheral blood or apheresis cones as described in Materials and Methods. These cells were then infected using BAC-derived strains of TB40/E. A) CD14 + monocytes latently infected with TB40/E SV40-mCherry IE2-2A-GFP stained by immunofluorescence for IFI16, MNDA, or HLA-DR as indicated at four d.p.i and imaged by widefield fluorescence microscopy. Top left image: Uninfected monocytes. Second from the left: Monocytes were treated +PMA (to permit lytic infection). mCherry (red) serves as a marker for infection and GFP (green) denotes expression from the IE2-2A-GFP cassette. Remaining panels: Monocytes were cultured in the absence of PMA. The absence of green fluorescence results from suppressed expression of the IE2-2A-GFP cassette and scored as IE negative. The magnification is indicated (40X or 20X). White arrows indicate corresponding cells in the upper and lower panels. B) Validation of experimental latency using TB40/E gfp virus. CD14 + Monocytes were infected and allowed to establish latency for 4 days (left panel, 10X magnification). Citrate wash buffer was used to remove externally bound virions. These latently infected cells were cultured -/+PMA for 3 days, and at 7 d.p.i, Hff-1 cells were added to the culture to demonstrate production of infectious virions. Transfer of virus to Hff-1 was monitored by fluorescence microscopy up to 13 d.p.i., and infected Hff-1 foci were counted and summed across the experiment (three wells of CD14 + monocytes per condition, graphed). C) CD14 + monocytes infected with TB40/E gfp stained by immunofluorescence for IFI16 at 24, 48, and 72 h.p.i. and imaged as before using 60X magnification. D) Quantification of IFI16 positive and negative monocytes in the uninfected and infected populations from two donors per time point. Raw numbers of cells are indicated in white text. Fisher’s exact test indicates a statistically significant difference between uninfected and infected populations for each time point (P
Figure Legend Snippet: IFI16, MNDA, and HLA-DR are downregulated in latently infected CD14 + monocytes. Primary CD14 + monocytes were isolated from peripheral blood or apheresis cones as described in Materials and Methods. These cells were then infected using BAC-derived strains of TB40/E. A) CD14 + monocytes latently infected with TB40/E SV40-mCherry IE2-2A-GFP stained by immunofluorescence for IFI16, MNDA, or HLA-DR as indicated at four d.p.i and imaged by widefield fluorescence microscopy. Top left image: Uninfected monocytes. Second from the left: Monocytes were treated +PMA (to permit lytic infection). mCherry (red) serves as a marker for infection and GFP (green) denotes expression from the IE2-2A-GFP cassette. Remaining panels: Monocytes were cultured in the absence of PMA. The absence of green fluorescence results from suppressed expression of the IE2-2A-GFP cassette and scored as IE negative. The magnification is indicated (40X or 20X). White arrows indicate corresponding cells in the upper and lower panels. B) Validation of experimental latency using TB40/E gfp virus. CD14 + Monocytes were infected and allowed to establish latency for 4 days (left panel, 10X magnification). Citrate wash buffer was used to remove externally bound virions. These latently infected cells were cultured -/+PMA for 3 days, and at 7 d.p.i, Hff-1 cells were added to the culture to demonstrate production of infectious virions. Transfer of virus to Hff-1 was monitored by fluorescence microscopy up to 13 d.p.i., and infected Hff-1 foci were counted and summed across the experiment (three wells of CD14 + monocytes per condition, graphed). C) CD14 + monocytes infected with TB40/E gfp stained by immunofluorescence for IFI16 at 24, 48, and 72 h.p.i. and imaged as before using 60X magnification. D) Quantification of IFI16 positive and negative monocytes in the uninfected and infected populations from two donors per time point. Raw numbers of cells are indicated in white text. Fisher’s exact test indicates a statistically significant difference between uninfected and infected populations for each time point (P

Techniques Used: Infection, Isolation, BAC Assay, Derivative Assay, Staining, Immunofluorescence, Fluorescence, Microscopy, Marker, Expressing, Cell Culture

IFI16 is rapidly downregulated in a US28-dependent manner during latent infection CD14 + monocytes were infected with either US28 WT TB40/E mCherry -US28-3XFLAG HCMV or the ΔUS28. A) Validation of the latent and lytic phenotypes associated with US28-3xF and ΔUS28 monocyte infections, respectively. At 7 d.p.i., supernatant from infected CD14 + cells (upper panel) were transferred to Hff1 cells (middle brightfield and lower mCherry panels) and formation of plaques was monitored and imaged at 20X magnification. B) Detection of US28-3XFLAG during the establishment of latency in CD14 + monocytes. At 2 d.p.i. US28-3xF or ΔUS28-infected CD14 + monocytes were fixed and stained by immunofluorescence for US28-3XFLAG using an anti-FLAG antibody and imaged at 40X magnification. C) US28-3xF and ΔUS28-infected monocytes were stained by immunofluorescence for IFI16 at the indicated times and imaged using 40X magnification. White arrows indicate corresp onding cells. D and E) IFI16 signal intensity in each nucleus was normalised to the average of the uninfected cells in a field of view. The results of three fields of view were then averaged to derive the resulting average signal intensities for each subpopulation of monocytes at the indicated time points infected with US28-3xF or ΔUS28 HCMV. Statistical significance was determined using one-way ANOVA. *** indicates P
Figure Legend Snippet: IFI16 is rapidly downregulated in a US28-dependent manner during latent infection CD14 + monocytes were infected with either US28 WT TB40/E mCherry -US28-3XFLAG HCMV or the ΔUS28. A) Validation of the latent and lytic phenotypes associated with US28-3xF and ΔUS28 monocyte infections, respectively. At 7 d.p.i., supernatant from infected CD14 + cells (upper panel) were transferred to Hff1 cells (middle brightfield and lower mCherry panels) and formation of plaques was monitored and imaged at 20X magnification. B) Detection of US28-3XFLAG during the establishment of latency in CD14 + monocytes. At 2 d.p.i. US28-3xF or ΔUS28-infected CD14 + monocytes were fixed and stained by immunofluorescence for US28-3XFLAG using an anti-FLAG antibody and imaged at 40X magnification. C) US28-3xF and ΔUS28-infected monocytes were stained by immunofluorescence for IFI16 at the indicated times and imaged using 40X magnification. White arrows indicate corresp onding cells. D and E) IFI16 signal intensity in each nucleus was normalised to the average of the uninfected cells in a field of view. The results of three fields of view were then averaged to derive the resulting average signal intensities for each subpopulation of monocytes at the indicated time points infected with US28-3xF or ΔUS28 HCMV. Statistical significance was determined using one-way ANOVA. *** indicates P

Techniques Used: Infection, Staining, Immunofluorescence

17) Product Images from "Mauve/LYST limits fusion of lysosome-related organelles and promotes centrosomal recruitment of microtubule nucleating proteins"

Article Title: Mauve/LYST limits fusion of lysosome-related organelles and promotes centrosomal recruitment of microtubule nucleating proteins

Journal: Developmental Cell

doi: 10.1016/j.devcel.2021.02.019

Mauve protein localizes around LROs in Drosophila oocytes and at mitotic spindles in early embryonic divisions (A) Localization of Mauve in stage 10 egg chambers stained to reveal Mv-mCherry, red; DNA, blue. Mv-mCherry has a polarized localization in follicular epithelial cells, enriched on the side facing the developing oocyte where yolk components are secreted before uptake into the oocyte. Scale bar, 50 μm; zoom, 10 μm. (B) Mauve localization in freshly dissected, unfixed mature eggs. YGs are autofluorescent in the green, but not, the red channel. Mv-mCherry localizes around the YGs. Scale bar, 10 μm. (C) Mv-mCherry localization in early embryonic divisions revealed by RFP-Booster Alexa Fluor 568 (Chromotek, gray), Dplp (red), and DAPI (blue). Mv-mCherry localizes all over the mitotic spindles and is enriched at the poles where it colocalizes with DPlp. Such localization was observed at all stages of mitotic division (data not shown). Or-R flies were used as controls (n = 50). Scale bar, 10 μm. See also Figure S4 A. (D) Mass spectrometric identification of proteins co-immunoprecipitating with Mv-mCherry from 0–3 h embryos (full datasets in Table S2 ).
Figure Legend Snippet: Mauve protein localizes around LROs in Drosophila oocytes and at mitotic spindles in early embryonic divisions (A) Localization of Mauve in stage 10 egg chambers stained to reveal Mv-mCherry, red; DNA, blue. Mv-mCherry has a polarized localization in follicular epithelial cells, enriched on the side facing the developing oocyte where yolk components are secreted before uptake into the oocyte. Scale bar, 50 μm; zoom, 10 μm. (B) Mauve localization in freshly dissected, unfixed mature eggs. YGs are autofluorescent in the green, but not, the red channel. Mv-mCherry localizes around the YGs. Scale bar, 10 μm. (C) Mv-mCherry localization in early embryonic divisions revealed by RFP-Booster Alexa Fluor 568 (Chromotek, gray), Dplp (red), and DAPI (blue). Mv-mCherry localizes all over the mitotic spindles and is enriched at the poles where it colocalizes with DPlp. Such localization was observed at all stages of mitotic division (data not shown). Or-R flies were used as controls (n = 50). Scale bar, 10 μm. See also Figure S4 A. (D) Mass spectrometric identification of proteins co-immunoprecipitating with Mv-mCherry from 0–3 h embryos (full datasets in Table S2 ).

Techniques Used: Staining

Mutations in mv result in enlarged YGs (A) Comparison of human LYST and Drosophila Mauve protein domains. Positions of known mv mutations are shown against the Drosophila protein ( mv 3 , red, is newly identified here). Downstream of a pleckstrin homology (PH) domain of approximately 100 residues lies a “beige and CHS” (BEACH) domain of about 300 residues and a series of WD40 repeats. (B) Deficiency mapping of 61F-63A indicating female fertility observed when deficiencies were heterozygous with mv ros and mv 3 alleles. The expanded interval shows all genes with mv in red. (C) Genomic region (green shading) encompassing mauve carried in the BAC CH322-23O09 used for genomic rescue. BAC recombineering introduced an in-frame mCherry or FLAG tag at the C-terminus of the coding sequence (CDS) followed by 4 stop codons and a kanamycin resistance gene (kana R ) upstream of the mv 3′UTR. (D) Examples of autofluorescent LROs (YGs) in Or-R and mv 3 /Df embryos. Scale bar, 50μm. (E) Diameters of LROs (YGs) in Or-R , mv/Df- derived embryos, and mv/Df- derived embryos with rescue transgenes, Mv-mCherry or UAS-Mv-GFP / P{matα4-GAL-VP16} : Or-R , 2.38 ± 0.09 μm; mv ros /Df , 6.96 ± 0.21 μm; mv 3 /Df , 7.39 ± 0.22 μm; Mv-mCherry ; mv ros /Df , 3.32 ± 0.05 μm; Mv-mCherry ; mv 3 /Df , 2.74 ± 0.07 μm; UAS-Mv-GFP mv 3 /Df , 2.68 ± 0.1 μm). n = 100, mean±SEM. Unpaired t test: ∗∗∗∗ p
Figure Legend Snippet: Mutations in mv result in enlarged YGs (A) Comparison of human LYST and Drosophila Mauve protein domains. Positions of known mv mutations are shown against the Drosophila protein ( mv 3 , red, is newly identified here). Downstream of a pleckstrin homology (PH) domain of approximately 100 residues lies a “beige and CHS” (BEACH) domain of about 300 residues and a series of WD40 repeats. (B) Deficiency mapping of 61F-63A indicating female fertility observed when deficiencies were heterozygous with mv ros and mv 3 alleles. The expanded interval shows all genes with mv in red. (C) Genomic region (green shading) encompassing mauve carried in the BAC CH322-23O09 used for genomic rescue. BAC recombineering introduced an in-frame mCherry or FLAG tag at the C-terminus of the coding sequence (CDS) followed by 4 stop codons and a kanamycin resistance gene (kana R ) upstream of the mv 3′UTR. (D) Examples of autofluorescent LROs (YGs) in Or-R and mv 3 /Df embryos. Scale bar, 50μm. (E) Diameters of LROs (YGs) in Or-R , mv/Df- derived embryos, and mv/Df- derived embryos with rescue transgenes, Mv-mCherry or UAS-Mv-GFP / P{matα4-GAL-VP16} : Or-R , 2.38 ± 0.09 μm; mv ros /Df , 6.96 ± 0.21 μm; mv 3 /Df , 7.39 ± 0.22 μm; Mv-mCherry ; mv ros /Df , 3.32 ± 0.05 μm; Mv-mCherry ; mv 3 /Df , 2.74 ± 0.07 μm; UAS-Mv-GFP mv 3 /Df , 2.68 ± 0.1 μm). n = 100, mean±SEM. Unpaired t test: ∗∗∗∗ p

Techniques Used: BAC Assay, FLAG-tag, Sequencing, Derivative Assay

18) Product Images from "The Acetylation of Lysine-376 of G3BP1 Regulates RNA Binding and Stress Granule Dynamics"

Article Title: The Acetylation of Lysine-376 of G3BP1 Regulates RNA Binding and Stress Granule Dynamics

Journal: Molecular and Cellular Biology

doi: 10.1128/MCB.00052-19

K376Q acetylation-mimicking mutation and the F380L/F382L RNA binding-deficient mutation impair SG formation. (A) The G3BP1-null 293T derivative mCherry-G3BP1 WT, K376Q, and F380L/F382L knock-in cell lines had G3BP1 levels comparable to that of 293T cells and each other, as shown in immunoblotting with the indicated antibodies. Symbols: #, the expected mCherry-tagged G3BP1 band; ##, mCherry-G3BP1 fusion protein with likely partially processed mCherry tag. See the text for details. (B) Representative confocal microscopic images of mCherry-G3BP1 fluorescence and TIA-1 immunofluorescence of the varied knock-in cell lines after 30 min of treatment with 0.5 mM sodium arsenite. The nuclei were visualized with DAPI stain. Scale bars, 20 μm. (C) The number ± SD of G3BP1 SGs per cell and the average size ± SD of G3BP1 SGs, as quantified in four independent experiments. The F380L/F382L error bars are shown semitransparent on the right to allow for distinction between the WT and K376Q error bars at 30 min. Significance compared to values for the WT is indicated by asterisks: *, 0.05 > P > 0.01; **, 0.01 > P > 0.001; ***, P
Figure Legend Snippet: K376Q acetylation-mimicking mutation and the F380L/F382L RNA binding-deficient mutation impair SG formation. (A) The G3BP1-null 293T derivative mCherry-G3BP1 WT, K376Q, and F380L/F382L knock-in cell lines had G3BP1 levels comparable to that of 293T cells and each other, as shown in immunoblotting with the indicated antibodies. Symbols: #, the expected mCherry-tagged G3BP1 band; ##, mCherry-G3BP1 fusion protein with likely partially processed mCherry tag. See the text for details. (B) Representative confocal microscopic images of mCherry-G3BP1 fluorescence and TIA-1 immunofluorescence of the varied knock-in cell lines after 30 min of treatment with 0.5 mM sodium arsenite. The nuclei were visualized with DAPI stain. Scale bars, 20 μm. (C) The number ± SD of G3BP1 SGs per cell and the average size ± SD of G3BP1 SGs, as quantified in four independent experiments. The F380L/F382L error bars are shown semitransparent on the right to allow for distinction between the WT and K376Q error bars at 30 min. Significance compared to values for the WT is indicated by asterisks: *, 0.05 > P > 0.01; **, 0.01 > P > 0.001; ***, P

Techniques Used: Mutagenesis, RNA Binding Assay, Knock-In, Fluorescence, Immunofluorescence, Staining

K376-acetylated G3BP1 localizes primarily outside SGs. Representative confocal microscopic images of mCherry-G3BP1 fluorescence and the K376-acetylated G3BP1 PLA foci (Ac-K376 PLA). The nuclei were visualized with DAPI stain. Acetylated G3BP1/total G3BP1 proximity ligation assay was performed on G3BP1-null 293T-derived cells stably expressing mCherry-WT G3BP1. The cells were pretreated with deacetylase inhibitors for 1 h, followed by SG induction with 0.5 mM sodium arsenite for 1 h and 1 or 3 h of recovery in fresh medium containing deacetylase inhibitors. The acetylated G3BP1 PLA foci were often found in the periphery of the disassembling SGs. The insets show magnifications of the boxed areas. Scale bars, 5 μm.
Figure Legend Snippet: K376-acetylated G3BP1 localizes primarily outside SGs. Representative confocal microscopic images of mCherry-G3BP1 fluorescence and the K376-acetylated G3BP1 PLA foci (Ac-K376 PLA). The nuclei were visualized with DAPI stain. Acetylated G3BP1/total G3BP1 proximity ligation assay was performed on G3BP1-null 293T-derived cells stably expressing mCherry-WT G3BP1. The cells were pretreated with deacetylase inhibitors for 1 h, followed by SG induction with 0.5 mM sodium arsenite for 1 h and 1 or 3 h of recovery in fresh medium containing deacetylase inhibitors. The acetylated G3BP1 PLA foci were often found in the periphery of the disassembling SGs. The insets show magnifications of the boxed areas. Scale bars, 5 μm.

Techniques Used: Fluorescence, Proximity Ligation Assay, Staining, Derivative Assay, Stable Transfection, Expressing, Histone Deacetylase Assay

19) Product Images from "Thalamus and claustrum control parallel layer 1 circuits in retrosplenial cortex"

Article Title: Thalamus and claustrum control parallel layer 1 circuits in retrosplenial cortex

Journal: eLife

doi: 10.7554/eLife.62207

Contralateral RSG projections drive RS, but not LR, neurons. ( A ) Left: Schematic of contralateral RSG (cRSG) ChR2-mCherry injection (opposite hemisphere compared to recordings). MCherry is pseudo colored as gray. Middle: Schematic of ipsilateral ADAV eYFP injection (same hemisphere as recordings). Right: Schematic of target recording region in RSG. ( B ) Confocal image of dual-injection expression with cRSG axons and terminal arbors in gray and ADAV axons and terminal arbors in green. Layers are demarcated by black lines. Note the precise targeting of L1c, L2, and L5 by cRSG axons and terminal arbors (gray) in contrast to the L1a and L3 targeting of anterior thalamic arbors (green). ( C ) Schematic showing cRSG inputs to patched LR and RS neurons being stimulated at L1/2 boundary. ( D ) Example traces of two sequential pairs of RS (blue) and LR (purple) neuron responses to the high power LED pulse. ( E ) Significantly larger EPSP amplitudes in RS (n = 5) cells compared to LR (n = 7) cells (p=0.0051; Wilcoxon rank sum test). Error bars are SEM.
Figure Legend Snippet: Contralateral RSG projections drive RS, but not LR, neurons. ( A ) Left: Schematic of contralateral RSG (cRSG) ChR2-mCherry injection (opposite hemisphere compared to recordings). MCherry is pseudo colored as gray. Middle: Schematic of ipsilateral ADAV eYFP injection (same hemisphere as recordings). Right: Schematic of target recording region in RSG. ( B ) Confocal image of dual-injection expression with cRSG axons and terminal arbors in gray and ADAV axons and terminal arbors in green. Layers are demarcated by black lines. Note the precise targeting of L1c, L2, and L5 by cRSG axons and terminal arbors (gray) in contrast to the L1a and L3 targeting of anterior thalamic arbors (green). ( C ) Schematic showing cRSG inputs to patched LR and RS neurons being stimulated at L1/2 boundary. ( D ) Example traces of two sequential pairs of RS (blue) and LR (purple) neuron responses to the high power LED pulse. ( E ) Significantly larger EPSP amplitudes in RS (n = 5) cells compared to LR (n = 7) cells (p=0.0051; Wilcoxon rank sum test). Error bars are SEM.

Techniques Used: Injection, Expressing

20) Product Images from "TMEM16K is an interorganelle regulator of endosomal sorting"

Article Title: TMEM16K is an interorganelle regulator of endosomal sorting

Journal: Nature Communications

doi: 10.1038/s41467-020-17016-8

Analysis of the endolysosomal pathway in the TMEM16K absence. RUSH assay 1st row: Scheme of the RUSH construct biosynthetic pathway. 2nd and 3rd rows: Representative images of WT and TMEM16K KO cells at the indicated time points. 4th row: Quantification at indicated time point. a RUSH assay with mCherry-GPI. Quantification of surface vs. total at 60 min. Two-tailed t -test, p value = 0.53 n.s. (three biological replicates, n = 53 WT and 56 TMEM16K KO cells) b RUSH assay with mCherry-TfR. Pearson’s correlation coefficient at 120 min. Two-tailed t -test, p value = 0.21 n.s. (three biological replicates, n = 129 WT, and 111 TMEM16K KO cells) c RUSH assay with GFP-CD-MPR. Pearson’s correlation coefficient at 60 min with GM130 (three biological replicates, n = 126 WT, and 127 TMEM16K KO cells, Two-tailed t -test, p value = 0.45 n.s.), and Pearson’s correlation coefficient at 120 min with mCherry-TfR RUSH (three biological replicates, n = 144 WT and 134 TMEM16K KO cells, Two-tailed t -test, p value = 0.17 n.s.) d Fluorescence intensity of transferrin at 60 min in the WT ( n = 100) and TMEM16K KO ( n = 50) cells from three biological replicates, Two-tailed t -test, p value = 0.63 n.s. e EGF-Alexa647 pulse-chase experiment was quantified for colocalization with endogenous Rab7, Two-tailed t -test between WT and KO at each measured time point (three biological replicates, n = 130 WT, and 117 TMEM16K KO cells at 10 min, p value = 0.75 n.s., 91 WT, and 103 KO cells at 15 min, p value = 0.88 n.s., 77 WT and 61 KO cells at 40 min, p value = 0.60 n.s., 89 WT, and 118 KO cells at 60 min, p value = 0.043*). f Fluorescence intensity of Lysosensor Green DNP-189 in WT ( n = 114) and TMEM16K KO ( n = 116) cells. Single factor ANOVA p value = 8E−25*** from three biological replicates. g Representative trace from three independent experiments of protonophore FCCP at a final concentration 2 µM added at 120 s to cells loaded with Lysosensor Green DNP-189. (WT slope is −0.0445, y = −0.0445 x + 128.45, R 2 = 0.9537; TMEM16K KO slope is −0.0305, y = −0.0305 x + 109.5, R 2 = 0.9581) h Evaluation of wild type and mutant TMEM16K cDNA ability to rescue acidification defect. WT or TMEM16K KO cells were co-transfected with mCherry-CAAX to visualize transfected cells, and TMEM16K wild type cDNA (TMEM16K-FLAG) or TMEM16K mutant cDNA (Ca5MUT-FLAG) and evaluated for acidification with Lysosensor Green D-189. Single factor ANOVA, p value = 1.46E−39 with post-test Bonferroni-corrected two sided t -test with pairwise comparison with WT + 16K wild type (three biological replicates, n = 50 WT + 16K wild type; 50 WT + 16K mutant, p value = 1.85E−07***; 40 KO + 16K wild type; 40 KO + 16K mutant cells, p value = 3.27E−19***). In the box and whiskers plot, the box includes the first quartile and the third quartile, with the central line representing the median. Whiskers represent the minimum and maximum values of data. X inside the box represents the mean of data. Source data are provided as a Source Data file.
Figure Legend Snippet: Analysis of the endolysosomal pathway in the TMEM16K absence. RUSH assay 1st row: Scheme of the RUSH construct biosynthetic pathway. 2nd and 3rd rows: Representative images of WT and TMEM16K KO cells at the indicated time points. 4th row: Quantification at indicated time point. a RUSH assay with mCherry-GPI. Quantification of surface vs. total at 60 min. Two-tailed t -test, p value = 0.53 n.s. (three biological replicates, n = 53 WT and 56 TMEM16K KO cells) b RUSH assay with mCherry-TfR. Pearson’s correlation coefficient at 120 min. Two-tailed t -test, p value = 0.21 n.s. (three biological replicates, n = 129 WT, and 111 TMEM16K KO cells) c RUSH assay with GFP-CD-MPR. Pearson’s correlation coefficient at 60 min with GM130 (three biological replicates, n = 126 WT, and 127 TMEM16K KO cells, Two-tailed t -test, p value = 0.45 n.s.), and Pearson’s correlation coefficient at 120 min with mCherry-TfR RUSH (three biological replicates, n = 144 WT and 134 TMEM16K KO cells, Two-tailed t -test, p value = 0.17 n.s.) d Fluorescence intensity of transferrin at 60 min in the WT ( n = 100) and TMEM16K KO ( n = 50) cells from three biological replicates, Two-tailed t -test, p value = 0.63 n.s. e EGF-Alexa647 pulse-chase experiment was quantified for colocalization with endogenous Rab7, Two-tailed t -test between WT and KO at each measured time point (three biological replicates, n = 130 WT, and 117 TMEM16K KO cells at 10 min, p value = 0.75 n.s., 91 WT, and 103 KO cells at 15 min, p value = 0.88 n.s., 77 WT and 61 KO cells at 40 min, p value = 0.60 n.s., 89 WT, and 118 KO cells at 60 min, p value = 0.043*). f Fluorescence intensity of Lysosensor Green DNP-189 in WT ( n = 114) and TMEM16K KO ( n = 116) cells. Single factor ANOVA p value = 8E−25*** from three biological replicates. g Representative trace from three independent experiments of protonophore FCCP at a final concentration 2 µM added at 120 s to cells loaded with Lysosensor Green DNP-189. (WT slope is −0.0445, y = −0.0445 x + 128.45, R 2 = 0.9537; TMEM16K KO slope is −0.0305, y = −0.0305 x + 109.5, R 2 = 0.9581) h Evaluation of wild type and mutant TMEM16K cDNA ability to rescue acidification defect. WT or TMEM16K KO cells were co-transfected with mCherry-CAAX to visualize transfected cells, and TMEM16K wild type cDNA (TMEM16K-FLAG) or TMEM16K mutant cDNA (Ca5MUT-FLAG) and evaluated for acidification with Lysosensor Green D-189. Single factor ANOVA, p value = 1.46E−39 with post-test Bonferroni-corrected two sided t -test with pairwise comparison with WT + 16K wild type (three biological replicates, n = 50 WT + 16K wild type; 50 WT + 16K mutant, p value = 1.85E−07***; 40 KO + 16K wild type; 40 KO + 16K mutant cells, p value = 3.27E−19***). In the box and whiskers plot, the box includes the first quartile and the third quartile, with the central line representing the median. Whiskers represent the minimum and maximum values of data. X inside the box represents the mean of data. Source data are provided as a Source Data file.

Techniques Used: Construct, Two Tailed Test, Fluorescence, Pulse Chase, Concentration Assay, Mutagenesis, Transfection

21) Product Images from "The SARS-CoV-2 spike (S) and the orthoreovirus p15 cause neuronal and glial fusion"

Article Title: The SARS-CoV-2 spike (S) and the orthoreovirus p15 cause neuronal and glial fusion

Journal: bioRxiv

doi: 10.1101/2021.09.01.458544

Expression of p15 induces fusion of murine neurons in culture. a , Representative images of fused neurons (upper row panels) identifiable with GFP (green) and mCherry (red) fluorescence appearing simultaneously in adjacent neurons (yellow in the merge panel), or non-fused control neurons (middle and lower row panels) with green and red fluorescence in adjacent neurons. Two populations of hippocampal neurons expressing either p15 and GFP, or empty vector and mCherry were cultured together for 7 days (7 DIV). In control conditions, p15 was substituted by the non-fusogenic mutant p15Δ21-22, or by the empty vector. Immunocytochemistry for nuclei (blue), neuronal MAP2 (magenta), GFP (green) and mCherry (red). b , Quantification of neuronal fusion as the percentage of transfected neurons that fuse (yellow) when two neurons are in proximity (≤ 200 μm). c , Representative images of neurons illustrating the propagation of fusion over time (upper panels). Hippocampal neurons were co-transfected at 7-10 DIV with p15 and GFP (or empty vector and GFP in control, lower panels), and were cultured for 1 day, 4 days or 7 days. Immunocytochemistry for nuclei (blue), MAP2 (magenta) and GFP (green). d , Quantification of neuronal syncytia as the percentage of interconnected neurons within a distance of ≤ 200 μm. e, Quantification of the average number of interconnected neurons per syncytium containing more than 5 neurons. Data in b are displayed as mean ± SEM, n > 150 neurons analyzed in 6 independent dishes from > 2 cultures, One-way ANOVA Kruskal-Wallis test followed by Dunn’s post hoc test in e comparing all groups to empty vector control. Data in d and e are displayed as mean ± SEM, n > 350 neurons analyzed in > 4 independent dishes from 4 cultures. Two-way ANOVA in d followed by Geisser-Greenhouse correction and the Šidák post hoc test comparing treatments (+ empty vector vs + p15) within each condition (days in culture). One-way ANOVA Kruskal-Wallis test followed by Dunn’s post hoc test in e comparing all groups to 1 day. * p
Figure Legend Snippet: Expression of p15 induces fusion of murine neurons in culture. a , Representative images of fused neurons (upper row panels) identifiable with GFP (green) and mCherry (red) fluorescence appearing simultaneously in adjacent neurons (yellow in the merge panel), or non-fused control neurons (middle and lower row panels) with green and red fluorescence in adjacent neurons. Two populations of hippocampal neurons expressing either p15 and GFP, or empty vector and mCherry were cultured together for 7 days (7 DIV). In control conditions, p15 was substituted by the non-fusogenic mutant p15Δ21-22, or by the empty vector. Immunocytochemistry for nuclei (blue), neuronal MAP2 (magenta), GFP (green) and mCherry (red). b , Quantification of neuronal fusion as the percentage of transfected neurons that fuse (yellow) when two neurons are in proximity (≤ 200 μm). c , Representative images of neurons illustrating the propagation of fusion over time (upper panels). Hippocampal neurons were co-transfected at 7-10 DIV with p15 and GFP (or empty vector and GFP in control, lower panels), and were cultured for 1 day, 4 days or 7 days. Immunocytochemistry for nuclei (blue), MAP2 (magenta) and GFP (green). d , Quantification of neuronal syncytia as the percentage of interconnected neurons within a distance of ≤ 200 μm. e, Quantification of the average number of interconnected neurons per syncytium containing more than 5 neurons. Data in b are displayed as mean ± SEM, n > 150 neurons analyzed in 6 independent dishes from > 2 cultures, One-way ANOVA Kruskal-Wallis test followed by Dunn’s post hoc test in e comparing all groups to empty vector control. Data in d and e are displayed as mean ± SEM, n > 350 neurons analyzed in > 4 independent dishes from 4 cultures. Two-way ANOVA in d followed by Geisser-Greenhouse correction and the Šidák post hoc test comparing treatments (+ empty vector vs + p15) within each condition (days in culture). One-way ANOVA Kruskal-Wallis test followed by Dunn’s post hoc test in e comparing all groups to 1 day. * p

Techniques Used: Expressing, Fluorescence, Plasmid Preparation, Cell Culture, Mutagenesis, Immunocytochemistry, Transfection

p15 and spike S induce fusion in human neurons and brain organoids. a , Representative images of 2D-cultured neurons illustrating fusion of cells into syncytia. Human neurons were co-transfected at 40-50 DIV with GFP and either p15, spike S or spike S-6P (or empty vector in controls), then cultured for 7 days. Immunocytochemistry for nuclei (blue), MAP2 (red) and GFP (green/white). b , Quantification of neuronal syncytia as the percentage of interconnected neurons within a distance of ≤ 200 μm. c , Quantification of the average number of interconnected neurons per syncytium containing more than one neuron. d , Representative images of 3D neuronal organoids illustrating fusion of cells into syncytia. Organoids were co-transfected at 43-50 DIV with mCherry and either p15, spike S or spike S-6P (or empty vector in controls), then cultured for 6 days. Immunocytochemistry for nuclei (blue), MAP2 (green) and mCherry (red/white). Regions of interest (ROIs) show higher magnification at positions indicated by broken lines. Arrowheads indicate clusters of fused neurons. e , Quantification of the average number of mCherry-positive cells per organoid section 6 days after transfection. Data in b , c and e are displayed as mean ± SEM, averages of n > 30 neurons analyzed in independent experiments for b and c , and n > 8 organoids analyzed in independent experiments for e . One-way ANOVA followed by Tukey post hoc test in b and c . * p
Figure Legend Snippet: p15 and spike S induce fusion in human neurons and brain organoids. a , Representative images of 2D-cultured neurons illustrating fusion of cells into syncytia. Human neurons were co-transfected at 40-50 DIV with GFP and either p15, spike S or spike S-6P (or empty vector in controls), then cultured for 7 days. Immunocytochemistry for nuclei (blue), MAP2 (red) and GFP (green/white). b , Quantification of neuronal syncytia as the percentage of interconnected neurons within a distance of ≤ 200 μm. c , Quantification of the average number of interconnected neurons per syncytium containing more than one neuron. d , Representative images of 3D neuronal organoids illustrating fusion of cells into syncytia. Organoids were co-transfected at 43-50 DIV with mCherry and either p15, spike S or spike S-6P (or empty vector in controls), then cultured for 6 days. Immunocytochemistry for nuclei (blue), MAP2 (green) and mCherry (red/white). Regions of interest (ROIs) show higher magnification at positions indicated by broken lines. Arrowheads indicate clusters of fused neurons. e , Quantification of the average number of mCherry-positive cells per organoid section 6 days after transfection. Data in b , c and e are displayed as mean ± SEM, averages of n > 30 neurons analyzed in independent experiments for b and c , and n > 8 organoids analyzed in independent experiments for e . One-way ANOVA followed by Tukey post hoc test in b and c . * p

Techniques Used: Cell Culture, Transfection, Plasmid Preparation, Immunocytochemistry

Expression of spike S and its receptor hACE2 induce fusion of murine neurons in culture. a , Representative images of fused neurons (first row), or non-fused control neurons (other rows). Two populations of hippocampal neurons expressing a combination of two plasmids as indicated on the left (spike S and GFP, hACE2 and mCherry, empty vector and GFP, or empty vector and mCherry) were cultured together for 7 days (7 DIV). Immunocytochemistry for nuclei (blue), MAP2 (magenta), GFP (green) and mCherry (red). Neuronal fusion only occurred (first row) when one population of neurons was transfected with spike S and GFP, and the other with hACE2 and mCherry, as visualized by the presence of GFP and mCherry in the same neurons (yellow in the merge panel). b , Quantification of neuronal fusion as the percentage of neurons that fuse (yellow) when two neurons are in proximity ( ≤ 200 μm). c , Representative images of fused neurons (first panel), or non-fused neurons (second and third panels). Two populations of hippocampal neurons expressing a combination of two plasmids as indicated above the images (spike S and GFP, hACE2 and mCherry, spike S-2P and GFP, or spike S-6P and GFP) were cultured together for 7 days (7 DIV). Immunocytochemistry for nuclei (blue), MAP2 (magenta), GFP (green) and mCherry (red). Neuronal fusion only occurred (first panel) when the full-length WT spike S protein was transfected, as visualized by the presence of GFP and mCherry in the same neurons (yellow in the panel), and not when any of the non-fusogenic mutants (spike S-2P, spike S-6P) were used (second and third panels). d , Quantification of neuronal fusion as the percentage of neurons that fuse (yellow) when two neurons are in proximity ( ≤ 200 μm). Data in b and d were displayed as mean ± SEM, n > 200 neurons analyzed in 4-6 independent dishes from 2 dissections, one-way ANOVA Kruskal-Wallis test in e followed by Dunn’s post hoc test comparing all groups to the group without spike S or hACE2. ** p
Figure Legend Snippet: Expression of spike S and its receptor hACE2 induce fusion of murine neurons in culture. a , Representative images of fused neurons (first row), or non-fused control neurons (other rows). Two populations of hippocampal neurons expressing a combination of two plasmids as indicated on the left (spike S and GFP, hACE2 and mCherry, empty vector and GFP, or empty vector and mCherry) were cultured together for 7 days (7 DIV). Immunocytochemistry for nuclei (blue), MAP2 (magenta), GFP (green) and mCherry (red). Neuronal fusion only occurred (first row) when one population of neurons was transfected with spike S and GFP, and the other with hACE2 and mCherry, as visualized by the presence of GFP and mCherry in the same neurons (yellow in the merge panel). b , Quantification of neuronal fusion as the percentage of neurons that fuse (yellow) when two neurons are in proximity ( ≤ 200 μm). c , Representative images of fused neurons (first panel), or non-fused neurons (second and third panels). Two populations of hippocampal neurons expressing a combination of two plasmids as indicated above the images (spike S and GFP, hACE2 and mCherry, spike S-2P and GFP, or spike S-6P and GFP) were cultured together for 7 days (7 DIV). Immunocytochemistry for nuclei (blue), MAP2 (magenta), GFP (green) and mCherry (red). Neuronal fusion only occurred (first panel) when the full-length WT spike S protein was transfected, as visualized by the presence of GFP and mCherry in the same neurons (yellow in the panel), and not when any of the non-fusogenic mutants (spike S-2P, spike S-6P) were used (second and third panels). d , Quantification of neuronal fusion as the percentage of neurons that fuse (yellow) when two neurons are in proximity ( ≤ 200 μm). Data in b and d were displayed as mean ± SEM, n > 200 neurons analyzed in 4-6 independent dishes from 2 dissections, one-way ANOVA Kruskal-Wallis test in e followed by Dunn’s post hoc test comparing all groups to the group without spike S or hACE2. ** p

Techniques Used: Expressing, Plasmid Preparation, Cell Culture, Immunocytochemistry, Transfection

22) Product Images from "Characterisation of the biochemical and cellular roles of native and pathogenic amelogenesis imperfecta mutants of FAM83H"

Article Title: Characterisation of the biochemical and cellular roles of native and pathogenic amelogenesis imperfecta mutants of FAM83H

Journal: Cellular Signalling

doi: 10.1016/j.cellsig.2020.109632

CK1 associated with FAM83H Q452X maintains kinase activity. A FLAG empty vector (vector), FLAG-FAM83H Q452X , FLAG-FAM83H D236A and FLAG-FAM83H WT were transiently transfected into U2OS FAM83H -/- cells. Cells were then lysed and lysates subjected to anti-FLAG immunoprecipitation. Cell extract (input) and immunoprecipitates (IP) were resolved by SDS-PAGE then immunoblotted (IB) with the antibodies indicated. n = 2. B As A but immunoprecipitates were incubated with recombinant GST (glutathione S-transferase)-PAWS1-His in the presence of [ γ 32 P]-ATP (adenosine 5′-triphosphate). Once the reaction was quenched, samples were resolved by SDS-PAGE. The gel was stained with InstantBlue (top) then dried and subjected to 32 P radiography (bottom). n = 2. C U2OS FAM83H -/- cells were transiently transfected with FLAG empty vector (vector), FLAG-FAM83H D236A , FLAG-FAM83H S287X , FLAG-FAM83H S287X, D236A and FLAG-FAM83H WT . Cells were lysed and lysates incubated with anti-FLAG beads for 16 h. Immunoprecipitates (IP) and cell extracts (input) were resolved by SDS-PAGE and proteins immunoblotted with the antibodies indicated. n = 2. D U2OS FAM83H -/- cells transiently expressing FLAG-FAM83H S287X , FLAG-FAM83H S287X, D236A , FLAG-FAM83H WT and mCherry-CK1α were stained with anti-FLAG and DNA stained with DAPI. n = 2
Figure Legend Snippet: CK1 associated with FAM83H Q452X maintains kinase activity. A FLAG empty vector (vector), FLAG-FAM83H Q452X , FLAG-FAM83H D236A and FLAG-FAM83H WT were transiently transfected into U2OS FAM83H -/- cells. Cells were then lysed and lysates subjected to anti-FLAG immunoprecipitation. Cell extract (input) and immunoprecipitates (IP) were resolved by SDS-PAGE then immunoblotted (IB) with the antibodies indicated. n = 2. B As A but immunoprecipitates were incubated with recombinant GST (glutathione S-transferase)-PAWS1-His in the presence of [ γ 32 P]-ATP (adenosine 5′-triphosphate). Once the reaction was quenched, samples were resolved by SDS-PAGE. The gel was stained with InstantBlue (top) then dried and subjected to 32 P radiography (bottom). n = 2. C U2OS FAM83H -/- cells were transiently transfected with FLAG empty vector (vector), FLAG-FAM83H D236A , FLAG-FAM83H S287X , FLAG-FAM83H S287X, D236A and FLAG-FAM83H WT . Cells were lysed and lysates incubated with anti-FLAG beads for 16 h. Immunoprecipitates (IP) and cell extracts (input) were resolved by SDS-PAGE and proteins immunoblotted with the antibodies indicated. n = 2. D U2OS FAM83H -/- cells transiently expressing FLAG-FAM83H S287X , FLAG-FAM83H S287X, D236A , FLAG-FAM83H WT and mCherry-CK1α were stained with anti-FLAG and DNA stained with DAPI. n = 2

Techniques Used: Activity Assay, Plasmid Preparation, Transfection, Immunoprecipitation, SDS Page, Incubation, Recombinant, Staining, Expressing

The interaction between FAM83H and CK1 can be detected at an endogenous level. A Schematic diagram to represent the mechanism by which A549 FAM83H GFP/GFP cells were generated using CRISPR/Cas9 genome editing technology. Arrows (^) represent guide RNAs. HA – homology arm. B GFP was immunoprecipitated from lysates of A549 FAM83H WT/WT transiently expressing GFP (WT + GFP) and A549 FAM83H GFP/GFP (KI) cells. Whole cell lysates (input) and immunoprecipitated proteins (IP) were resolved by SDS-PAGE then analysed by immunoblotting with the antibodies shown. n = 1. C As B , but after resolving samples by SDS-PAGE, the gel was stained in InstantBlue, the gel pieces were excised then processed and proteins digested with trypsin and peptides analysed by mass spectrometry. The table shows total spectral counts of FAM83H, CK1α, CK1δ and CK1ε. n = 1. D Schematic representation of mCherry-CK1 knockin proteins. E U2OS CK1 mCherry/mCherry cells or control U2OS wild type cells (−) were lysed, resolved by SDS-PAGE, stained in InstantBlue then gel pieces excised then processed and proteins digested with trypsin. Peptides were then analysed by mass spectrometry and total spectral counts are shown in the table. n = 1. F Cells from E were lysed and mCherry immunoprecipitated from cell lysates. Immunoblot shows cell extracts (input), immunoprecipitate (IP) and flow through (FT) samples. n = 2.
Figure Legend Snippet: The interaction between FAM83H and CK1 can be detected at an endogenous level. A Schematic diagram to represent the mechanism by which A549 FAM83H GFP/GFP cells were generated using CRISPR/Cas9 genome editing technology. Arrows (^) represent guide RNAs. HA – homology arm. B GFP was immunoprecipitated from lysates of A549 FAM83H WT/WT transiently expressing GFP (WT + GFP) and A549 FAM83H GFP/GFP (KI) cells. Whole cell lysates (input) and immunoprecipitated proteins (IP) were resolved by SDS-PAGE then analysed by immunoblotting with the antibodies shown. n = 1. C As B , but after resolving samples by SDS-PAGE, the gel was stained in InstantBlue, the gel pieces were excised then processed and proteins digested with trypsin and peptides analysed by mass spectrometry. The table shows total spectral counts of FAM83H, CK1α, CK1δ and CK1ε. n = 1. D Schematic representation of mCherry-CK1 knockin proteins. E U2OS CK1 mCherry/mCherry cells or control U2OS wild type cells (−) were lysed, resolved by SDS-PAGE, stained in InstantBlue then gel pieces excised then processed and proteins digested with trypsin. Peptides were then analysed by mass spectrometry and total spectral counts are shown in the table. n = 1. F Cells from E were lysed and mCherry immunoprecipitated from cell lysates. Immunoblot shows cell extracts (input), immunoprecipitate (IP) and flow through (FT) samples. n = 2.

Techniques Used: Generated, CRISPR, Immunoprecipitation, Expressing, SDS Page, Staining, Mass Spectrometry, Knock-In

Wild type FAM83H and disease mutants co-localise with CK1, but mutants do not colocalise with NCK1. A U2OS FAM83H -/- cells transiently expressing GFP, GFP-FAM83H, mCherry-NCK1 or mCherry-NCK2, as indicated, were fixed and stained with 4′,6-diamidino-2-phenylindole (DAPI). Cells were imaged using the DeltaVision widefield fluorescence microscope then images were deconvolved and processed using OMERO (12). Scale bar represents 10 μm. n = 3 B As A but U2OS FAM83H -/- cells were transiently transfected with FLAG-FAM83H WT (WT), FLAG-FAM83H S287X (S287X), FLAG-FAM83H Q452X (Q452X), FLAG-FAM83H E694X (E694X), or no FLAG construct (−) and co-transfected with mCherry-CK1α. Scale bar represents 10 μm. n = 3 C As B but with mCherry-NCK1 instead of mCherry-CK1α. n = 2 D U2OS FAM83H -/- cells transiently expressing FLAG (vector), FLAG-FAM83H S342T , FLAG-FAM83H G557C , FLAG-FAM83H WT and mCherry-CK1α were fixed and stained with anti-FLAG antibodies and DNA stained with DAPI. Cells were imaged using DeltaVision widefield fluorescence microscope. Scale bar represents 10 μm. n = 2 E As D but mCherry-NCK1 was used instead of mCherry-CK1α. Scale bar represents 10 μm. n = 1
Figure Legend Snippet: Wild type FAM83H and disease mutants co-localise with CK1, but mutants do not colocalise with NCK1. A U2OS FAM83H -/- cells transiently expressing GFP, GFP-FAM83H, mCherry-NCK1 or mCherry-NCK2, as indicated, were fixed and stained with 4′,6-diamidino-2-phenylindole (DAPI). Cells were imaged using the DeltaVision widefield fluorescence microscope then images were deconvolved and processed using OMERO (12). Scale bar represents 10 μm. n = 3 B As A but U2OS FAM83H -/- cells were transiently transfected with FLAG-FAM83H WT (WT), FLAG-FAM83H S287X (S287X), FLAG-FAM83H Q452X (Q452X), FLAG-FAM83H E694X (E694X), or no FLAG construct (−) and co-transfected with mCherry-CK1α. Scale bar represents 10 μm. n = 3 C As B but with mCherry-NCK1 instead of mCherry-CK1α. n = 2 D U2OS FAM83H -/- cells transiently expressing FLAG (vector), FLAG-FAM83H S342T , FLAG-FAM83H G557C , FLAG-FAM83H WT and mCherry-CK1α were fixed and stained with anti-FLAG antibodies and DNA stained with DAPI. Cells were imaged using DeltaVision widefield fluorescence microscope. Scale bar represents 10 μm. n = 2 E As D but mCherry-NCK1 was used instead of mCherry-CK1α. Scale bar represents 10 μm. n = 1

Techniques Used: Expressing, Staining, Fluorescence, Microscopy, Transfection, Construct, Plasmid Preparation

FAM83H interacts with NCK adaptor proteins. A Schematic diagram showing FAM83 proteins interact with CK1 isoforms and have unique interactors. B HEK-293 Flp-In T-REx cells that express GFP-FAM83 proteins under a doxycycline inducible promoter were treated with 20 ng/mL doxycycline for 24 h and GFP was pulled down from lysates then cell extracts (input) and pull downs (IP) were analysed by immunoblotting (IB). n = 3. C U2OS FAM83H -/- cells were transiently transfected with vectors encoding GFP, GFP-FAM83H and/or mCherry-UNC45A and lysates subjected to anti-GFP pull downs. Cell extracts (input) and pull downs (IP) were then analysed by Western blotting. n = 2. D Empty FLAG vector (vector), FLAG-FAM83H S287X , FLAG-FAM83H Q452X , FLAG-FAM83H E694X and FLAG-FAM83H WT were transiently transfected into U2OS FAM83H -/- cells. Untransfected U2OS FAM83H -/- cells (−) were used as an additional control. Anti-FLAG immunoprecipitations were performed on lysates and cell extracts (input) and immunoprecipitates (IP) were analysed by Western blotting. n = 3. E FLAG empty vector (vector), FLAG-FAM83H S342T , FLAG-FAM83H G557C , FLAG-FAM83H E694X and FLAG-FAM83H WT were transiently transfected into HEK-293 wild type cells. Anti-FLAG pull downs were performed on lysates then cell extracts (input) and immunoprecipitates (IP) were analysed by Western blotting. n = 1.
Figure Legend Snippet: FAM83H interacts with NCK adaptor proteins. A Schematic diagram showing FAM83 proteins interact with CK1 isoforms and have unique interactors. B HEK-293 Flp-In T-REx cells that express GFP-FAM83 proteins under a doxycycline inducible promoter were treated with 20 ng/mL doxycycline for 24 h and GFP was pulled down from lysates then cell extracts (input) and pull downs (IP) were analysed by immunoblotting (IB). n = 3. C U2OS FAM83H -/- cells were transiently transfected with vectors encoding GFP, GFP-FAM83H and/or mCherry-UNC45A and lysates subjected to anti-GFP pull downs. Cell extracts (input) and pull downs (IP) were then analysed by Western blotting. n = 2. D Empty FLAG vector (vector), FLAG-FAM83H S287X , FLAG-FAM83H Q452X , FLAG-FAM83H E694X and FLAG-FAM83H WT were transiently transfected into U2OS FAM83H -/- cells. Untransfected U2OS FAM83H -/- cells (−) were used as an additional control. Anti-FLAG immunoprecipitations were performed on lysates and cell extracts (input) and immunoprecipitates (IP) were analysed by Western blotting. n = 3. E FLAG empty vector (vector), FLAG-FAM83H S342T , FLAG-FAM83H G557C , FLAG-FAM83H E694X and FLAG-FAM83H WT were transiently transfected into HEK-293 wild type cells. Anti-FLAG pull downs were performed on lysates then cell extracts (input) and immunoprecipitates (IP) were analysed by Western blotting. n = 1.

Techniques Used: Transfection, Western Blot, Plasmid Preparation

23) Product Images from "Exostosin glycosyltransferase 1 reduces porcine reproductive and respiratory syndrome virus infection through proteasomal degradation of nsp3 and nsp5"

Article Title: Exostosin glycosyltransferase 1 reduces porcine reproductive and respiratory syndrome virus infection through proteasomal degradation of nsp3 and nsp5

Journal: The Journal of Biological Chemistry

doi: 10.1016/j.jbc.2021.101548

EXT1 promotes K48-linked polyubiquitination of nsp3 and nsp5. A and B , 293T cells were cotransfected with mCherry-tagged nsp3 or nsp5 together with HA-tagged ubiquitin for 24 h and were treated with MG132 (10 μM) for another 6 h. Then the cells were lysed for coimmunoprecipitation (co-IP) using an antibody against mCherry. The immunoprecipitates were analyzed by Western blot with antibodies against HA and mCherry. C and D , co-IP and Western blot analysis of 293T cells transfected with various combinations of plasmids encoding Myc-tagged EXT1-WT, Myc-tagged EXT1-D1, mCherry-tagged nsp3 ( C ) or nsp5 ( D ), and HA-tagged ubiquitin and treated with MG132 (10 μM). E and G , co-IP and Western blot analysis of 293T cells transfected with various combinations of plasmids encoding Myc-tagged EXT1-WT, Myc-tagged EXT1-D1, mCherry-tagged nsp3 and HA-tagged K48-linked ( E ) or K63-linked ( G ) ubiquitin, and treated with MG132 (10 μM). F and H , co-IP and Western blot analysis of 293T cells transfected with various combinations of plasmids encoding Myc-tagged EXT1-WT, Myc-tagged EXT1-D1, mCherry-tagged nsp5 and HA-tagged K48-linked ( F ) or K63-linked ( H ) ubiquitin, and treated with MG132 (10 μM). EXT1, exostosin glycosyltransferase 1; HA, hemagglutinin; nsp, nonstructural protein.
Figure Legend Snippet: EXT1 promotes K48-linked polyubiquitination of nsp3 and nsp5. A and B , 293T cells were cotransfected with mCherry-tagged nsp3 or nsp5 together with HA-tagged ubiquitin for 24 h and were treated with MG132 (10 μM) for another 6 h. Then the cells were lysed for coimmunoprecipitation (co-IP) using an antibody against mCherry. The immunoprecipitates were analyzed by Western blot with antibodies against HA and mCherry. C and D , co-IP and Western blot analysis of 293T cells transfected with various combinations of plasmids encoding Myc-tagged EXT1-WT, Myc-tagged EXT1-D1, mCherry-tagged nsp3 ( C ) or nsp5 ( D ), and HA-tagged ubiquitin and treated with MG132 (10 μM). E and G , co-IP and Western blot analysis of 293T cells transfected with various combinations of plasmids encoding Myc-tagged EXT1-WT, Myc-tagged EXT1-D1, mCherry-tagged nsp3 and HA-tagged K48-linked ( E ) or K63-linked ( G ) ubiquitin, and treated with MG132 (10 μM). F and H , co-IP and Western blot analysis of 293T cells transfected with various combinations of plasmids encoding Myc-tagged EXT1-WT, Myc-tagged EXT1-D1, mCherry-tagged nsp5 and HA-tagged K48-linked ( F ) or K63-linked ( H ) ubiquitin, and treated with MG132 (10 μM). EXT1, exostosin glycosyltransferase 1; HA, hemagglutinin; nsp, nonstructural protein.

Techniques Used: Co-Immunoprecipitation Assay, Western Blot, Transfection

EXT1 interacts with PRRSV nsp3 and nsp5 via its N-terminal cytoplasmic tail. A , colocalization of EXT1 protein with PRRSV nsp3 or nsp5 in 293T cells. 293T cells were cotransfected with Myc-tagged EXT1 and mCherry-tagged nsp3 or nsp5; 36 h later, the cells were fixed for immunofluorescent staining of EXT1-Myc ( green ), mCherry-nsp3 or nsp5 ( red ), and Golgi marker 58k or ER marker calnexin ( purple ). Nuclei were stained with DAPI ( blue ). Images of cells were acquired by laser-scanning fluorescent confocal microscopy. The bar represents 10 μm. B , 293T cells were cotransfected with Myc-tagged EXT1 and different mCherry-tagged PRRSV nsp-expressing plasmids, and 36 h later, cells were lysed for IP to analyze the interactions between EXT1 and these nsps. The asterisks indicate the specific nsp protein bands. C , Marc-145 cells were transiently transfected with Myc-tagged EXT1 plasmid for 36 h and then infected with PRRSV at an MOI of 0.5. The cells were lysed for IP to detect the interaction between EXT1 and nsp3. D , schematic representations of full-length or truncated EXT1. E – H , 293T cells were transiently cotransfected with plasmids expressing different EXT1 truncations and nsp3 or nsp5; 36 h later, cells were lysed and subjected to IP using a mouse anti-Myc monoclonal antibody. Immunoprecipitates were analyzed by Western blot with the indicated antibodies. DAPI, 4′,6-diamidino-2-phenylindole; ER, endoplasmic reticulum; EXT1, exostosin glycosyltransferase 1; IP, immunoprecipitation; nsp, nonstructural protein; PRRSV, porcine reproductive and respiratory syndrome virus.
Figure Legend Snippet: EXT1 interacts with PRRSV nsp3 and nsp5 via its N-terminal cytoplasmic tail. A , colocalization of EXT1 protein with PRRSV nsp3 or nsp5 in 293T cells. 293T cells were cotransfected with Myc-tagged EXT1 and mCherry-tagged nsp3 or nsp5; 36 h later, the cells were fixed for immunofluorescent staining of EXT1-Myc ( green ), mCherry-nsp3 or nsp5 ( red ), and Golgi marker 58k or ER marker calnexin ( purple ). Nuclei were stained with DAPI ( blue ). Images of cells were acquired by laser-scanning fluorescent confocal microscopy. The bar represents 10 μm. B , 293T cells were cotransfected with Myc-tagged EXT1 and different mCherry-tagged PRRSV nsp-expressing plasmids, and 36 h later, cells were lysed for IP to analyze the interactions between EXT1 and these nsps. The asterisks indicate the specific nsp protein bands. C , Marc-145 cells were transiently transfected with Myc-tagged EXT1 plasmid for 36 h and then infected with PRRSV at an MOI of 0.5. The cells were lysed for IP to detect the interaction between EXT1 and nsp3. D , schematic representations of full-length or truncated EXT1. E – H , 293T cells were transiently cotransfected with plasmids expressing different EXT1 truncations and nsp3 or nsp5; 36 h later, cells were lysed and subjected to IP using a mouse anti-Myc monoclonal antibody. Immunoprecipitates were analyzed by Western blot with the indicated antibodies. DAPI, 4′,6-diamidino-2-phenylindole; ER, endoplasmic reticulum; EXT1, exostosin glycosyltransferase 1; IP, immunoprecipitation; nsp, nonstructural protein; PRRSV, porcine reproductive and respiratory syndrome virus.

Techniques Used: Staining, Marker, Confocal Microscopy, Expressing, Transfection, Plasmid Preparation, Infection, Western Blot, Immunoprecipitation

EXT1 mediates PRRSV nsp3 and nsp5 reduction through proteasome degradation. A and B , 293T cells were cotransfected with Myc-tagged EXT1-WT or EXT1-D1 along with different mCherry-tagged PRRSV nsp-expressing plasmids, and 36 h later, cells were lysed for Western blot to analyze the abundance of PRRSV nsps. The abundance of PRRSV nsp3 and nsp5 in A was quantified by ImageJ software. The asterisks indicate the specific nsp protein bands. C , Marc-145 cells were transiently transfected with Myc-tagged EXT1-WT or EXT1-D1 plasmid for 36 h and then infected with PRRSV at an MOI of 1 for 18 h. The cells were lysed for Western blot to examine PRRSV nsp3. D , 293T cells were cotransfected with Myc-tagged EXT1-WT or EXT1-D1 together with mCherry-tagged PRRSV nsp3- or nsp5-expressing plasmids; 24 h later, cells were treated with MG132 (10 μM) for another 6 h and harvested to detect the nsp3 ( left ) or nsp5 ( right ) protein level. E , Myc-tagged EXT1-WT- or EXT1-D1-transfected Marc-145 cells were infected with PRRSV for 12 h at an MOI of 1 and then treated with MG132 (10 μM) for 6 h. The cells were lysed for Western blot using antibody against PRRSV nsp3. F , 293T cells were cotransfected with Myc-tagged EXT1-WT or EXT1-D1 and mCherry-tagged PRRSV nsp3- or nsp5-expressing plasmids for 24 h and treated with leupeptin (200 μM) for another 6 h. Cells were harvested to determine the nsp3 ( left ) or nsp5 ( right ) protein level. G , Myc-tagged EXT1-WT- or EXT1-D1-transfected Marc-145 cells were infected with PRRSV at an MOI of 1 for 12 h and then treated with leupeptin (200 μM) for 6 h. The cells were lysed for Western blot using antibody against PRRSV nsp3. EXT1, exostosin glycosyltransferase 1; MOI, multiplicity of infection; nsp, nonstructural protein; PRRSV, porcine reproductive and respiratory syndrome virus.
Figure Legend Snippet: EXT1 mediates PRRSV nsp3 and nsp5 reduction through proteasome degradation. A and B , 293T cells were cotransfected with Myc-tagged EXT1-WT or EXT1-D1 along with different mCherry-tagged PRRSV nsp-expressing plasmids, and 36 h later, cells were lysed for Western blot to analyze the abundance of PRRSV nsps. The abundance of PRRSV nsp3 and nsp5 in A was quantified by ImageJ software. The asterisks indicate the specific nsp protein bands. C , Marc-145 cells were transiently transfected with Myc-tagged EXT1-WT or EXT1-D1 plasmid for 36 h and then infected with PRRSV at an MOI of 1 for 18 h. The cells were lysed for Western blot to examine PRRSV nsp3. D , 293T cells were cotransfected with Myc-tagged EXT1-WT or EXT1-D1 together with mCherry-tagged PRRSV nsp3- or nsp5-expressing plasmids; 24 h later, cells were treated with MG132 (10 μM) for another 6 h and harvested to detect the nsp3 ( left ) or nsp5 ( right ) protein level. E , Myc-tagged EXT1-WT- or EXT1-D1-transfected Marc-145 cells were infected with PRRSV for 12 h at an MOI of 1 and then treated with MG132 (10 μM) for 6 h. The cells were lysed for Western blot using antibody against PRRSV nsp3. F , 293T cells were cotransfected with Myc-tagged EXT1-WT or EXT1-D1 and mCherry-tagged PRRSV nsp3- or nsp5-expressing plasmids for 24 h and treated with leupeptin (200 μM) for another 6 h. Cells were harvested to determine the nsp3 ( left ) or nsp5 ( right ) protein level. G , Myc-tagged EXT1-WT- or EXT1-D1-transfected Marc-145 cells were infected with PRRSV at an MOI of 1 for 12 h and then treated with leupeptin (200 μM) for 6 h. The cells were lysed for Western blot using antibody against PRRSV nsp3. EXT1, exostosin glycosyltransferase 1; MOI, multiplicity of infection; nsp, nonstructural protein; PRRSV, porcine reproductive and respiratory syndrome virus.

Techniques Used: Expressing, Western Blot, Software, Transfection, Plasmid Preparation, Infection

24) Product Images from "Role of Nuclear Lamin A/C in the Regulation of Nav1.5 Channel and Microtubules: Lesson From the Pathogenic Lamin A/C Variant Q517X"

Article Title: Role of Nuclear Lamin A/C in the Regulation of Nav1.5 Channel and Microtubules: Lesson From the Pathogenic Lamin A/C Variant Q517X

Journal: Frontiers in Cell and Developmental Biology

doi: 10.3389/fcell.2022.918760

(A) Left panel: representative western blot of p-ERK1/2 and ERK1/2 in lysates from LMNA WT and LMNA Q517X-expressing HL-1 cells; right panel: normalized densitometric analysis of p-ERK1/2 immunoreactive bands in LMNA WT and Q517X-expressing HL-1 cells. (B) Left panel: representative western blot of p-AKT and AKT in lysates from LMNA WT and LMNA Q517X-expressing HL-1 cells; right panel: normalized densitometric analysis of p-AKT immunoreactive bands in LMNA WT and Q517X-expressing HL-1 cells. (C) Left panel: representative western blot of acetylated α-tubulin (acet α-tub) and α-tubulin (tub) in lysates from LMNA WT and LMNA Q517X-expressing HL-1 cells; right panel: normalized densitometric analysis of acetylated α-tubulin immunoreactive bands in LMNA WT and Q517X-expressing HL-1 cells. (D) Left panel: representative western blot of mCherry-tagged LMNA in lysates from LMNA WT and LMNA Q517X-expressing HL-1 cells; right panel: normalized densitometric analysis of m-Cherry tagged LMNA immunoreactive bands. The data are means of 3 independent experiments. **** p
Figure Legend Snippet: (A) Left panel: representative western blot of p-ERK1/2 and ERK1/2 in lysates from LMNA WT and LMNA Q517X-expressing HL-1 cells; right panel: normalized densitometric analysis of p-ERK1/2 immunoreactive bands in LMNA WT and Q517X-expressing HL-1 cells. (B) Left panel: representative western blot of p-AKT and AKT in lysates from LMNA WT and LMNA Q517X-expressing HL-1 cells; right panel: normalized densitometric analysis of p-AKT immunoreactive bands in LMNA WT and Q517X-expressing HL-1 cells. (C) Left panel: representative western blot of acetylated α-tubulin (acet α-tub) and α-tubulin (tub) in lysates from LMNA WT and LMNA Q517X-expressing HL-1 cells; right panel: normalized densitometric analysis of acetylated α-tubulin immunoreactive bands in LMNA WT and Q517X-expressing HL-1 cells. (D) Left panel: representative western blot of mCherry-tagged LMNA in lysates from LMNA WT and LMNA Q517X-expressing HL-1 cells; right panel: normalized densitometric analysis of m-Cherry tagged LMNA immunoreactive bands. The data are means of 3 independent experiments. **** p

Techniques Used: Western Blot, Expressing

25) Product Images from "Plasticity in prefrontal cortex induced by coordinated nucleus reuniens and hippocampal synaptic transmission"

Article Title: Plasticity in prefrontal cortex induced by coordinated nucleus reuniens and hippocampal synaptic transmission

Journal: bioRxiv

doi: 10.1101/2020.07.11.197798

Electrophysiological characterisation of L5 pyramidal cells receiving optogenetically activated nucleus reuniens synapses. A - Representative widefield-fluorescence image showing neuronal transduction following injection of AAV9:CaMKii:hChR2(E123T/T159C):mCherry (red) into nucleus reuniens (Re) and DAPI (blue). VRe, ventral reuniens; Rh, rhomboid; Xi, xiphoid; PaXi, paraxiphoid; CM, central medial; AM, anteromedial; VM, ventromedial; MD, mediodorsal; Sub, submedius thalamic nuclei; mt, mammillothalamic tract. B - Monochrome image of mCherry positive fibres in PFC following AAV injection into nucleus reuniens. Dotted lines denote the boundaries of prelimbic cortex. mCherry signal is amplified with anti-mCherry antibody. Cg1 = cingulate cortex, IL = infralimbic cortex, PrL = prelimbic cortex. Scale bar = 500 µm. C - Schematic of acute mPFC slice with whole-cell recording from layer 5 pyramidal neuron in PrL, light activation of soma and proximal dendrites via microscope objective (blue) and stimulation of hippocampal fibre bundle using conventional stimulating electrode. D - Representative NRe (blue) and HPC (black) EPSPs. Blue arrow denotes light activation. E – Proportion of cells receiving different permutations of NRe and HPC inputs, 187 cells from 65 animals. Passive membrane properties measured from −100 pA current injection split by synaptic input. Resting membrane potential plotted as mean ± SD, one-way ANOVA F (3,183) = 1.2, p = 0.32. Other parameters one or more column failed Shapiro-Wilk test for normality, box plots show median and interquartile range, whiskers max and min data points. Kruskal-Wallis test p values: Tau = 0.074, Rinput = 0.031, Sag % = 0.0036, sag + rebound = 0.84. */** = p
Figure Legend Snippet: Electrophysiological characterisation of L5 pyramidal cells receiving optogenetically activated nucleus reuniens synapses. A - Representative widefield-fluorescence image showing neuronal transduction following injection of AAV9:CaMKii:hChR2(E123T/T159C):mCherry (red) into nucleus reuniens (Re) and DAPI (blue). VRe, ventral reuniens; Rh, rhomboid; Xi, xiphoid; PaXi, paraxiphoid; CM, central medial; AM, anteromedial; VM, ventromedial; MD, mediodorsal; Sub, submedius thalamic nuclei; mt, mammillothalamic tract. B - Monochrome image of mCherry positive fibres in PFC following AAV injection into nucleus reuniens. Dotted lines denote the boundaries of prelimbic cortex. mCherry signal is amplified with anti-mCherry antibody. Cg1 = cingulate cortex, IL = infralimbic cortex, PrL = prelimbic cortex. Scale bar = 500 µm. C - Schematic of acute mPFC slice with whole-cell recording from layer 5 pyramidal neuron in PrL, light activation of soma and proximal dendrites via microscope objective (blue) and stimulation of hippocampal fibre bundle using conventional stimulating electrode. D - Representative NRe (blue) and HPC (black) EPSPs. Blue arrow denotes light activation. E – Proportion of cells receiving different permutations of NRe and HPC inputs, 187 cells from 65 animals. Passive membrane properties measured from −100 pA current injection split by synaptic input. Resting membrane potential plotted as mean ± SD, one-way ANOVA F (3,183) = 1.2, p = 0.32. Other parameters one or more column failed Shapiro-Wilk test for normality, box plots show median and interquartile range, whiskers max and min data points. Kruskal-Wallis test p values: Tau = 0.074, Rinput = 0.031, Sag % = 0.0036, sag + rebound = 0.84. */** = p

Techniques Used: Fluorescence, Transduction, Injection, Amplification, Activation Assay, Microscopy

Nucleus reuniens input to prelimbic cortex depresses at theta frequency A - NRe inputs to L5 pyramidal neurons undergo strong short-term depression, and show different plasticity pattern to HPC inputs at 5Hz (repeated measures two-way ANOVA: main effect of pathway F (1,11) = 8.5, p = 0.014; main effect of response number F (1.9,20.6) = 5.1; p = 0.018, interaction F (3.1, 34.1) =4.4, p = 0.0095)and 10 Hz (pathway F (1,11) = 5.0, p = 0.048; response number F (3.2,35.4) = 27.9, p = 1.1 × 10 −9 ; interaction F (4.3,47.0) = 8.5, p=0.00002; Greenhouse-Geisser correction applied, both frequencies). N= 12 cells from 11 animals. B – NRe inputs to L2/3 pyramidal cells show equal degree of short-term depression as inputs to L5 at 5 Hz (Repeated-measures two-way ANOVA: main effect of layer F (1,19) = 0.71, p = 0.41; main effect of response number F (8,61.0) = 15.3, p = 2.6 × 10 −16 ; interaction F (3.2,61) = 0.45, p = 0.73) and 10Hz (main effect of layer: F (1,19) = 0.12, p = 0.73; main effect of response number F (8,62.7) = 30.3, p = 6.8 × 10 −28 ; interaction F (3.3,62.7) = 0.9, p = 0.44; Greenhouse-Geisser correction applied, both frequencies. L2/3 n = 9 cells from 5 animals; L5 data repeated from Fig 3A ). C – Following injection of AAV9-CaMKii-hChETATC-mCherry into intermediate/ventral HPC, transmission evoked by electrical and optogenetic stimulation were compared. EPSPs evoked by ChETA TC and electrical stimulation were of similar amplitude (paired t-test, t (12) = 0.78, p = 0.45). No difference in short-term plasticity was observed at 5 (main effect of stimulation method 5 Hz: F (1,12) = 0.07, p = 0.79; main effect of response number F (2.1,30.0) = 0.45, p = 0.65; interaction F (3.1,36.9) = 2.2, p = 0.10) or 10 Hz stimulation frequency (stimulation method F (1,12) = 0.38, p = 0.55; response number F (2.1,24.7) = 4.6, p = 0.02, interaction F (2.6,31.7) = 1.1, p =0.38). Greenhouse-Geisser corrections applied. N = 13 cells from 5 animals.
Figure Legend Snippet: Nucleus reuniens input to prelimbic cortex depresses at theta frequency A - NRe inputs to L5 pyramidal neurons undergo strong short-term depression, and show different plasticity pattern to HPC inputs at 5Hz (repeated measures two-way ANOVA: main effect of pathway F (1,11) = 8.5, p = 0.014; main effect of response number F (1.9,20.6) = 5.1; p = 0.018, interaction F (3.1, 34.1) =4.4, p = 0.0095)and 10 Hz (pathway F (1,11) = 5.0, p = 0.048; response number F (3.2,35.4) = 27.9, p = 1.1 × 10 −9 ; interaction F (4.3,47.0) = 8.5, p=0.00002; Greenhouse-Geisser correction applied, both frequencies). N= 12 cells from 11 animals. B – NRe inputs to L2/3 pyramidal cells show equal degree of short-term depression as inputs to L5 at 5 Hz (Repeated-measures two-way ANOVA: main effect of layer F (1,19) = 0.71, p = 0.41; main effect of response number F (8,61.0) = 15.3, p = 2.6 × 10 −16 ; interaction F (3.2,61) = 0.45, p = 0.73) and 10Hz (main effect of layer: F (1,19) = 0.12, p = 0.73; main effect of response number F (8,62.7) = 30.3, p = 6.8 × 10 −28 ; interaction F (3.3,62.7) = 0.9, p = 0.44; Greenhouse-Geisser correction applied, both frequencies. L2/3 n = 9 cells from 5 animals; L5 data repeated from Fig 3A ). C – Following injection of AAV9-CaMKii-hChETATC-mCherry into intermediate/ventral HPC, transmission evoked by electrical and optogenetic stimulation were compared. EPSPs evoked by ChETA TC and electrical stimulation were of similar amplitude (paired t-test, t (12) = 0.78, p = 0.45). No difference in short-term plasticity was observed at 5 (main effect of stimulation method 5 Hz: F (1,12) = 0.07, p = 0.79; main effect of response number F (2.1,30.0) = 0.45, p = 0.65; interaction F (3.1,36.9) = 2.2, p = 0.10) or 10 Hz stimulation frequency (stimulation method F (1,12) = 0.38, p = 0.55; response number F (2.1,24.7) = 4.6, p = 0.02, interaction F (2.6,31.7) = 1.1, p =0.38). Greenhouse-Geisser corrections applied. N = 13 cells from 5 animals.

Techniques Used: Injection, Transmission Assay

26) Product Images from "Fast 3D Clear: A Fast, Aqueous-Based, Reversible Three-Day Tissue Clearing Method for Adult and Embryonic Mouse Brain and Whole Body"

Article Title: Fast 3D Clear: A Fast, Aqueous-Based, Reversible Three-Day Tissue Clearing Method for Adult and Embryonic Mouse Brain and Whole Body

Journal: bioRxiv

doi: 10.1101/2021.06.25.449994

Fast 3D Clear is reversible and compatible with immunohistochemistry. Control (A-D) and (I-L) and stained sections (E-H) and ((M-P) with mCherry and doublecortin DCX antibodies from the basolateral amygdala (BLA) and perirhinal cortex from cFos-Cre ERT2 -tdTomato animals. (Scale bars 20 μm for 20x magnification).
Figure Legend Snippet: Fast 3D Clear is reversible and compatible with immunohistochemistry. Control (A-D) and (I-L) and stained sections (E-H) and ((M-P) with mCherry and doublecortin DCX antibodies from the basolateral amygdala (BLA) and perirhinal cortex from cFos-Cre ERT2 -tdTomato animals. (Scale bars 20 μm for 20x magnification).

Techniques Used: Immunohistochemistry, Staining

Fast 3D Clear is reversible and compatible with immunohistochemistry. Brain sections after reverse clearing. A-C) Sections from cFos-Cre ERT2 -tdTomato animals incubated only with secondary antibodies Alexa 488 and 647. Only the endogenous tdTomato (B) can be detected. D-F) Sections from the same animals stained with antibodies against mCherry (488) (D), tdTomato (E) and doublecortin (DCX) (F) (647). G) 60x magnification of the dentate gyrus (DG) stained for mCherry and DCX. H-I) Co- localization of mCherry antibody (488) with the endogenous transgenic tdTomato signal in basolateral amygdala (BLA) and in the Perirhinal Cortex, respectively. Doublecortin staining is completely absent from these regions. (Scale bars 20 μm for 20x and 5 μm for 60x magnification).
Figure Legend Snippet: Fast 3D Clear is reversible and compatible with immunohistochemistry. Brain sections after reverse clearing. A-C) Sections from cFos-Cre ERT2 -tdTomato animals incubated only with secondary antibodies Alexa 488 and 647. Only the endogenous tdTomato (B) can be detected. D-F) Sections from the same animals stained with antibodies against mCherry (488) (D), tdTomato (E) and doublecortin (DCX) (F) (647). G) 60x magnification of the dentate gyrus (DG) stained for mCherry and DCX. H-I) Co- localization of mCherry antibody (488) with the endogenous transgenic tdTomato signal in basolateral amygdala (BLA) and in the Perirhinal Cortex, respectively. Doublecortin staining is completely absent from these regions. (Scale bars 20 μm for 20x and 5 μm for 60x magnification).

Techniques Used: Immunohistochemistry, Incubation, Staining, Transgenic Assay

Fast 3D Clear preserves virally-delivered fluorescent proteins and injected dyes. A-D) Fluorescent images at 10x magnification showing labeling in the entorhinal cortex (EC) from animals injected with Fast Blue dye (A), AAV GCaMP6-CaMK2 (control) (B), retroAAV2 Arch-tdTomato (C), and retroAAV2 IRF670 (D). E-F) Dorsal hippocampus (E) and Entorhinal/Perirhinal Cortex (F) labelled with GFP and tdTomato fluorophores. G-H) Higher magnifications of the dentate gyrus (G) and entorhinal/perirhinal cortex (H). GCaMP3 homozygous animals injected with tdTomato-Cre virus. I-J) GFP activation in the dorsal hippocampus (I) and Entorhinal/Perirhinal Cortex (J) with cells labelled with both GFP and tdTomato fluorophores. K-L) Higher magnifications of the Dentate gyrus (K) and Entorhinal/Perirhinal cortex (L). (N=4). M) Injection of AAV hSyn-DIO-hM3D(Gq)-mCherry virus (DREADD) in the CA3 hippocampal region of CaMK2-Cre animals and N) higher magnification (N=2). (Scale bars 200 μm for 4x and 80 μm for 10x magnification).
Figure Legend Snippet: Fast 3D Clear preserves virally-delivered fluorescent proteins and injected dyes. A-D) Fluorescent images at 10x magnification showing labeling in the entorhinal cortex (EC) from animals injected with Fast Blue dye (A), AAV GCaMP6-CaMK2 (control) (B), retroAAV2 Arch-tdTomato (C), and retroAAV2 IRF670 (D). E-F) Dorsal hippocampus (E) and Entorhinal/Perirhinal Cortex (F) labelled with GFP and tdTomato fluorophores. G-H) Higher magnifications of the dentate gyrus (G) and entorhinal/perirhinal cortex (H). GCaMP3 homozygous animals injected with tdTomato-Cre virus. I-J) GFP activation in the dorsal hippocampus (I) and Entorhinal/Perirhinal Cortex (J) with cells labelled with both GFP and tdTomato fluorophores. K-L) Higher magnifications of the Dentate gyrus (K) and Entorhinal/Perirhinal cortex (L). (N=4). M) Injection of AAV hSyn-DIO-hM3D(Gq)-mCherry virus (DREADD) in the CA3 hippocampal region of CaMK2-Cre animals and N) higher magnification (N=2). (Scale bars 200 μm for 4x and 80 μm for 10x magnification).

Techniques Used: Injection, Labeling, Activation Assay

27) Product Images from "Plasticity in Prefrontal Cortex Induced by Coordinated Synaptic Transmission Arising from Reuniens/Rhomboid Nuclei and Hippocampus"

Article Title: Plasticity in Prefrontal Cortex Induced by Coordinated Synaptic Transmission Arising from Reuniens/Rhomboid Nuclei and Hippocampus

Journal: Cerebral Cortex Communications

doi: 10.1093/texcom/tgab029

Electrophysiological characterization of L5 pyramidal cells receiving optogenetically activated nucleus reuniens/rhomboid synapses. ( A ) Representative widefield-fluorescence image showing neuronal transduction of nucleus reuniens (Re) and rhomboid nucleus (Rh) following injection of AAV9:CaMKii:hChR2 (E123T/T159C):mCherry (red) and DAPI (blue). VRe, ventral reuniens; Xi, xiphoid; PaXi, paraxiphoid; CM, central medial; AM, anteromedial; VM, ventromedial; MD, mediodorsal; Sub, submedius thalamic nuclei; mt, mammillothalamic tract. ( B ) Monochrome image of mCherry positive fibers in PFC following AAV injection into ReRh. Dotted lines denote the boundaries of prelimbic cortex. mCherry signal is amplified with anti-mCherry antibody. Cg1, cingulate cortex; IL, infralimbic cortex; PrL, prelimbic cortex. Scale bar = 500 μm. ( C ) Schematic of acute mPFC slice with whole-cell recording from layer 5 pyramidal neuron in PrL, light activation of soma and proximal dendrites via microscope objective (blue) and stimulation of hippocampal fiber bundle using conventional stimulating electrode. ( D ) Representative ReRh (blue) and HPC (black) EPSPs. Blue arrow denotes light activation. ( E ) Proportion of cells receiving different permutations of ReRh and HPC inputs, 187 cells from 65 animals. Passive membrane properties measured from −100 pA current injection split by synaptic input. RMP plotted as mean ± standard deviation, one-way ANOVA F (3,183) = 1.2, P = 0.32. Other parameters one or more column failed Shapiro–Wilk test for normality, box plots show median and interquartile range, whiskers max and min data points. Kruskal–Wallis test P values: Tau = 0.074, Rinput = 0.031, Sag % = 0.0036, sag + rebound = 0.84. * / * * = P
Figure Legend Snippet: Electrophysiological characterization of L5 pyramidal cells receiving optogenetically activated nucleus reuniens/rhomboid synapses. ( A ) Representative widefield-fluorescence image showing neuronal transduction of nucleus reuniens (Re) and rhomboid nucleus (Rh) following injection of AAV9:CaMKii:hChR2 (E123T/T159C):mCherry (red) and DAPI (blue). VRe, ventral reuniens; Xi, xiphoid; PaXi, paraxiphoid; CM, central medial; AM, anteromedial; VM, ventromedial; MD, mediodorsal; Sub, submedius thalamic nuclei; mt, mammillothalamic tract. ( B ) Monochrome image of mCherry positive fibers in PFC following AAV injection into ReRh. Dotted lines denote the boundaries of prelimbic cortex. mCherry signal is amplified with anti-mCherry antibody. Cg1, cingulate cortex; IL, infralimbic cortex; PrL, prelimbic cortex. Scale bar = 500 μm. ( C ) Schematic of acute mPFC slice with whole-cell recording from layer 5 pyramidal neuron in PrL, light activation of soma and proximal dendrites via microscope objective (blue) and stimulation of hippocampal fiber bundle using conventional stimulating electrode. ( D ) Representative ReRh (blue) and HPC (black) EPSPs. Blue arrow denotes light activation. ( E ) Proportion of cells receiving different permutations of ReRh and HPC inputs, 187 cells from 65 animals. Passive membrane properties measured from −100 pA current injection split by synaptic input. RMP plotted as mean ± standard deviation, one-way ANOVA F (3,183) = 1.2, P = 0.32. Other parameters one or more column failed Shapiro–Wilk test for normality, box plots show median and interquartile range, whiskers max and min data points. Kruskal–Wallis test P values: Tau = 0.074, Rinput = 0.031, Sag % = 0.0036, sag + rebound = 0.84. * / * * = P

Techniques Used: Fluorescence, Transduction, Injection, Amplification, Activation Assay, Microscopy, Standard Deviation

Reuniens/rhomboid inputs to prelimbic cortex depress at theta frequency. ( A ) ReRh inputs to L5 pyramidal neurons undergo strong short-term depression, and show different plasticity pattern to HPC inputs at 5 Hz (repeated measures 2-way ANOVA: main effect of pathway F (1,11) = 8.5, P = 0.014; main effect of response number F (1.9,20.6) = 5.1; P = 0.018, interaction F (3.1, 34.1) = 4.4, P = 0.0095) and 10 Hz (pathway F (1,11) = 5.0, P = 0.048; response number F (3.2,35.4) = 27.9, P = 1.1 × 10 −9 ; interaction F (4.3,47.0) = 8.5, P = 0.00002; Greenhouse–Geisser correction applied, both frequencies). N = 12 cells from 11 animals. Scale bars = 0.3 mV/200 ms. ( B ) ReRh inputs to L2/3 pyramidal cells show equal degree of short-term depression as inputs to L5 at 5 Hz (Repeated-measures 2-way ANOVA: main effect of layer F (1,19) = 0.71, P = 0.41; main effect of response number F (8,61.0) = 15.3, P = 2.6 × 10 −16 ; interaction F (3.2,61) = 0.45, P = 0.73) and 10 Hz (main effect of layer: F (1,19) = 0.12, P = 0.73; main effect of response number F (8,62.7) = 30.3, P = 6.8 × 10 −28 ; interaction F (3.3,62.7) = 0.9, P = 0.44; Greenhouse–Geisser correction applied, both frequencies. L2/3 n = 9 cells from 5 animals; L5 data repeated from Fig 3 A ). ( C ) Following injection of AAV9-CaMKii-hChETA TC -mCherry into intermediate/ventral HPC, acute mPFC slices were made and HPC-mPFC transmission evoked by electrical and optogenetic stimulation were compared. EPSPs evoked by ChETA TC and electrical stimulation were of similar amplitude (paired t -test, t (12) = 0.78, P = 0.45). No difference in short-term plasticity was observed at 5 (main effect of stimulation method 5 Hz: F (1,12) = 0.07, P = 0.79; main effect of response number F (2.1,30.0) = 0.45, P = 0.65; interaction F (3.1,36.9) = 2.2, P = 0.10) or 10 Hz stimulation frequency (stimulation method F (1,12) = 0.38, P = 0.55; response number F (2.1,24.7) = 4.6, P = 0.02, interaction F (2.6,31.7) = 1.1, P = 0.38). Greenhouse–Geisser corrections applied. N = 13 cells from 5 animals.
Figure Legend Snippet: Reuniens/rhomboid inputs to prelimbic cortex depress at theta frequency. ( A ) ReRh inputs to L5 pyramidal neurons undergo strong short-term depression, and show different plasticity pattern to HPC inputs at 5 Hz (repeated measures 2-way ANOVA: main effect of pathway F (1,11) = 8.5, P = 0.014; main effect of response number F (1.9,20.6) = 5.1; P = 0.018, interaction F (3.1, 34.1) = 4.4, P = 0.0095) and 10 Hz (pathway F (1,11) = 5.0, P = 0.048; response number F (3.2,35.4) = 27.9, P = 1.1 × 10 −9 ; interaction F (4.3,47.0) = 8.5, P = 0.00002; Greenhouse–Geisser correction applied, both frequencies). N = 12 cells from 11 animals. Scale bars = 0.3 mV/200 ms. ( B ) ReRh inputs to L2/3 pyramidal cells show equal degree of short-term depression as inputs to L5 at 5 Hz (Repeated-measures 2-way ANOVA: main effect of layer F (1,19) = 0.71, P = 0.41; main effect of response number F (8,61.0) = 15.3, P = 2.6 × 10 −16 ; interaction F (3.2,61) = 0.45, P = 0.73) and 10 Hz (main effect of layer: F (1,19) = 0.12, P = 0.73; main effect of response number F (8,62.7) = 30.3, P = 6.8 × 10 −28 ; interaction F (3.3,62.7) = 0.9, P = 0.44; Greenhouse–Geisser correction applied, both frequencies. L2/3 n = 9 cells from 5 animals; L5 data repeated from Fig 3 A ). ( C ) Following injection of AAV9-CaMKii-hChETA TC -mCherry into intermediate/ventral HPC, acute mPFC slices were made and HPC-mPFC transmission evoked by electrical and optogenetic stimulation were compared. EPSPs evoked by ChETA TC and electrical stimulation were of similar amplitude (paired t -test, t (12) = 0.78, P = 0.45). No difference in short-term plasticity was observed at 5 (main effect of stimulation method 5 Hz: F (1,12) = 0.07, P = 0.79; main effect of response number F (2.1,30.0) = 0.45, P = 0.65; interaction F (3.1,36.9) = 2.2, P = 0.10) or 10 Hz stimulation frequency (stimulation method F (1,12) = 0.38, P = 0.55; response number F (2.1,24.7) = 4.6, P = 0.02, interaction F (2.6,31.7) = 1.1, P = 0.38). Greenhouse–Geisser corrections applied. N = 13 cells from 5 animals.

Techniques Used: Injection, Transmission Assay

28) Product Images from "Experimental considerations for study of C. elegans lysosomal proteins"

Article Title: Experimental considerations for study of C. elegans lysosomal proteins

Journal: bioRxiv

doi: 10.1101/2022.06.30.498309

Rigid proline linkers and truncated mCherry does not prevent cleavage from a NUC-1 fusion. Animals of the indicated genotype were synchronized and harvested in mid L4 (48 hours postrelease), late L4 (56 hours post-release), and adulthood (72 hours post-release) for immunoblotting (A) and imaging (B). We performed anti-alpha tubulin, anti-FLAG, and anti-mCherry immunoblots on lysates from the indicated genotypes (A). The blots and images are representative of three experimental replicates. Marker size (in kDa) is provided. Full-length NUC-1::mCherry fusions and cleavage products are indicated by arrows. Non-specific background bands are indicated by asterisks (*). Note that in the adult samples an adult-specific background band not seen in L4 larvae appears. These background bands are indicated by double asterisks (**). (B) Animals of the indicated genotype were imaged at mid L4 and late L4. DIC and mCherry images are provided for each strain and time point. Scale bars=20 μm. Images are representative of 50 animals examined per genotype in two independent experiments.
Figure Legend Snippet: Rigid proline linkers and truncated mCherry does not prevent cleavage from a NUC-1 fusion. Animals of the indicated genotype were synchronized and harvested in mid L4 (48 hours postrelease), late L4 (56 hours post-release), and adulthood (72 hours post-release) for immunoblotting (A) and imaging (B). We performed anti-alpha tubulin, anti-FLAG, and anti-mCherry immunoblots on lysates from the indicated genotypes (A). The blots and images are representative of three experimental replicates. Marker size (in kDa) is provided. Full-length NUC-1::mCherry fusions and cleavage products are indicated by arrows. Non-specific background bands are indicated by asterisks (*). Note that in the adult samples an adult-specific background band not seen in L4 larvae appears. These background bands are indicated by double asterisks (**). (B) Animals of the indicated genotype were imaged at mid L4 and late L4. DIC and mCherry images are provided for each strain and time point. Scale bars=20 μm. Images are representative of 50 animals examined per genotype in two independent experiments.

Techniques Used: Imaging, Western Blot, Marker

Gamillus is not quenched in lysosomes and does not disrupt localization of his-72, lmn-1 , and glh-1 fusion proteins. Mixed stage animals carrying extrachromosomal arrays of hsp-16.41::nuc-1::Gamillus::mCherry (A) and hsp-16.41::nuc-1::Gamillus::mScarlet (B) were heat-shocked for 30 minutes at 34°C and adult animals were images 24 hours later. A merge of green and red fluorescent channels is provided. Three biological replicates were performed. Images are representative of 33 animals for JDW288 and JDW304, 49 animals for JDW305, and 8 animals for JDW289. The extrachromosomal array in JDW289 transmits at low rates, hence the lower number of animals scored. Scale bars =5 μm. Gamillus::HIS-72 and DIC image of adult head (C) and embryos (D). Images are representative of 20 animals in two independent replicates. Scale bars=20μm. Gamillus::LMN-1 and DIC image of adult head (E) and embryos (F). Images are representative of 20 animals in two independent replicates. Scale bars=20μm. GFP::GLH-1 and Gamillus::GLH-1 germline images along with DIC overlays (G). A processed Gamillus::GLH-1 image where fluorescence was increased 100% is provided. Scale bars=20μm.
Figure Legend Snippet: Gamillus is not quenched in lysosomes and does not disrupt localization of his-72, lmn-1 , and glh-1 fusion proteins. Mixed stage animals carrying extrachromosomal arrays of hsp-16.41::nuc-1::Gamillus::mCherry (A) and hsp-16.41::nuc-1::Gamillus::mScarlet (B) were heat-shocked for 30 minutes at 34°C and adult animals were images 24 hours later. A merge of green and red fluorescent channels is provided. Three biological replicates were performed. Images are representative of 33 animals for JDW288 and JDW304, 49 animals for JDW305, and 8 animals for JDW289. The extrachromosomal array in JDW289 transmits at low rates, hence the lower number of animals scored. Scale bars =5 μm. Gamillus::HIS-72 and DIC image of adult head (C) and embryos (D). Images are representative of 20 animals in two independent replicates. Scale bars=20μm. Gamillus::LMN-1 and DIC image of adult head (E) and embryos (F). Images are representative of 20 animals in two independent replicates. Scale bars=20μm. GFP::GLH-1 and Gamillus::GLH-1 germline images along with DIC overlays (G). A processed Gamillus::GLH-1 image where fluorescence was increased 100% is provided. Scale bars=20μm.

Techniques Used: Fluorescence

29) Product Images from "Kinesin-binding protein remodels the kinesin motor to prevent microtubule-binding"

Article Title: Kinesin-binding protein remodels the kinesin motor to prevent microtubule-binding

Journal: bioRxiv

doi: 10.1101/2021.06.02.446814

Mutations in KIFBP-L1 and KIFBP-L14 disrupt KlFBP-regulation of KIF18A localization. (A) KIF18A localization in MG132 arrested HeLa Kyoto cells overexpressing mCherry or indicated mCherry-KIFBP construct. Hed is used as a marker for the kinetochore. Scale bar 2 mm. (B) Line scan analyses of KIF18A distribution along kinetochore microtubules. Fluorescence values were normalized, aligned by peak Hed intensity, and averaged across multiple line scans. Hed, blue; KIF18A, green; Tubulin, magenta. Solid lines indicate the means, shaded areas indicate standard deviation. A.U. indicates arbitrary units. The following cell numbers and line scans were analyzed for the mCherry and mCherry-KIFBP constructs: (1) mCherry (control) = 40 cells (64 lines), (2) mCherry-KIFBP-WT = 34 cells (64 lines), (3) rnCherry-KIFBP-L1 m = 34 cells (64 lines), (4) rnCherry-KIFBP-L14 m = 32 cells (68 lines), (5) rnCherry-KIFBP-HP9b m = 33 cells (63 lines).
Figure Legend Snippet: Mutations in KIFBP-L1 and KIFBP-L14 disrupt KlFBP-regulation of KIF18A localization. (A) KIF18A localization in MG132 arrested HeLa Kyoto cells overexpressing mCherry or indicated mCherry-KIFBP construct. Hed is used as a marker for the kinetochore. Scale bar 2 mm. (B) Line scan analyses of KIF18A distribution along kinetochore microtubules. Fluorescence values were normalized, aligned by peak Hed intensity, and averaged across multiple line scans. Hed, blue; KIF18A, green; Tubulin, magenta. Solid lines indicate the means, shaded areas indicate standard deviation. A.U. indicates arbitrary units. The following cell numbers and line scans were analyzed for the mCherry and mCherry-KIFBP constructs: (1) mCherry (control) = 40 cells (64 lines), (2) mCherry-KIFBP-WT = 34 cells (64 lines), (3) rnCherry-KIFBP-L1 m = 34 cells (64 lines), (4) rnCherry-KIFBP-L14 m = 32 cells (68 lines), (5) rnCherry-KIFBP-HP9b m = 33 cells (63 lines).

Techniques Used: Construct, Marker, Fluorescence, Standard Deviation

Mutations in KIFBP-L1 and KIFBP-L14 diminish KlFBP-mediated regulation of spindle length and chromosome alignment during mitosis. (A) MG132 arrested HeLa Kyoto cells overexpressing mCherry or indicated mCherry-KlFBP construct. Scale bar 2 mm. (B) Top: Graph of spindle lengths measured in cells overexpressing mCherry or indicated mCherry-KlFBP construct. Each dot represents a single cell. Mean ± standard deviation is displayed. Statistical results are shown fora one-wayANOVAwith Tukey’s Multiple Comparisons test. N.s. indicates not significant, *”* indicates adjusted p-value
Figure Legend Snippet: Mutations in KIFBP-L1 and KIFBP-L14 diminish KlFBP-mediated regulation of spindle length and chromosome alignment during mitosis. (A) MG132 arrested HeLa Kyoto cells overexpressing mCherry or indicated mCherry-KlFBP construct. Scale bar 2 mm. (B) Top: Graph of spindle lengths measured in cells overexpressing mCherry or indicated mCherry-KlFBP construct. Each dot represents a single cell. Mean ± standard deviation is displayed. Statistical results are shown fora one-wayANOVAwith Tukey’s Multiple Comparisons test. N.s. indicates not significant, *”* indicates adjusted p-value

Techniques Used: Construct, Standard Deviation

30) Product Images from "SAC-1 ensures epithelial endocytic recycling by restricting ARF-6 activity"

Article Title: SAC-1 ensures epithelial endocytic recycling by restricting ARF-6 activity

Journal: The Journal of Cell Biology

doi: 10.1083/jcb.201711065

The recycling transport of hTAC-GFP is impaired in sac-1 mutants. (A and A′) In the top focal plane, IL-2 receptor α chain hTAC-GFP–labeled tubular structures were severely perturbed in sac-1(ycx18) mutants. In the middle focal plane, hTAC-GFP overaccumulated on the cytosolic endosomal structures. An approximately 5.4-fold escalation of total hTAC-GFP intensity was observed in sac-1 animals. Also, in mCherry-SAC-1–overexpressing intestinal cells, hTAC-GFP accumulated on the enlarged structures. RNAi-mediated knockdown of PPK-1 failed to alleviate the overaccumulation phenotype of hTAC-GFP in sac-1(ycx18) mutants. Asterisks in the panels indicate intestinal lumen. Error bars represent SEM ( n = 18 each), and asterisks indicate significant differences in the one-tailed Student’s t test (***, P
Figure Legend Snippet: The recycling transport of hTAC-GFP is impaired in sac-1 mutants. (A and A′) In the top focal plane, IL-2 receptor α chain hTAC-GFP–labeled tubular structures were severely perturbed in sac-1(ycx18) mutants. In the middle focal plane, hTAC-GFP overaccumulated on the cytosolic endosomal structures. An approximately 5.4-fold escalation of total hTAC-GFP intensity was observed in sac-1 animals. Also, in mCherry-SAC-1–overexpressing intestinal cells, hTAC-GFP accumulated on the enlarged structures. RNAi-mediated knockdown of PPK-1 failed to alleviate the overaccumulation phenotype of hTAC-GFP in sac-1(ycx18) mutants. Asterisks in the panels indicate intestinal lumen. Error bars represent SEM ( n = 18 each), and asterisks indicate significant differences in the one-tailed Student’s t test (***, P

Techniques Used: Labeling, One-tailed Test

Loss of SAC-1 leads to increased PI(4,5)P2 levels and overaccumulation of actin structures. (A and A′) Compared with wild-type animals, the labeling intensity of Tb R332H -GFP in the basolateral endosomes was significantly increased (by ∼3.3-fold) in sac-1(ycx18) mutants. However, in mCherry-SAC-1–overexpressing intestinal cells, Tb R332H -GFP labeling in puncta and tubules decreased remarkably. Furthermore, RNAi-mediated knockdown of PPK-1 (PIP5 kinase) significantly reversed the intensity of Tb R332H -GFP in sac-1 mutants. Error bars represent SEM ( n = 18 each), and asterisks indicate significant differences in the one-tailed Student’s t test (*, P
Figure Legend Snippet: Loss of SAC-1 leads to increased PI(4,5)P2 levels and overaccumulation of actin structures. (A and A′) Compared with wild-type animals, the labeling intensity of Tb R332H -GFP in the basolateral endosomes was significantly increased (by ∼3.3-fold) in sac-1(ycx18) mutants. However, in mCherry-SAC-1–overexpressing intestinal cells, Tb R332H -GFP labeling in puncta and tubules decreased remarkably. Furthermore, RNAi-mediated knockdown of PPK-1 (PIP5 kinase) significantly reversed the intensity of Tb R332H -GFP in sac-1 mutants. Error bars represent SEM ( n = 18 each), and asterisks indicate significant differences in the one-tailed Student’s t test (*, P

Techniques Used: Labeling, One-tailed Test

SAC-1 interacts with the active and inactive mutant forms of ARF-6, and the residence of SAC-1 in basolateral puncta requires ARF-6. (A) Western blot showing GST pull-down with in vitro–translated HA-tagged proteins. Glutathione beads loaded with GST and GST-SAC-1 were incubated with in vitro–expressed HA-tagged RAB-5(Q78L), RAB-7(Q68L), RAB-8(Q67L), RAB-10(Q68L), RAB-11(Q70L), ARF-6(Q67L), and ARF-6(T27N). Eluted proteins were separated on the SDS-PAGE gel and analyzed by Western blotting using anti–HA antibody. Input lanes contain in vitro–expressed HA-tagged proteins in the binding assays (5%). (B and B′) SAC-1 colocalized well with ARF-6–, RAB-5–, or RAB-10–labeled endosomes. In addition, SAC-1 overlaps significantly with the TGN marker AMAN-2 and the ER marker SP12. Nevertheless, SAC-1 displayed little colocalization with the late endosome marker RAB-7. Arrowheads indicate positive overlap. Pearson’s correlation coefficients for GFP and mCherry signals were calculated ( n = 6 animals). (C and C′) Compared with wild-type animals, the labeling of GFP-SAC-1 in basolateral puncta was significantly reduced in the absence of ARF-6 (top focal plane). Transgenic expression of ARF-6(Q67L)-mCherry rescued the puncta labeling GFP-SAC-1, and the transgenic expression of ARF-6(T27N)-mCherry failed to restore the endosomal localization of GFP-SAC-1. Asterisks in the panels indicate intestinal lumen. Error bars represent SEM ( n = 18 each), and asterisks indicate the significant differences in the one-tailed Student’s t test (ns, no significance; **, P
Figure Legend Snippet: SAC-1 interacts with the active and inactive mutant forms of ARF-6, and the residence of SAC-1 in basolateral puncta requires ARF-6. (A) Western blot showing GST pull-down with in vitro–translated HA-tagged proteins. Glutathione beads loaded with GST and GST-SAC-1 were incubated with in vitro–expressed HA-tagged RAB-5(Q78L), RAB-7(Q68L), RAB-8(Q67L), RAB-10(Q68L), RAB-11(Q70L), ARF-6(Q67L), and ARF-6(T27N). Eluted proteins were separated on the SDS-PAGE gel and analyzed by Western blotting using anti–HA antibody. Input lanes contain in vitro–expressed HA-tagged proteins in the binding assays (5%). (B and B′) SAC-1 colocalized well with ARF-6–, RAB-5–, or RAB-10–labeled endosomes. In addition, SAC-1 overlaps significantly with the TGN marker AMAN-2 and the ER marker SP12. Nevertheless, SAC-1 displayed little colocalization with the late endosome marker RAB-7. Arrowheads indicate positive overlap. Pearson’s correlation coefficients for GFP and mCherry signals were calculated ( n = 6 animals). (C and C′) Compared with wild-type animals, the labeling of GFP-SAC-1 in basolateral puncta was significantly reduced in the absence of ARF-6 (top focal plane). Transgenic expression of ARF-6(Q67L)-mCherry rescued the puncta labeling GFP-SAC-1, and the transgenic expression of ARF-6(T27N)-mCherry failed to restore the endosomal localization of GFP-SAC-1. Asterisks in the panels indicate intestinal lumen. Error bars represent SEM ( n = 18 each), and asterisks indicate the significant differences in the one-tailed Student’s t test (ns, no significance; **, P

Techniques Used: Mutagenesis, Western Blot, In Vitro, Incubation, SDS Page, Binding Assay, Labeling, Marker, Transgenic Assay, Expressing, One-tailed Test

SAC-1 competes with ARF-6(T27N) for binding to the Sec7 domain of BRIS-1. (A–A″) SAC-1 outcompeted ARF-6(T27N) for interaction with the Sec7 domain in a concentration-dependent manner, as determined by GST pull-down. Three independent experiments are represented in the histogram, error bars represent SEM ( n = 3 each), and P values are indicated above. (B and B′) In wild-type animals, mCherry-BRIS-1 mainly labeled basolateral tubules and puncta and partially colocalized with ARF-6-GFP (Pearson’s coefficient, ∼72.4%). In sac-1(ycx18) mutants, mCherry-BRIS-1 and ARF-6-GFP were concentrated in the intracellular aggregates, and their colocalization level was substantially promoted (Pearson’s coefficient, ∼85.4%). Arrowheads indicate positive overlap. Pearson’s correlation coefficients for GFP and mCherry signals were calculated ( n = 6 animals). Asterisks indicate significant differences in the one-tailed Student’s t test (**, P
Figure Legend Snippet: SAC-1 competes with ARF-6(T27N) for binding to the Sec7 domain of BRIS-1. (A–A″) SAC-1 outcompeted ARF-6(T27N) for interaction with the Sec7 domain in a concentration-dependent manner, as determined by GST pull-down. Three independent experiments are represented in the histogram, error bars represent SEM ( n = 3 each), and P values are indicated above. (B and B′) In wild-type animals, mCherry-BRIS-1 mainly labeled basolateral tubules and puncta and partially colocalized with ARF-6-GFP (Pearson’s coefficient, ∼72.4%). In sac-1(ycx18) mutants, mCherry-BRIS-1 and ARF-6-GFP were concentrated in the intracellular aggregates, and their colocalization level was substantially promoted (Pearson’s coefficient, ∼85.4%). Arrowheads indicate positive overlap. Pearson’s correlation coefficients for GFP and mCherry signals were calculated ( n = 6 animals). Asterisks indicate significant differences in the one-tailed Student’s t test (**, P

Techniques Used: Binding Assay, Concentration Assay, Labeling, One-tailed Test

31) Product Images from "Heparan Sulfate Structure Affects Autophagy, Lifespan, Responses to Oxidative Stress, and Cell Degeneration in Drosophila parkin Mutants"

Article Title: Heparan Sulfate Structure Affects Autophagy, Lifespan, Responses to Oxidative Stress, and Cell Degeneration in Drosophila parkin Mutants

Journal: G3: Genes|Genomes|Genetics

doi: 10.1534/g3.119.400730

Reduction of heparan sulfate biosynthetic function prevents increases of ubiquitin-modified protein in the brains of ROS exposed animals. Accumulation of insoluble ubiquitinated proteins (IUPs) was examined in the triton-X100 insoluble fraction of total head proteins. UAS-mCherry RNAi, an shRNAi with no predicted targets in the Drosophila genome, was used as a control. All plus/minus oxidant pairs are from the same blot. All lanes are from the same experiment, with the same standard sample loaded on all gels from this one experiment for standardization (see Supplemental Figure 3 for original blots). Overexpression of Atg8a ( UAS-Atg8a ) was used as a positive control for enhancement of autophagy. The UAS-constructs were expressed under control of elav -GAL4 with UAS-dcrII to enhance RNAi efficacy. Lanes from anti-ubiquitin stained membranes are shown in control vs. oxidant-exposed pairs. The ratio depicted is the average density of anti-ubiquitin staining, normalized to loading, in samples with peroxide (+) divided by samples without peroxide (-). Two different sfl RNAi lines were tested. Two additional replicate experiments gave average peroxide/no peroxide ratios of 1.23 for control, and 0.8 for sfl RNAi-HMS . Two replicates of ttv RNAi also showed lower levels of ubiquitin-insoluble material upon peroxide exposure (ratio of 0.7).
Figure Legend Snippet: Reduction of heparan sulfate biosynthetic function prevents increases of ubiquitin-modified protein in the brains of ROS exposed animals. Accumulation of insoluble ubiquitinated proteins (IUPs) was examined in the triton-X100 insoluble fraction of total head proteins. UAS-mCherry RNAi, an shRNAi with no predicted targets in the Drosophila genome, was used as a control. All plus/minus oxidant pairs are from the same blot. All lanes are from the same experiment, with the same standard sample loaded on all gels from this one experiment for standardization (see Supplemental Figure 3 for original blots). Overexpression of Atg8a ( UAS-Atg8a ) was used as a positive control for enhancement of autophagy. The UAS-constructs were expressed under control of elav -GAL4 with UAS-dcrII to enhance RNAi efficacy. Lanes from anti-ubiquitin stained membranes are shown in control vs. oxidant-exposed pairs. The ratio depicted is the average density of anti-ubiquitin staining, normalized to loading, in samples with peroxide (+) divided by samples without peroxide (-). Two different sfl RNAi lines were tested. Two additional replicate experiments gave average peroxide/no peroxide ratios of 1.23 for control, and 0.8 for sfl RNAi-HMS . Two replicates of ttv RNAi also showed lower levels of ubiquitin-insoluble material upon peroxide exposure (ratio of 0.7).

Techniques Used: Modification, Over Expression, Positive Control, Construct, Staining

Autophagy-dependent cleavage of mCherry-Atg8a is increased in the CNS upon RNA interference of sfl or ttv . Heads from adult animals bearing transgene constructs expressing an mCherry-Atg8a fusion protein in neurons were obtained and mCherry-Atg8a protein detected by SDS-PAGE and western blotting with anti-mCherry antibody. The large fusion protein is cleaved during autophagosome maturation to a relatively stable product that contains the mCherry epitope. The ratio of the parental and cleavage products provides a measure of autophagy-dependent activity in the CNS. UAS-w RNAi was used as the control sample. Activation of autophagy in neurons with overexpression of Atg8a shows increased relative levels of the mCherry-bearing cleavage product. Knockdown of either sfl or ttv using RNAi also produces an increase in the relative levels of mCherry-Atg8a cleavage in the brain. Ratios provided below the image are averages of 4 samples, 2 replicates in two separate experiments. QPCR of sfl mRNA showed reductions to 42% of wild type levels (data not shown).
Figure Legend Snippet: Autophagy-dependent cleavage of mCherry-Atg8a is increased in the CNS upon RNA interference of sfl or ttv . Heads from adult animals bearing transgene constructs expressing an mCherry-Atg8a fusion protein in neurons were obtained and mCherry-Atg8a protein detected by SDS-PAGE and western blotting with anti-mCherry antibody. The large fusion protein is cleaved during autophagosome maturation to a relatively stable product that contains the mCherry epitope. The ratio of the parental and cleavage products provides a measure of autophagy-dependent activity in the CNS. UAS-w RNAi was used as the control sample. Activation of autophagy in neurons with overexpression of Atg8a shows increased relative levels of the mCherry-bearing cleavage product. Knockdown of either sfl or ttv using RNAi also produces an increase in the relative levels of mCherry-Atg8a cleavage in the brain. Ratios provided below the image are averages of 4 samples, 2 replicates in two separate experiments. QPCR of sfl mRNA showed reductions to 42% of wild type levels (data not shown).

Techniques Used: Construct, Expressing, SDS Page, Western Blot, Activity Assay, Activation Assay, Over Expression, Real-time Polymerase Chain Reaction

32) Product Images from "The E3 ligase TRIM1 ubiquitinates LRRK2 and controls its localization, degradation, and toxicity"

Article Title: The E3 ligase TRIM1 ubiquitinates LRRK2 and controls its localization, degradation, and toxicity

Journal: bioRxiv

doi: 10.1101/2020.10.21.336578

TRIM1 mediates proteasomal degradation of PD-mutant LRRK2 G2019S to rescue its toxicity. (A) Live-cell confocal microscopy of GFP-LRRK2 G2019S and mcherry-TRIM1 transiently transfected into H1299 cells. (B) Co-immunoprecipitation and ubiquitination of GFP-LRRK2 G2019S with myc-TRIM1 in the presence of HA-ubiquitin in HEK-293T cells. (C) Flow cytometric assay on dox-inducible GFP-LRRK2 G2019S HEK-293T cells in the presence and absence of TRIM1 and the proteasome inhibitor MG132; bars show median green fluorescence intensity with error bars showing twice the standard error of the mean. (D) Representative dox-inducible LRRK2 G2019S PC-12 cells transfected with mCherry-TRIM1 or mCherry alone vector and GFP and differentiated with NGF for 5 days in the presence and absence of 1 μg/ml doxycycline. (E) Quantification of the fraction of neurite-bearing PC-12 cells in the presence and absence of LRRK2 G2019S and the presence and absence of TRIM1; bars show average of three independent experiments of 150-250 cells each, error bars show standard error of the mean. (F) Quantification of average neurite length on PC-12 cells with neurites in the presence and absence of LRRK2 G2019S and the presence and absence of TRIM1; bars show average of three independent experiments, error bars show standard error of the mean.
Figure Legend Snippet: TRIM1 mediates proteasomal degradation of PD-mutant LRRK2 G2019S to rescue its toxicity. (A) Live-cell confocal microscopy of GFP-LRRK2 G2019S and mcherry-TRIM1 transiently transfected into H1299 cells. (B) Co-immunoprecipitation and ubiquitination of GFP-LRRK2 G2019S with myc-TRIM1 in the presence of HA-ubiquitin in HEK-293T cells. (C) Flow cytometric assay on dox-inducible GFP-LRRK2 G2019S HEK-293T cells in the presence and absence of TRIM1 and the proteasome inhibitor MG132; bars show median green fluorescence intensity with error bars showing twice the standard error of the mean. (D) Representative dox-inducible LRRK2 G2019S PC-12 cells transfected with mCherry-TRIM1 or mCherry alone vector and GFP and differentiated with NGF for 5 days in the presence and absence of 1 μg/ml doxycycline. (E) Quantification of the fraction of neurite-bearing PC-12 cells in the presence and absence of LRRK2 G2019S and the presence and absence of TRIM1; bars show average of three independent experiments of 150-250 cells each, error bars show standard error of the mean. (F) Quantification of average neurite length on PC-12 cells with neurites in the presence and absence of LRRK2 G2019S and the presence and absence of TRIM1; bars show average of three independent experiments, error bars show standard error of the mean.

Techniques Used: Mutagenesis, Confocal Microscopy, Transfection, Immunoprecipitation, Flow Cytometry, Fluorescence, Plasmid Preparation

TRIM1 co-expression recruits LRRK2 to microtubules. (A) Live-cell confocal microscopy of GFP-LRRK2 and mCherry-tubulin or mCherry-TRIM1 transiently transfected into H1299 cells. In the presence of mCherry-tubulin, GFP-LRRK2 is diffusely cytoplasmic. From left to right: mCherry-tubulin, GFP-LRRK2, merged image. Inset shows higher magnification of region identified by box in merged image. (B) In the presence of mCherry-TRIM1, GFP-LRRK2 localizes to microtubules. From left to right: mCherry-TRIM1, GFP-LRRK2, merged image. Inset shows higher magnification of region identified by box in merged image. (C) In the presence of mcherry-TRIM18, GFP-LRRK2 is diffusely cytoplasmic. From left to right: mCherry-TRIM18, GFP-LRRK2, merged image. Inset shows higher magnification of region identified by box in merged image.
Figure Legend Snippet: TRIM1 co-expression recruits LRRK2 to microtubules. (A) Live-cell confocal microscopy of GFP-LRRK2 and mCherry-tubulin or mCherry-TRIM1 transiently transfected into H1299 cells. In the presence of mCherry-tubulin, GFP-LRRK2 is diffusely cytoplasmic. From left to right: mCherry-tubulin, GFP-LRRK2, merged image. Inset shows higher magnification of region identified by box in merged image. (B) In the presence of mCherry-TRIM1, GFP-LRRK2 localizes to microtubules. From left to right: mCherry-TRIM1, GFP-LRRK2, merged image. Inset shows higher magnification of region identified by box in merged image. (C) In the presence of mcherry-TRIM18, GFP-LRRK2 is diffusely cytoplasmic. From left to right: mCherry-TRIM18, GFP-LRRK2, merged image. Inset shows higher magnification of region identified by box in merged image.

Techniques Used: Expressing, Confocal Microscopy, Transfection

Characterization of LRRK2 co-expressed with 14-3-3 or cytoplasmic TRIM1. (A) Live-cell microscopy in the presence of mCherry-tubulin, GFP-LRRK2, and EBFP-14-3-3 in H1299 cells. (B) Co-immunoprecipitation and ubiquitination of GFP-LRRK2 with myc-TRIM1 C in the presence of HA-ubiquitin in HEK-293T cells. (C) Livecell confocal microscopy of GFP-LRRK2 and mCherry-TRIM1 C transiently transfected into H1299 cells. From left to right, mCherry-TRIM1 C, GFP-LRRK2, merged image. (D) Immunoblot of Rab29 phosphorylation with LRRK2 R1441G in the absence of TRIM1 or with overexpression of Myc-tagged WT TRIM1, TRIM1 C, or TRIM1ΔRF.
Figure Legend Snippet: Characterization of LRRK2 co-expressed with 14-3-3 or cytoplasmic TRIM1. (A) Live-cell microscopy in the presence of mCherry-tubulin, GFP-LRRK2, and EBFP-14-3-3 in H1299 cells. (B) Co-immunoprecipitation and ubiquitination of GFP-LRRK2 with myc-TRIM1 C in the presence of HA-ubiquitin in HEK-293T cells. (C) Livecell confocal microscopy of GFP-LRRK2 and mCherry-TRIM1 C transiently transfected into H1299 cells. From left to right, mCherry-TRIM1 C, GFP-LRRK2, merged image. (D) Immunoblot of Rab29 phosphorylation with LRRK2 R1441G in the absence of TRIM1 or with overexpression of Myc-tagged WT TRIM1, TRIM1 C, or TRIM1ΔRF.

Techniques Used: Microscopy, Immunoprecipitation, Confocal Microscopy, Transfection, Over Expression

Additional characterization of the LRRK2-TRIM1 interaction. Live-cell confocal microscopy of GFP-LRRK2 and mCherry-tubulin or mCherry-TRIM1 transiently transfected into (A) A549 cells, (B) SK-N-SH cells, or (C) HEK 293T cells. From left to right, each row shows mCherry-tubulin or mCherry-TRIM1, GFP-LRRK2, merged image. In all lines examined, in the presence of mCherry-tubulin, GFP-LRRK2 is diffusely cytoplasmic, but microtubule localized in the presence of mCherry-TRIM1. (D) Alignment of TRIM18 with TRIM1. Domains labeled above alignment. Red line designates region required for TRIM1 interaction with LRRK2. Double red line designates region of least homology in TRIM1 and TRIM18 dual B-box domain. DualAAA motifs below the sequence designate the mutated amino acids used to make cytoplasmic TRIM1 C variant. (E) Immunoprecipitation of GFP-LRRK2, which fails to co-immunoprecipitate with myc-TRIM18 in HEK293T cells. (F) Immunoprecipitation of GFP-LRRK2, which fails to co-immunoprecipitate with HA-TRIM9 in HEK-293T cells.
Figure Legend Snippet: Additional characterization of the LRRK2-TRIM1 interaction. Live-cell confocal microscopy of GFP-LRRK2 and mCherry-tubulin or mCherry-TRIM1 transiently transfected into (A) A549 cells, (B) SK-N-SH cells, or (C) HEK 293T cells. From left to right, each row shows mCherry-tubulin or mCherry-TRIM1, GFP-LRRK2, merged image. In all lines examined, in the presence of mCherry-tubulin, GFP-LRRK2 is diffusely cytoplasmic, but microtubule localized in the presence of mCherry-TRIM1. (D) Alignment of TRIM18 with TRIM1. Domains labeled above alignment. Red line designates region required for TRIM1 interaction with LRRK2. Double red line designates region of least homology in TRIM1 and TRIM18 dual B-box domain. DualAAA motifs below the sequence designate the mutated amino acids used to make cytoplasmic TRIM1 C variant. (E) Immunoprecipitation of GFP-LRRK2, which fails to co-immunoprecipitate with myc-TRIM18 in HEK293T cells. (F) Immunoprecipitation of GFP-LRRK2, which fails to co-immunoprecipitate with HA-TRIM9 in HEK-293T cells.

Techniques Used: Confocal Microscopy, Transfection, Labeling, Sequencing, Variant Assay, Immunoprecipitation

TRIM1 binds an N-terminal LRRK2 regulatory loop region via its B-box domain. (A) Co-immunoprecipitation of full-length myc-LRRK2 with GFP-TRIM1 domain constructs in HEK-293T cells (ΔBB: TRIM1 construct lacking both B-box domains; ΔCT: TRIM1 lacking C-terminal domain, ΔRF: TRIM1 lacking ring-finger domain; ΔCC: TRIM1 lacking coiled coil domain; ΔFN3: TRIM1 lacking fibronectin III domain; details of constructs in 75 ). (B) Co-immunoprecipitation of full-length myc-LRRK2 with GFP-TRIM1 B-box domain constructs in HEK-293T cells (ΔRF denotes TRIM1 60-715 ; linker denotes TRIM1 60-117 ; BB1 denotes TRIM1 60-164 ; BB1,2 denotes TRIM1 60-212 ). (C) Co-immunoprecipitation of full-length myc-TRIM1 with GFP-LRRK2 domain constructs in HEK-293T cells. LRRK2 822-892 is necessary and sufficient for interaction with TRIM1. (D) Co-immunoprecipitation of full-length myc-TRIM1 with GFP-LRRK2 with alanine mutagenesis within the RL. (E) Live-cell confocal microscopy of GFP-LRRK2 822-982 and mCherry-TRIM1 transiently transfected into H1299 cells. (F) Schematic of LRRK2-TRIM1 domain interaction mediated by the LRRK2 Regulatory Loop (LRRK2 822-982 (green)), labelled “RL” and TRIM1 BBOX1 (red). All co-immunoprecipitation experiments are a representative image of at least three independent experiments.
Figure Legend Snippet: TRIM1 binds an N-terminal LRRK2 regulatory loop region via its B-box domain. (A) Co-immunoprecipitation of full-length myc-LRRK2 with GFP-TRIM1 domain constructs in HEK-293T cells (ΔBB: TRIM1 construct lacking both B-box domains; ΔCT: TRIM1 lacking C-terminal domain, ΔRF: TRIM1 lacking ring-finger domain; ΔCC: TRIM1 lacking coiled coil domain; ΔFN3: TRIM1 lacking fibronectin III domain; details of constructs in 75 ). (B) Co-immunoprecipitation of full-length myc-LRRK2 with GFP-TRIM1 B-box domain constructs in HEK-293T cells (ΔRF denotes TRIM1 60-715 ; linker denotes TRIM1 60-117 ; BB1 denotes TRIM1 60-164 ; BB1,2 denotes TRIM1 60-212 ). (C) Co-immunoprecipitation of full-length myc-TRIM1 with GFP-LRRK2 domain constructs in HEK-293T cells. LRRK2 822-892 is necessary and sufficient for interaction with TRIM1. (D) Co-immunoprecipitation of full-length myc-TRIM1 with GFP-LRRK2 with alanine mutagenesis within the RL. (E) Live-cell confocal microscopy of GFP-LRRK2 822-982 and mCherry-TRIM1 transiently transfected into H1299 cells. (F) Schematic of LRRK2-TRIM1 domain interaction mediated by the LRRK2 Regulatory Loop (LRRK2 822-982 (green)), labelled “RL” and TRIM1 BBOX1 (red). All co-immunoprecipitation experiments are a representative image of at least three independent experiments.

Techniques Used: Immunoprecipitation, Construct, Mutagenesis, Confocal Microscopy, Transfection

TRIM1 binds LRRK2-RL to cause LRRK2 microtubule localization. Schematic of GFP-LRRK2 constructs (above) with corresponding live-cell microscopy in the presence of mCherry-TRIM1 in H1299 cells (below). Each panel shows only fluorescence at 488 (GFP) to illustrate the subcellular localization of each GFP-LRRK2 construct in the presence of mCherry-TRIM1, which is always localized to the microtubule network.
Figure Legend Snippet: TRIM1 binds LRRK2-RL to cause LRRK2 microtubule localization. Schematic of GFP-LRRK2 constructs (above) with corresponding live-cell microscopy in the presence of mCherry-TRIM1 in H1299 cells (below). Each panel shows only fluorescence at 488 (GFP) to illustrate the subcellular localization of each GFP-LRRK2 construct in the presence of mCherry-TRIM1, which is always localized to the microtubule network.

Techniques Used: Construct, Microscopy, Fluorescence

TRIM1 competes with 14-3-3 to bind LRRK2’s regulatory loop and recruit LRRK2 to microtubules. (A) Co-immunoprecipitation of GFP-LRRK2 wild type (WT) and Ser910Ala Ser935Ala (SA) with V5-14-3-3 theta in the presence and absence of myc-TRIM1 in HEK-293T cells. (B) Quantification of (A) showing mean value from three independent experiments with error bars showing the standard error of the mean. (C) Co-immunoprecipitation of GFP-LRRK2 with either V5-14-3-3 theta or myc-TRIM1 in HEK-293T cells. Overlaid immunoblots in color show relative ratio of total to phospho-LRRK2 (total LRRK2 in green, antibody is NeuroMab clone N241A/34; phospho-LRRK2 is in red, antibodies are phospho-Ser910 (Abcam, UDD 1 15(3)) and phospho-Ser935 (Abcam, UDD 2 10(12)). (D) Quantification of (C) showing mean value from three independent experiments with error bars showing the standard error of the mean. (E) Live-cell confocal microscopy of GFP-LRRK2 in the presence of mCherry-TRIM1 and EBPF2-14-3-3 theta. Inset shows GFP-LRRK2 and mCherry-TRIM1 at microtubules with EBFP-14-3-3 theta diffusely cytoplasmic. All live cell imaging and co-immunoprecipitation experiments are a representative image of at least three independent experiments.
Figure Legend Snippet: TRIM1 competes with 14-3-3 to bind LRRK2’s regulatory loop and recruit LRRK2 to microtubules. (A) Co-immunoprecipitation of GFP-LRRK2 wild type (WT) and Ser910Ala Ser935Ala (SA) with V5-14-3-3 theta in the presence and absence of myc-TRIM1 in HEK-293T cells. (B) Quantification of (A) showing mean value from three independent experiments with error bars showing the standard error of the mean. (C) Co-immunoprecipitation of GFP-LRRK2 with either V5-14-3-3 theta or myc-TRIM1 in HEK-293T cells. Overlaid immunoblots in color show relative ratio of total to phospho-LRRK2 (total LRRK2 in green, antibody is NeuroMab clone N241A/34; phospho-LRRK2 is in red, antibodies are phospho-Ser910 (Abcam, UDD 1 15(3)) and phospho-Ser935 (Abcam, UDD 2 10(12)). (D) Quantification of (C) showing mean value from three independent experiments with error bars showing the standard error of the mean. (E) Live-cell confocal microscopy of GFP-LRRK2 in the presence of mCherry-TRIM1 and EBPF2-14-3-3 theta. Inset shows GFP-LRRK2 and mCherry-TRIM1 at microtubules with EBFP-14-3-3 theta diffusely cytoplasmic. All live cell imaging and co-immunoprecipitation experiments are a representative image of at least three independent experiments.

Techniques Used: Immunoprecipitation, Western Blot, Confocal Microscopy, Live Cell Imaging

33) Product Images from "DDX3X acts as a live-or-die checkpoint in stressed cells by regulating NLRP3 inflammasome"

Article Title: DDX3X acts as a live-or-die checkpoint in stressed cells by regulating NLRP3 inflammasome

Journal: Nature

doi: 10.1038/s41586-019-1551-2

Loss of DDX3X in BMDMs leads to the specific inhibition of NLRP3 inflammasome activation. a – d , CASP1 cleavage in stimulated and unstimulated BMDMs and IL-1β and IL-18 release from BMDMs stimulated to activate the NLRP3, NLRC4, AIM2 and PYRIN inflammasomes by using LPS and nigericin ( a ) (ELISA, n > 10); flagellin ( b ) (ELISA, n = 2); poly(dA:dT) ( c ) (ELISA, n = 2); and Clostridium difficile toxin B (TcdB) ( d ) (ELISA, n = 2). P values in a (from left to right): * P = 0.0170, * P = 0.0116 (unpaired two-sided t- test); n.d., not detected. ELISA data are mean ± s.e.m. Representative blots ( n = 3 biologically independent experiments each). e , Immunoblot analysis of CASP1 cleavage in wild-type BMDMs expressing a doxycycline-inducible DDX3X–mCherry construct and treated with LPS and nigericin. Representative blots ( n = 2 biologically independent experiments). f , Immunoblot analysis of CASP1 cleavage in wild-type BMDMs and Ddx3x fl/fl LysM cre BMDMs that constitutively express DDX3X–YFP from a cytomegalovirus promoter, treated with LPS and nigericin. Representative blots ( n = 2 biologically independent experiments).
Figure Legend Snippet: Loss of DDX3X in BMDMs leads to the specific inhibition of NLRP3 inflammasome activation. a – d , CASP1 cleavage in stimulated and unstimulated BMDMs and IL-1β and IL-18 release from BMDMs stimulated to activate the NLRP3, NLRC4, AIM2 and PYRIN inflammasomes by using LPS and nigericin ( a ) (ELISA, n > 10); flagellin ( b ) (ELISA, n = 2); poly(dA:dT) ( c ) (ELISA, n = 2); and Clostridium difficile toxin B (TcdB) ( d ) (ELISA, n = 2). P values in a (from left to right): * P = 0.0170, * P = 0.0116 (unpaired two-sided t- test); n.d., not detected. ELISA data are mean ± s.e.m. Representative blots ( n = 3 biologically independent experiments each). e , Immunoblot analysis of CASP1 cleavage in wild-type BMDMs expressing a doxycycline-inducible DDX3X–mCherry construct and treated with LPS and nigericin. Representative blots ( n = 2 biologically independent experiments). f , Immunoblot analysis of CASP1 cleavage in wild-type BMDMs and Ddx3x fl/fl LysM cre BMDMs that constitutively express DDX3X–YFP from a cytomegalovirus promoter, treated with LPS and nigericin. Representative blots ( n = 2 biologically independent experiments).

Techniques Used: Inhibition, Activation Assay, Enzyme-linked Immunosorbent Assay, Expressing, Construct

Identification of the region of DDX3X required for its interaction with NLRP3, prediction of disordered regions using PONDR and PSIPRED, and effect of DDX3X helicase activity inhibition on NLRP3 inflammasome activation. a , Schematic of C-terminal mCherry-tagged DDX3X domain-deletion expression constructs. b , Immunoblot analysis of the input lysates used for immunoprecipitation of Flag–NLRP3-FL and DDX3X–mCherry constructs. Representative blots ( n = 2 biologically independent experiments). c – f , Immunoblot analysis of immunoprecipitates with Flag–NLRP3-FL and mCherry-tagged DDX3X domain-deletion expression constructs: full-length DDX3X ( c ), DDX3X with an N-terminal deletion ( d ), DDX3X with a C-terminal deletion ( e ) and the DDX3X helicase domain ( f ). Red asterisk indicates a non-specific band. Representative blots ( n = 2 biologically independent experiments each). g , Overlay of the predicted disordered regions in NLRP3 from PONDR and PSIPRED. h , Overlay of the predicted disordered regions in DDX3X from PONDR and PSIPRED. i , Immunoblot analysis of CASP1 cleavage in BMDMs treated with LPS; LPS and nigericin; LPS and RK-33; LPS, RK-33 and arsenite; LPS, arsenite and nigericin; LPS, RK-33 and nigericin; and LPS, RK-33, arsenite and nigericin. Representative blots ( n = 2 biologically independent experiments).
Figure Legend Snippet: Identification of the region of DDX3X required for its interaction with NLRP3, prediction of disordered regions using PONDR and PSIPRED, and effect of DDX3X helicase activity inhibition on NLRP3 inflammasome activation. a , Schematic of C-terminal mCherry-tagged DDX3X domain-deletion expression constructs. b , Immunoblot analysis of the input lysates used for immunoprecipitation of Flag–NLRP3-FL and DDX3X–mCherry constructs. Representative blots ( n = 2 biologically independent experiments). c – f , Immunoblot analysis of immunoprecipitates with Flag–NLRP3-FL and mCherry-tagged DDX3X domain-deletion expression constructs: full-length DDX3X ( c ), DDX3X with an N-terminal deletion ( d ), DDX3X with a C-terminal deletion ( e ) and the DDX3X helicase domain ( f ). Red asterisk indicates a non-specific band. Representative blots ( n = 2 biologically independent experiments each). g , Overlay of the predicted disordered regions in NLRP3 from PONDR and PSIPRED. h , Overlay of the predicted disordered regions in DDX3X from PONDR and PSIPRED. i , Immunoblot analysis of CASP1 cleavage in BMDMs treated with LPS; LPS and nigericin; LPS and RK-33; LPS, RK-33 and arsenite; LPS, arsenite and nigericin; LPS, RK-33 and nigericin; and LPS, RK-33, arsenite and nigericin. Representative blots ( n = 2 biologically independent experiments).

Techniques Used: Activity Assay, Inhibition, Activation Assay, Expressing, Construct, Immunoprecipitation

DDX3X interacts with NLRP3 but not ASC and CASP1, and its loss does not lead to a decrease in the levels of core components of the NLRP3 inflammasome. a , Immunoblot analysis of the levels of NLRP3, ASC, CASP1, pro-IL-1β, NEK7, DDX3X and GAPDH proteins in Ddx3x fl/fl , Ddx3x fl/fl LysM cre and Nlrp3 −/− BMDMs after LPS priming. Representative blots ( n = 3 biologically independent experiments). b , Immunoblot analysis of NLRP3 and DDX3X immunoprecipitation from HEK293T cells transfected with Flag–NLRP3 and DDX3X–mCherry expression constructs. S.E., short exposure; L.E., long exposure. Representative blots ( n = 3 biologically independent experiments). c , Immunoblot analysis of ASC and DDX3X immunoprecipitates from HEK293T cells transfected with ASC and DDX3X–mCherry expression constructs. Representative blots ( n = 3 biologically independent experiments). d , Immunoblot analysis of CASP1 and DDX3X immunoprecipitates from HEK293T cells transfected with CASP1 and DDX3X–mCherry expression constructs. Representative blots ( n = 2 biologically independent experiments). e , Quantification of the percentage of cells that contain ASC specks in Ddx3x fl/fl and Ddx3x fl/fl LysM cre BMDMs. **** P ≤ 0.0001 (unpaired two-sided t- test). Data are mean ± s.e.m. ( n = 48). f , Quantification of the percentage of cells that contain ASC specks in wild-type and Ddx3x fl/fl LysM cre peritoneal macrophages (PMs). **** P ≤ 0.0001 (unpaired two-sided t- test). Data are mean ± s.e.m. ( n = 48). g , Confocal microscopy imaging of wild-type and Ddx3x fl/fl LysM cre peritoneal macrophages treated with LPS and nigericin to visualize ASC and DDX3X. Scale bar, 10 μm.
Figure Legend Snippet: DDX3X interacts with NLRP3 but not ASC and CASP1, and its loss does not lead to a decrease in the levels of core components of the NLRP3 inflammasome. a , Immunoblot analysis of the levels of NLRP3, ASC, CASP1, pro-IL-1β, NEK7, DDX3X and GAPDH proteins in Ddx3x fl/fl , Ddx3x fl/fl LysM cre and Nlrp3 −/− BMDMs after LPS priming. Representative blots ( n = 3 biologically independent experiments). b , Immunoblot analysis of NLRP3 and DDX3X immunoprecipitation from HEK293T cells transfected with Flag–NLRP3 and DDX3X–mCherry expression constructs. S.E., short exposure; L.E., long exposure. Representative blots ( n = 3 biologically independent experiments). c , Immunoblot analysis of ASC and DDX3X immunoprecipitates from HEK293T cells transfected with ASC and DDX3X–mCherry expression constructs. Representative blots ( n = 3 biologically independent experiments). d , Immunoblot analysis of CASP1 and DDX3X immunoprecipitates from HEK293T cells transfected with CASP1 and DDX3X–mCherry expression constructs. Representative blots ( n = 2 biologically independent experiments). e , Quantification of the percentage of cells that contain ASC specks in Ddx3x fl/fl and Ddx3x fl/fl LysM cre BMDMs. **** P ≤ 0.0001 (unpaired two-sided t- test). Data are mean ± s.e.m. ( n = 48). f , Quantification of the percentage of cells that contain ASC specks in wild-type and Ddx3x fl/fl LysM cre peritoneal macrophages (PMs). **** P ≤ 0.0001 (unpaired two-sided t- test). Data are mean ± s.e.m. ( n = 48). g , Confocal microscopy imaging of wild-type and Ddx3x fl/fl LysM cre peritoneal macrophages treated with LPS and nigericin to visualize ASC and DDX3X. Scale bar, 10 μm.

Techniques Used: Immunoprecipitation, Transfection, Expressing, Construct, Confocal Microscopy, Imaging

The stress granule component DDX3X interacts with NLRP3 and is required for NLRP3 inflammasome activation. a , Co-immunoprecipitation (IP) of NLRP3 and DDX3X in BMDMs treated with LPS with or without nigericin. Representative blots ( n = 3). b , Immunoblot analysis of CASP1 cleavage in wild-type (WT), Ddx3x fl/fl , Ddx3x fl/fl LysM cre and Nlrp3 −/− BMDMs treated with LPS and nigericin. Representative blots ( n > 3). c , Immunoblots of DDX3X expression and CASP1 cleavage after siRNA-mediated knockdown of Ddx3x in BMDMs treated with LPS with or without nigericin. Representative blots ( n = 2). d , Confocal microscopy imaging of ASC specks in BMDMs treated with LPS with or without nigericin, to visualize the subcellular localization of DDX3X, NLRP3 and ASC. Scale bars, 10 μm. Representative images ( n = 3). e , Schematic of N-terminal Flag-tagged NLRP3 expression constructs: full-length NLRP3 (Flag-NLRP3-FL), the pyrin domain of NLRP3 (Flag-PYD; amino acids 1–90 of NLRP3), the NACHT domain of NLRP3 (Flag-NACHT; amino acids 91–710 of NLRP3) and the LRR domain of NLRP3 (Flag-LRR; amino acids 711–1034 of NLRP3). f, Immunoblot (IB) analysis of input lysates used for co-immunoprecipitation of Flag-tagged NLRP3 constructs and DDX3X-mCherry. Representative blots ( n > 3). g , Immunoblot analysis of immunoprecipitation of DDX3X-mCherry and the indicated NLRP3 constructs. Red asterisk indicates the antibody light chain. Representative blots ( n > 3).
Figure Legend Snippet: The stress granule component DDX3X interacts with NLRP3 and is required for NLRP3 inflammasome activation. a , Co-immunoprecipitation (IP) of NLRP3 and DDX3X in BMDMs treated with LPS with or without nigericin. Representative blots ( n = 3). b , Immunoblot analysis of CASP1 cleavage in wild-type (WT), Ddx3x fl/fl , Ddx3x fl/fl LysM cre and Nlrp3 −/− BMDMs treated with LPS and nigericin. Representative blots ( n > 3). c , Immunoblots of DDX3X expression and CASP1 cleavage after siRNA-mediated knockdown of Ddx3x in BMDMs treated with LPS with or without nigericin. Representative blots ( n = 2). d , Confocal microscopy imaging of ASC specks in BMDMs treated with LPS with or without nigericin, to visualize the subcellular localization of DDX3X, NLRP3 and ASC. Scale bars, 10 μm. Representative images ( n = 3). e , Schematic of N-terminal Flag-tagged NLRP3 expression constructs: full-length NLRP3 (Flag-NLRP3-FL), the pyrin domain of NLRP3 (Flag-PYD; amino acids 1–90 of NLRP3), the NACHT domain of NLRP3 (Flag-NACHT; amino acids 91–710 of NLRP3) and the LRR domain of NLRP3 (Flag-LRR; amino acids 711–1034 of NLRP3). f, Immunoblot (IB) analysis of input lysates used for co-immunoprecipitation of Flag-tagged NLRP3 constructs and DDX3X-mCherry. Representative blots ( n > 3). g , Immunoblot analysis of immunoprecipitation of DDX3X-mCherry and the indicated NLRP3 constructs. Red asterisk indicates the antibody light chain. Representative blots ( n > 3).

Techniques Used: Activation Assay, Immunoprecipitation, Western Blot, Expressing, Confocal Microscopy, Imaging, Construct

34) Product Images from "Protein exchange is reduced in calcium-independent epithelial junctions"

Article Title: Protein exchange is reduced in calcium-independent epithelial junctions

Journal: The Journal of Cell Biology

doi: 10.1083/jcb.201906153

Dsg3 order factor is not impacted by DP-S8249G-mCherry transfection. (A and B) Representative images of HaCaT cells cotransfected with (A) Dsg3-ΔEA-GFP and DP-wt-mCherry or (B) Dsg3-ΔEA-GFP and DP-S2849G-mCherry and maintained in normal-Ca 2+ medium. Scale bar = 5 µm. (C) Order factor of Dsg3-ΔEA-GFP in cells expressing DP-wt-mCherry ( n = 27) or DP S2849G-mCherry ( n = 26; ns, not significant, P > 0.05; Student’s t test). (D) Quantification of adhesive strength by dispase fragmentation assay in HaCaT cells expressing either wt or mutant DP with Gö6976 and Ca 2+ conditions indicated ( n = 3; mean ± SD; **, P ≤ 0.01; Student’s t test).
Figure Legend Snippet: Dsg3 order factor is not impacted by DP-S8249G-mCherry transfection. (A and B) Representative images of HaCaT cells cotransfected with (A) Dsg3-ΔEA-GFP and DP-wt-mCherry or (B) Dsg3-ΔEA-GFP and DP-S2849G-mCherry and maintained in normal-Ca 2+ medium. Scale bar = 5 µm. (C) Order factor of Dsg3-ΔEA-GFP in cells expressing DP-wt-mCherry ( n = 27) or DP S2849G-mCherry ( n = 26; ns, not significant, P > 0.05; Student’s t test). (D) Quantification of adhesive strength by dispase fragmentation assay in HaCaT cells expressing either wt or mutant DP with Gö6976 and Ca 2+ conditions indicated ( n = 3; mean ± SD; **, P ≤ 0.01; Student’s t test).

Techniques Used: Transfection, Expressing, Mutagenesis

Plaque protein exchange is reduced in hyperadhesive desmosomes. (A–D) FRAP experiments were conducted on HaCaT cells transfected with DP-mCherry and mock treated in normal-Ca 2+ medium. (A) Representative cell (inverted intensity) with bleach region of interest (ROI; red). (B) Individual puncta over the time course underscored by dashed lines. (C) Linescans through the bleach region indicate relative intensities before bleaching (black), immediately after bleaching (gray), and at 20 min after bleaching (red). (D) Fluorescence intensity over time and mobile fraction of the bleach ROI (MF). (E–H) Representative cell from FRAP experiments conducted on HaCaT cells transfected with DP-mCherry, treated with Gö6976, and switched into low-Ca 2+ medium for 90 min. (I–L) Representative cell from FRAP experiments conducted on HaCaT cells transfected with PG-mEmerald and mock treated in normal-Ca 2+ medium. (M–P) Representative cell from FRAP experiments conducted on HaCaT cells transfected with PG-mEmerald, treated with Gö6976, and switched into low-Ca 2+ medium for 90 min. ( A, E, I, M ) Scale bar = 5 µm. ( B, F, J, N ) Scale bar = 1 µm. (Q and R) FRAP recovery and fit to one-phase association curves (mean ± SD; Q) and mobile fraction (mean ± SD; R) of PG-mEmerald in mock ( n = 9) and Gö6976 ( n = 8) treated cells and DP-mCherry in mock ( n = 14) and Gö6976 ( n = 7) treated cells. All data from three independent experiments. (ns, not significant, P > 0.05; *, P ≤ 0.05; **, P ≤ 0.01; ANOVA).
Figure Legend Snippet: Plaque protein exchange is reduced in hyperadhesive desmosomes. (A–D) FRAP experiments were conducted on HaCaT cells transfected with DP-mCherry and mock treated in normal-Ca 2+ medium. (A) Representative cell (inverted intensity) with bleach region of interest (ROI; red). (B) Individual puncta over the time course underscored by dashed lines. (C) Linescans through the bleach region indicate relative intensities before bleaching (black), immediately after bleaching (gray), and at 20 min after bleaching (red). (D) Fluorescence intensity over time and mobile fraction of the bleach ROI (MF). (E–H) Representative cell from FRAP experiments conducted on HaCaT cells transfected with DP-mCherry, treated with Gö6976, and switched into low-Ca 2+ medium for 90 min. (I–L) Representative cell from FRAP experiments conducted on HaCaT cells transfected with PG-mEmerald and mock treated in normal-Ca 2+ medium. (M–P) Representative cell from FRAP experiments conducted on HaCaT cells transfected with PG-mEmerald, treated with Gö6976, and switched into low-Ca 2+ medium for 90 min. ( A, E, I, M ) Scale bar = 5 µm. ( B, F, J, N ) Scale bar = 1 µm. (Q and R) FRAP recovery and fit to one-phase association curves (mean ± SD; Q) and mobile fraction (mean ± SD; R) of PG-mEmerald in mock ( n = 9) and Gö6976 ( n = 8) treated cells and DP-mCherry in mock ( n = 14) and Gö6976 ( n = 7) treated cells. All data from three independent experiments. (ns, not significant, P > 0.05; *, P ≤ 0.05; **, P ≤ 0.01; ANOVA).

Techniques Used: Transfection, Fluorescence

Tagged proteins are expressed at the expected molecular weights. Cells were transiently transfected with plasmids as indicated. 48 h after transfection, cells were harvested for SDS-PAGE and Western blot analysis. Each blot includes an untransfected control. (A) Cos7 cells transfected with GFP, Dsg3-ΔEA-GFP, or Dsg3-ΔEA-W2A-GFP blot were probed for Dsg3 or GFP. Tubulin was used as a loading control. (B) Cos7 cells transfected with mCherry, DP-mCherry, or DP-S2849-mCherry. Blot was probed for DP or mCherry with tubulin as a loading control. (C) A431 cells transfected with E-cad-GFP. Blot was probed with GFP and tubulin. (D) A431 cells transfected with PG-mEmerald. Blot was probed with GFP, and tubulin was used as a loading control. (E) A431 cells transfected with Dsg2-mCherry. Blot was probed with mCherry, and GAPDH was used as a loading control.
Figure Legend Snippet: Tagged proteins are expressed at the expected molecular weights. Cells were transiently transfected with plasmids as indicated. 48 h after transfection, cells were harvested for SDS-PAGE and Western blot analysis. Each blot includes an untransfected control. (A) Cos7 cells transfected with GFP, Dsg3-ΔEA-GFP, or Dsg3-ΔEA-W2A-GFP blot were probed for Dsg3 or GFP. Tubulin was used as a loading control. (B) Cos7 cells transfected with mCherry, DP-mCherry, or DP-S2849-mCherry. Blot was probed for DP or mCherry with tubulin as a loading control. (C) A431 cells transfected with E-cad-GFP. Blot was probed with GFP and tubulin. (D) A431 cells transfected with PG-mEmerald. Blot was probed with GFP, and tubulin was used as a loading control. (E) A431 cells transfected with Dsg2-mCherry. Blot was probed with mCherry, and GAPDH was used as a loading control.

Techniques Used: Transfection, SDS Page, Western Blot

Dsg3 trans binding is critical in cadherin order and dynamics. (A) HaCaT cells were transfected with Dsg3-ΔEA-GFP (Dsg3-wt) or Dsg3-ΔEA-GFP-W2A (Dsg3-W2A) and imaged with fluorescence polarization microscopy. (B) Mean order factor (Dsg3-W2A n = 7; Dsg3-wt n = 19; mean ± SD; **, P ≤ 0.01; Student’s t test). (C and D) FRAP experiments were conducted on HaCaT cells cotransfected with Dsg3-W2A and DP-mCherry and mock treated in normal-Ca 2+ medium. (C) Representative cell and time points of region of interest (ROIs). (D) Fluorescence recovery over time and mobile fraction of the ROI. (E and F) FRAP experiments were conducted on HaCaT cells cotransfected Dsg3-W2A and DP-mCherry, treated with Gö6976, and switched to low-Ca 2+ medium. (E) Representative cell and time points of ROI. (F) Fluorescence recovery over time and mobile fraction of the ROI. (G and H) FRAP recovery curves (mean ± SD; G) and mobile fractions (mean ± SD; H) for cells cotransfected with both Dsg3-W2A and DP-mCherry with mock ( n = 9) or Gö6976 ( n = 11) treatment (ns, not significant, P > 0.05; *, P ≤ 0.05; **, P ≤ 0.01; ANOVA). (I) Schematic of how trans binding changes cadherin mobility in hyperadhesive desmosomes. Scale bars = 5 µm; ROI scale bars =1 µm.
Figure Legend Snippet: Dsg3 trans binding is critical in cadherin order and dynamics. (A) HaCaT cells were transfected with Dsg3-ΔEA-GFP (Dsg3-wt) or Dsg3-ΔEA-GFP-W2A (Dsg3-W2A) and imaged with fluorescence polarization microscopy. (B) Mean order factor (Dsg3-W2A n = 7; Dsg3-wt n = 19; mean ± SD; **, P ≤ 0.01; Student’s t test). (C and D) FRAP experiments were conducted on HaCaT cells cotransfected with Dsg3-W2A and DP-mCherry and mock treated in normal-Ca 2+ medium. (C) Representative cell and time points of region of interest (ROIs). (D) Fluorescence recovery over time and mobile fraction of the ROI. (E and F) FRAP experiments were conducted on HaCaT cells cotransfected Dsg3-W2A and DP-mCherry, treated with Gö6976, and switched to low-Ca 2+ medium. (E) Representative cell and time points of ROI. (F) Fluorescence recovery over time and mobile fraction of the ROI. (G and H) FRAP recovery curves (mean ± SD; G) and mobile fractions (mean ± SD; H) for cells cotransfected with both Dsg3-W2A and DP-mCherry with mock ( n = 9) or Gö6976 ( n = 11) treatment (ns, not significant, P > 0.05; *, P ≤ 0.05; **, P ≤ 0.01; ANOVA). (I) Schematic of how trans binding changes cadherin mobility in hyperadhesive desmosomes. Scale bars = 5 µm; ROI scale bars =1 µm.

Techniques Used: Binding Assay, Transfection, Fluorescence, Microscopy

35) Product Images from "Global and transcription-coupled repair of 8-oxoG is initiated by nucleotide excision repair proteins"

Article Title: Global and transcription-coupled repair of 8-oxoG is initiated by nucleotide excision repair proteins

Journal: Nature Communications

doi: 10.1038/s41467-022-28642-9

DDB2 facilitates 8-oxoG repair and is rapidly recruited to sites of 8-oxoG within telomeric DNA. a , b Immunofluorescence and quantification of 8-oxoG in cells transfected with control, DDB2 or OGG1 siRNA. c Schematic of the repair enzyme-based assay for 8-oxoG quantification in DNA. Genomic DNA containing 8-oxoG is treated with FPG to convert 8-oxoG to one nucleotide gaps. Treating with S1 nuclease converts the gaps to double stranded breaks (DSBs). The cleaved DNA is subjected to pulse field gel electrophoresis (PFGE) to track repair, as damaged DNA migrates faster than repaired DNA. d Quantification of 8-oxoG repair in U2OS cells transfected with control or DDB2 siRNA and treated with KBrO3. e Clonogenic cell survival curves in U2OS WT and DDB2 knockout (KO) cells treated with a range of concentrations of KBrO3. f Schematic of dye plus light treatment. Cells stably expressing FAP-TRF1 were treated with dye (100 nM, 15 min) plus light (660 nm, 10 min) to introduce 8-oxoG lesions at telomeres. g (left) Recruitment of DDB2-mCherry to 8-oxoG sites at telomeres in untreated, dye alone, light alone, and dye plus light treated cells. (right) Percentage telomeres colocalized with DDB2-mCherry. h Proximity ligation assay (PLA) for DDB2-mCherry and TRF1 in untreated cells and cells treated with dye (100 nM, 15 min) plus light (660 nm, 10 min). Data ( a , b , d , g , h ) represent mean ± SEM from two to three independent experiments. “ n ” represents the number of cells scored for each condition. Data ( e ) shows one representative experiment (performed in triplicate) from three independent experiments, mean ± SD. One-way ANOVA (Sidak multiple comparison test) ( b , g ), Student’s two-tailed Student’s t -test ( h ) and two-way ANOVA (Sidak multiple comparison test) ( d , e ) were performed for statistical analysis: * p
Figure Legend Snippet: DDB2 facilitates 8-oxoG repair and is rapidly recruited to sites of 8-oxoG within telomeric DNA. a , b Immunofluorescence and quantification of 8-oxoG in cells transfected with control, DDB2 or OGG1 siRNA. c Schematic of the repair enzyme-based assay for 8-oxoG quantification in DNA. Genomic DNA containing 8-oxoG is treated with FPG to convert 8-oxoG to one nucleotide gaps. Treating with S1 nuclease converts the gaps to double stranded breaks (DSBs). The cleaved DNA is subjected to pulse field gel electrophoresis (PFGE) to track repair, as damaged DNA migrates faster than repaired DNA. d Quantification of 8-oxoG repair in U2OS cells transfected with control or DDB2 siRNA and treated with KBrO3. e Clonogenic cell survival curves in U2OS WT and DDB2 knockout (KO) cells treated with a range of concentrations of KBrO3. f Schematic of dye plus light treatment. Cells stably expressing FAP-TRF1 were treated with dye (100 nM, 15 min) plus light (660 nm, 10 min) to introduce 8-oxoG lesions at telomeres. g (left) Recruitment of DDB2-mCherry to 8-oxoG sites at telomeres in untreated, dye alone, light alone, and dye plus light treated cells. (right) Percentage telomeres colocalized with DDB2-mCherry. h Proximity ligation assay (PLA) for DDB2-mCherry and TRF1 in untreated cells and cells treated with dye (100 nM, 15 min) plus light (660 nm, 10 min). Data ( a , b , d , g , h ) represent mean ± SEM from two to three independent experiments. “ n ” represents the number of cells scored for each condition. Data ( e ) shows one representative experiment (performed in triplicate) from three independent experiments, mean ± SD. One-way ANOVA (Sidak multiple comparison test) ( b , g ), Student’s two-tailed Student’s t -test ( h ) and two-way ANOVA (Sidak multiple comparison test) ( d , e ) were performed for statistical analysis: * p

Techniques Used: Immunofluorescence, Transfection, Enzymatic Assay, Nucleic Acid Electrophoresis, Knock-Out, Stable Transfection, Expressing, Introduce, Proximity Ligation Assay, Two Tailed Test

DDB2 is required for efficient OGG1 recruitment to 8-oxoG. a DDB2-mCherry and OGG1-GFP associate at 8-oxoG sites as shown by PLA after dye (100 nM, 15 min) plus light (660 nm, 10 min) treatment, over a period of 3 h. Antibodies against mCherry and GFP were used. b Quantification of PLA. c Accumulation of DDB2-mCherry at telomeric 8-oxoG 30 min post dye plus light treatment in U2OS-FAP-TRF1 cells transfected with control or OGG1 siRNA. d Percent telomeres colocalized with DDB2-mCherry as shown in (c). e Recruitment of OGG1-GFP at damaged telomeres in cells transfected with control or DDB2 siRNA. f Percent telomeres colocalized with OGG1-GFP as shown in ( e ). Data ( a – f ) represents mean ± SEM from two independent experiments. “ n ” represents the number of cells scored for each condition. One-way ANOVA (Sidak multiple comparison test): * p
Figure Legend Snippet: DDB2 is required for efficient OGG1 recruitment to 8-oxoG. a DDB2-mCherry and OGG1-GFP associate at 8-oxoG sites as shown by PLA after dye (100 nM, 15 min) plus light (660 nm, 10 min) treatment, over a period of 3 h. Antibodies against mCherry and GFP were used. b Quantification of PLA. c Accumulation of DDB2-mCherry at telomeric 8-oxoG 30 min post dye plus light treatment in U2OS-FAP-TRF1 cells transfected with control or OGG1 siRNA. d Percent telomeres colocalized with DDB2-mCherry as shown in (c). e Recruitment of OGG1-GFP at damaged telomeres in cells transfected with control or DDB2 siRNA. f Percent telomeres colocalized with OGG1-GFP as shown in ( e ). Data ( a – f ) represents mean ± SEM from two independent experiments. “ n ” represents the number of cells scored for each condition. One-way ANOVA (Sidak multiple comparison test): * p

Techniques Used: Proximity Ligation Assay, Transfection

DDB2 binds sparse telomeric 8-oxoG independently of the DDB1-Cul4A-RBX1 E3 ligase. a Representative images showing recruitment of DDB2-mCherry to telomeric 8-oxoG in cells transfected with control, DDB1 or Cul4A siRNA. b Quantification of a. c , e DDB2-mCherry and GFP-DDB1 ( c ) or DDB2-mCherry and GFP-Cul4A ( e ) accumulation at 8-oxoG sites after dye (100 nM, 15 min) plus light (660 nm, 10 min) treatment. d , f Quantification of c and e respectively. g Western blot for DDB2 in U2OS-FAP-TRF1 cells treated with UVC, potassium bromate (KBrO 3 ) or dye plus light at indicated doses. Independent experiments are represented by black circles. h Colocalization of DDB2-mCherry and GFP-Cul4A at damaged telomeres in U2OS-FAP-TRF1 cells transfected with control or OGG1 siRNA. i Quantification of h. Data ( a – h ) represents mean ± SEM from two independent experiments. ‘n’ represents the number of cells scored for each condition. One-way ANOVA (Sidak multiple comparison test) ( b , i ) was performed for statistical analysis: * p
Figure Legend Snippet: DDB2 binds sparse telomeric 8-oxoG independently of the DDB1-Cul4A-RBX1 E3 ligase. a Representative images showing recruitment of DDB2-mCherry to telomeric 8-oxoG in cells transfected with control, DDB1 or Cul4A siRNA. b Quantification of a. c , e DDB2-mCherry and GFP-DDB1 ( c ) or DDB2-mCherry and GFP-Cul4A ( e ) accumulation at 8-oxoG sites after dye (100 nM, 15 min) plus light (660 nm, 10 min) treatment. d , f Quantification of c and e respectively. g Western blot for DDB2 in U2OS-FAP-TRF1 cells treated with UVC, potassium bromate (KBrO 3 ) or dye plus light at indicated doses. Independent experiments are represented by black circles. h Colocalization of DDB2-mCherry and GFP-Cul4A at damaged telomeres in U2OS-FAP-TRF1 cells transfected with control or OGG1 siRNA. i Quantification of h. Data ( a – h ) represents mean ± SEM from two independent experiments. ‘n’ represents the number of cells scored for each condition. One-way ANOVA (Sidak multiple comparison test) ( b , i ) was performed for statistical analysis: * p

Techniques Used: Transfection, Western Blot

36) Product Images from "Inducible cell-specific mouse models for paired epigenetic and transcriptomic studies of microglia and astroglia"

Article Title: Inducible cell-specific mouse models for paired epigenetic and transcriptomic studies of microglia and astroglia

Journal: bioRxiv

doi: 10.1101/862722

Validation of astrocytic nuclei and epigenome enrichment in the Aldh1l1-NuTRAP mouse brain by INTACT-BSAS. A) Representative confocal fluorescent microscopy images from input, negative, and positive INTACT nuclei fractions. B) Purity of astrocytic nuclei, expressed as average percentage ± SEM mCherry + / Biotin + nuclei in the positive fraction, compared to percentage ± SEM mCherry + nuclei in the input demonstrates a high degree of specificity to the INTACT isolation (n=3/group, ***p
Figure Legend Snippet: Validation of astrocytic nuclei and epigenome enrichment in the Aldh1l1-NuTRAP mouse brain by INTACT-BSAS. A) Representative confocal fluorescent microscopy images from input, negative, and positive INTACT nuclei fractions. B) Purity of astrocytic nuclei, expressed as average percentage ± SEM mCherry + / Biotin + nuclei in the positive fraction, compared to percentage ± SEM mCherry + nuclei in the input demonstrates a high degree of specificity to the INTACT isolation (n=3/group, ***p

Techniques Used: Microscopy, Isolation

Flow cytometry and immunohistochemical validation of the Cx3cr1-NuTRAP mouse brain. After Tam treatment, brains from Cx3cr1-NuTRAP and cre negative NuTRAP + (control) mice were harvested and single hemispheres assessed by flow cytometry (FC) and immunohistochemistry (IHC). A) Representative FC plots of immunostained single-cell suspensions showed a distinct population of brain EGFP + cells, identified as CD11b + cells (microglia lineage), based on gating strategy for EGFP and CD11b co-expression in Cx3cr1-NuTRAP samples upon cre-mediated recombination but not in the controls. B) Analysis of absolute cell counts from FC quantitation expressed as mean cell count/brain sample ±SEM. C) Representative confocal fluorescent microscopy images of sagittal brain sections. EGFP expression (green signal) was found in cells that co-expressed mCherry (red signal) and CD11b (blue signal) in Cx3cr1-NuTRAP + brains. * p
Figure Legend Snippet: Flow cytometry and immunohistochemical validation of the Cx3cr1-NuTRAP mouse brain. After Tam treatment, brains from Cx3cr1-NuTRAP and cre negative NuTRAP + (control) mice were harvested and single hemispheres assessed by flow cytometry (FC) and immunohistochemistry (IHC). A) Representative FC plots of immunostained single-cell suspensions showed a distinct population of brain EGFP + cells, identified as CD11b + cells (microglia lineage), based on gating strategy for EGFP and CD11b co-expression in Cx3cr1-NuTRAP samples upon cre-mediated recombination but not in the controls. B) Analysis of absolute cell counts from FC quantitation expressed as mean cell count/brain sample ±SEM. C) Representative confocal fluorescent microscopy images of sagittal brain sections. EGFP expression (green signal) was found in cells that co-expressed mCherry (red signal) and CD11b (blue signal) in Cx3cr1-NuTRAP + brains. * p

Techniques Used: Flow Cytometry, Immunohistochemistry, Mouse Assay, Expressing, Quantitation Assay, Cell Counting, Microscopy

Flow cytometry and immunohistochemical validation of the Aldh1l1-NuTRAP transgene expression. One week after Tam treatment, brains were harvested from Aldh1l1-NuTRAP and cre negative NuTRAP + (control) mice for flow cytometry (FC) and immunohistochemistry (IHC) purposes. A) Representative FC plots of immunostained single-cell suspensions showed a distinct population of brain EGFP + cells, identified as Aldh1l1 + cells (astrocyte lineage), based on gating strategy for EGFP and ACSA-2 co-expression in Aldh1l1-NuTRAP samples but not in the controls. B) Analysis of absolute cell counts from FC quantitation expressed as mean cell count/brain sample ±SEM. C) Representative confocal fluorescent microscopy images of sagittal brain sections show EGFP expression (green signal) in cells that co-expressed mCherry (red signal) and GFAP (blue signal) in Aldh1l1-NuTRAP brains but did not colocalize with other cell type marker expression. *** p
Figure Legend Snippet: Flow cytometry and immunohistochemical validation of the Aldh1l1-NuTRAP transgene expression. One week after Tam treatment, brains were harvested from Aldh1l1-NuTRAP and cre negative NuTRAP + (control) mice for flow cytometry (FC) and immunohistochemistry (IHC) purposes. A) Representative FC plots of immunostained single-cell suspensions showed a distinct population of brain EGFP + cells, identified as Aldh1l1 + cells (astrocyte lineage), based on gating strategy for EGFP and ACSA-2 co-expression in Aldh1l1-NuTRAP samples but not in the controls. B) Analysis of absolute cell counts from FC quantitation expressed as mean cell count/brain sample ±SEM. C) Representative confocal fluorescent microscopy images of sagittal brain sections show EGFP expression (green signal) in cells that co-expressed mCherry (red signal) and GFAP (blue signal) in Aldh1l1-NuTRAP brains but did not colocalize with other cell type marker expression. *** p

Techniques Used: Flow Cytometry, Immunohistochemistry, Expressing, Mouse Assay, Quantitation Assay, Cell Counting, Microscopy, Marker

Validation of microglial epigenome enrichment in the Cx3cr1-NuTRAP mouse brain by INTACT-BSAS. A) Representative confocal fluorescent microscopy images from input, negative, and positive INTACT nuclei fractions. B) Purity of microglial nuclei expressed as average percentage ± SEM mCherry + / Biotin + nuclei in the positive fraction, and percentage ± SEM mCherry + nuclei in the input and average percentage ± SEM mCherry + /Biotin + nuclei in the input demonstrates a high degree of specificity to the INTACT isolation (n=5/group, ***p
Figure Legend Snippet: Validation of microglial epigenome enrichment in the Cx3cr1-NuTRAP mouse brain by INTACT-BSAS. A) Representative confocal fluorescent microscopy images from input, negative, and positive INTACT nuclei fractions. B) Purity of microglial nuclei expressed as average percentage ± SEM mCherry + / Biotin + nuclei in the positive fraction, and percentage ± SEM mCherry + nuclei in the input and average percentage ± SEM mCherry + /Biotin + nuclei in the input demonstrates a high degree of specificity to the INTACT isolation (n=5/group, ***p

Techniques Used: Microscopy, Isolation

37) Product Images from "A hidden gene in astroviruses encodes a cell-permeabilizing protein involved in virus release"

Article Title: A hidden gene in astroviruses encodes a cell-permeabilizing protein involved in virus release

Journal: bioRxiv

doi: 10.1101/661579

Cellular localization and membrane topology of XP. HeLa cells were electroporated with pCAG-mCherry, pCAG-mCherry-XP or pCAG-XP-mCherry. (A) Representative confocal images of fixed and permeabilized cells were visualized for mCherry (red) and stained for nuclei (Hoechst, blue). The images are averaged single plane scans. (B) Cell lysates were fractionated and whole cell lysate (WCL), cytoplasmic (Cyto), membrane (Mem) and soluble nuclear (Nucl) fractions were analyzed by immunoblotting with antibodies to mCherry, tubulin, VDAC or Lamin A+C as indicated. See Fig. S13 for complete images. (C) Live cells after electroporation were probed with anti-mCherry antibody, fixed, and visualized using confocal microscopy. The images are averaged single plane scans. (D) Zoom-in image of punctate structures seen in C. All scale bars are 10 µm (A, C, D).
Figure Legend Snippet: Cellular localization and membrane topology of XP. HeLa cells were electroporated with pCAG-mCherry, pCAG-mCherry-XP or pCAG-XP-mCherry. (A) Representative confocal images of fixed and permeabilized cells were visualized for mCherry (red) and stained for nuclei (Hoechst, blue). The images are averaged single plane scans. (B) Cell lysates were fractionated and whole cell lysate (WCL), cytoplasmic (Cyto), membrane (Mem) and soluble nuclear (Nucl) fractions were analyzed by immunoblotting with antibodies to mCherry, tubulin, VDAC or Lamin A+C as indicated. See Fig. S13 for complete images. (C) Live cells after electroporation were probed with anti-mCherry antibody, fixed, and visualized using confocal microscopy. The images are averaged single plane scans. (D) Zoom-in image of punctate structures seen in C. All scale bars are 10 µm (A, C, D).

Techniques Used: Staining, Electroporation, Confocal Microscopy

XP from HAstV1 and related astroviruses has viroporin-like activity. (A) HAstV1 XP sequence and helical wheel representation of amino acids 92–109. (B) Kyte-Doolittle hydropathy plots of HAstV XPs (see Fig. S16 for individual plots). (C) Membrane permeabilization in BSR cells at 8 h post RNA electroporation with Sindbis virus replicons (SINV repC) expressing HAstV1 XP, enterovirus Strep-2B (positive control) or mCherry (negative control). Ongoing protein synthesis was labelled with 1 mM AHA in the presence or absence of 1 mM HB as a translation inhibitor. Cells were lysed and AHA-bearing proteins were ligated to the fluorescent reporter IRDye800CW Alkyne by click chemistry, separated by SDS-PAGE, and visualized by in-gel fluorescence. The numbers below each pair of samples indicate protein synthesis quantified for HB-treated cells relative to the values obtained for untreated cells. (D) Statistical analysis of membrane permeabilization caused by XP and the indicated control proteins in BSR cells. Bars indicate the amount of protein synthesis in HB-treated cells relative to untreated cells (mean ± s.d.; n = 3 biologically independent experiments). (E,G,I) E. coli lysis assay for HAstV1 XP (E) and indicated mutants (G) or XPs derived from other astrovirus species (I) (mean ± s.d.; n = 3 biologically independent experiments). (F) Amino acid mutations used in G. (H) Astrovirus XP sequences tested in I. (J) Western blots for E, G and I confirming expression of the relevant products (see Fig. S17 for full images).
Figure Legend Snippet: XP from HAstV1 and related astroviruses has viroporin-like activity. (A) HAstV1 XP sequence and helical wheel representation of amino acids 92–109. (B) Kyte-Doolittle hydropathy plots of HAstV XPs (see Fig. S16 for individual plots). (C) Membrane permeabilization in BSR cells at 8 h post RNA electroporation with Sindbis virus replicons (SINV repC) expressing HAstV1 XP, enterovirus Strep-2B (positive control) or mCherry (negative control). Ongoing protein synthesis was labelled with 1 mM AHA in the presence or absence of 1 mM HB as a translation inhibitor. Cells were lysed and AHA-bearing proteins were ligated to the fluorescent reporter IRDye800CW Alkyne by click chemistry, separated by SDS-PAGE, and visualized by in-gel fluorescence. The numbers below each pair of samples indicate protein synthesis quantified for HB-treated cells relative to the values obtained for untreated cells. (D) Statistical analysis of membrane permeabilization caused by XP and the indicated control proteins in BSR cells. Bars indicate the amount of protein synthesis in HB-treated cells relative to untreated cells (mean ± s.d.; n = 3 biologically independent experiments). (E,G,I) E. coli lysis assay for HAstV1 XP (E) and indicated mutants (G) or XPs derived from other astrovirus species (I) (mean ± s.d.; n = 3 biologically independent experiments). (F) Amino acid mutations used in G. (H) Astrovirus XP sequences tested in I. (J) Western blots for E, G and I confirming expression of the relevant products (see Fig. S17 for full images).

Techniques Used: Activity Assay, Sequencing, Electroporation, Expressing, Positive Control, Negative Control, SDS Page, Fluorescence, Lysis, Derivative Assay, Western Blot

38) Product Images from "Off-Target Expression of Cre-Dependent Adeno-Associated Viruses in Wild-Type C57BL/6J Mice"

Article Title: Off-Target Expression of Cre-Dependent Adeno-Associated Viruses in Wild-Type C57BL/6J Mice

Journal: eNeuro

doi: 10.1523/ENEURO.0363-21.2021

Antibody amplification of Cre-dependent viral expression. A , Representative images from a TH-Cre mouse injected in the VTA with AAV5-EF1a-DIO-mCherry show a similar pattern of expression between nonamplified and amplified fluorescence (yellow and white arrows). B , Long-range VTA–NAc/DS projections are easier to visualize following mCherry amplification (yellow vs white arrow). C , Similarly, nonamplified fluorescence of VTA to mPFC projections was generally weak (yellow arrows), and the fluorescence signal was significantly improved following mCherry amplification (white arrows). D , E , Representative images from PV-Cre mice injected with AAV5-EF1a-DIO-EYFP ( D ) or AAV5-EF1a-DIO-mCherry ( E ). The nonamplified fluorescence signal was similar between eYFP and mCherry constructs. Moreover, fluorescence signal amplification is similar to the nonamplified signal (yellow arrows) but is brighter and easier to visualize (white arrows), especially the dendrites in the ML. F , Representative images from a C57BL/6J mouse injected with AAV5-EF1a-DIO-mCherry show minimal nonamplified fluorescence (yellow arrow). Remarkably, amplification of adjacent sections from the same mouse revealed mCherry expression within the DG (white arrows). Scale bars: Panels A – B : 200 μm; Panels C – F : 100 μm.
Figure Legend Snippet: Antibody amplification of Cre-dependent viral expression. A , Representative images from a TH-Cre mouse injected in the VTA with AAV5-EF1a-DIO-mCherry show a similar pattern of expression between nonamplified and amplified fluorescence (yellow and white arrows). B , Long-range VTA–NAc/DS projections are easier to visualize following mCherry amplification (yellow vs white arrow). C , Similarly, nonamplified fluorescence of VTA to mPFC projections was generally weak (yellow arrows), and the fluorescence signal was significantly improved following mCherry amplification (white arrows). D , E , Representative images from PV-Cre mice injected with AAV5-EF1a-DIO-EYFP ( D ) or AAV5-EF1a-DIO-mCherry ( E ). The nonamplified fluorescence signal was similar between eYFP and mCherry constructs. Moreover, fluorescence signal amplification is similar to the nonamplified signal (yellow arrows) but is brighter and easier to visualize (white arrows), especially the dendrites in the ML. F , Representative images from a C57BL/6J mouse injected with AAV5-EF1a-DIO-mCherry show minimal nonamplified fluorescence (yellow arrow). Remarkably, amplification of adjacent sections from the same mouse revealed mCherry expression within the DG (white arrows). Scale bars: Panels A – B : 200 μm; Panels C – F : 100 μm.

Techniques Used: Amplification, Expressing, Injection, Fluorescence, Mouse Assay, Construct

Amplified expression of DIO-mCherry in the hippocampus of WT C57BL/6J mice. A , B , Experimental design and timeline. AAV5-EF1a-DIO-mCherry was injected into the anterior and posterior hippocampi of C57BL/6J mice ( n = 8) and perfused 2–3 weeks later. Brains were sectioned in the coronal plane, and viral signal was amplified with rabbit anti-mCherry and goat anti-rabbit 568 antibodies. C , Representative immunofluorescence of mCherry throughout the relatively dorsal (top) and caudal (bottom) DG. Expression of mCherry was primarily observed in the GCL and dendrites extending into the ML (putative dentate GCs). The amplified mCherry signal also resulted in labeling of mossy fibers and cells in the hilus. D , Quantification of mCherry + cells indicated that somatic expression was restricted to the injected hemisphere. Female (clear circles) and male (dotted circles) data points are identified, but no sex differences were found. *** p
Figure Legend Snippet: Amplified expression of DIO-mCherry in the hippocampus of WT C57BL/6J mice. A , B , Experimental design and timeline. AAV5-EF1a-DIO-mCherry was injected into the anterior and posterior hippocampi of C57BL/6J mice ( n = 8) and perfused 2–3 weeks later. Brains were sectioned in the coronal plane, and viral signal was amplified with rabbit anti-mCherry and goat anti-rabbit 568 antibodies. C , Representative immunofluorescence of mCherry throughout the relatively dorsal (top) and caudal (bottom) DG. Expression of mCherry was primarily observed in the GCL and dendrites extending into the ML (putative dentate GCs). The amplified mCherry signal also resulted in labeling of mossy fibers and cells in the hilus. D , Quantification of mCherry + cells indicated that somatic expression was restricted to the injected hemisphere. Female (clear circles) and male (dotted circles) data points are identified, but no sex differences were found. *** p

Techniques Used: Amplification, Expressing, Mouse Assay, Injection, Immunofluorescence, Labeling

Nonamplified fluorescence of DIO constructs in WT C57BL/6J mice. A , B , Representative photomicrographs of nonamplified fluorescence signal in C57BL/6J mice injected with AAV5-EF1a-DIO-eYFP ( A ) or AAV5-EF1a-DIO-mCherry ( B ). Nonamplified immunofluorescence was generally weak and primarily restricted to the soma (yellow arrows; see insets) of the injected hemisphere only. We hypothesize that the weak nonamplified immunofluorescence in these cells is significantly enhanced after antibody amplification. In addition, a very small number of cells with bright immunofluorescence throughout the cell body and its processes were observed (white arrows; see insets). Scale bars: 10× objective, 100 μm; 20× objective, insets, 25 μm.
Figure Legend Snippet: Nonamplified fluorescence of DIO constructs in WT C57BL/6J mice. A , B , Representative photomicrographs of nonamplified fluorescence signal in C57BL/6J mice injected with AAV5-EF1a-DIO-eYFP ( A ) or AAV5-EF1a-DIO-mCherry ( B ). Nonamplified immunofluorescence was generally weak and primarily restricted to the soma (yellow arrows; see insets) of the injected hemisphere only. We hypothesize that the weak nonamplified immunofluorescence in these cells is significantly enhanced after antibody amplification. In addition, a very small number of cells with bright immunofluorescence throughout the cell body and its processes were observed (white arrows; see insets). Scale bars: 10× objective, 100 μm; 20× objective, insets, 25 μm.

Techniques Used: Fluorescence, Construct, Mouse Assay, Injection, Immunofluorescence, Amplification

The hM3Dq agonist C21 does not affect fear behavior in C57BL/6J mice injected with DIO-mCherry or DIO-hM3Dq-mCherry in the DG. A , B , Experimental design and timeline. Adult C57BL/6J mice underw ent surgery to receive intrahippocampal injections of AAV-EF1a-DIO-mCherry or AAV-hSyn-DIO-hM3Dq-mCherry. After a 2 week recovery period, mice were injected with the hM3Dq agonist C21 1 h before contextual fear training. C , Mice were then placed in a fear-conditioning chamber. Baseline activity was assessed over 2 min, followed by five footshocks (0.5 mA) spaced 1 min apart. D , Minute-by-minute analysis of the training session revealed that freezing behavior did not differ between EF1a-DIO-mCherry or hSyn-DIO-hM3Dq-mCherry groups. E , The average postshock freezing did not differ between the EF1a-DIO-mCherry and hSyn-DIO-hM3Dq-mCherry groups. F , Mice were returned to the same operant chamber 24 h later to test contextual fear memory. Notably, C21 was not administered a second time before the contextual memory test. G , Minute-by-minute analysis revealed that conditioned freezing did not differ between the EF1a-DIO-mCherry or hSyn-DIO-hM3Dq-mCherry groups. H , Average freezing during the memory test did not differ between groups. Female (clear points) and male (dotted points) data points are identified, but no sex differences were found.
Figure Legend Snippet: The hM3Dq agonist C21 does not affect fear behavior in C57BL/6J mice injected with DIO-mCherry or DIO-hM3Dq-mCherry in the DG. A , B , Experimental design and timeline. Adult C57BL/6J mice underw ent surgery to receive intrahippocampal injections of AAV-EF1a-DIO-mCherry or AAV-hSyn-DIO-hM3Dq-mCherry. After a 2 week recovery period, mice were injected with the hM3Dq agonist C21 1 h before contextual fear training. C , Mice were then placed in a fear-conditioning chamber. Baseline activity was assessed over 2 min, followed by five footshocks (0.5 mA) spaced 1 min apart. D , Minute-by-minute analysis of the training session revealed that freezing behavior did not differ between EF1a-DIO-mCherry or hSyn-DIO-hM3Dq-mCherry groups. E , The average postshock freezing did not differ between the EF1a-DIO-mCherry and hSyn-DIO-hM3Dq-mCherry groups. F , Mice were returned to the same operant chamber 24 h later to test contextual fear memory. Notably, C21 was not administered a second time before the contextual memory test. G , Minute-by-minute analysis revealed that conditioned freezing did not differ between the EF1a-DIO-mCherry or hSyn-DIO-hM3Dq-mCherry groups. H , Average freezing during the memory test did not differ between groups. Female (clear points) and male (dotted points) data points are identified, but no sex differences were found.

Techniques Used: Mouse Assay, Injection, Activity Assay

mCherry and c-Fos immunofluorescence following C21 home-cage challenge. A , B , Experimental design and timeline. Mice underwent surgery for AAV injection and were allowed 2 weeks for recovery. Mice underwent behavioral testing and were then given a 3 d washout period. Mice were then injected with C21 (1.5 mg/kg) in their home cage and were euthanized 90 min later to evaluate the immediate early gene c-Fos. C , The percentage of colocalization of c-Fos + and mCherry + cells following C21 challenge was significantly lower in C57BL/6J mice injected with DIO-mCherry (7 c-Fos + mCherry + /497 mCherry + cells = 1.41%) or DIO-hM3Dq-mCherry (23 c-Fos + mCherry + /1062 mCherry + cells = 2.17%) compared with PV-Cre + mice injected with DIO-hM3Dq-mCherry (267 c-Fos + mCherry/367 mCherry + cells = 72.75%). D–F , Representative images show that C57BL/6J mice lacked the clear elevation of c-Fos (green) in mCherry + cells seen in PV-Cre + mice (yellow; white arrows). **** p
Figure Legend Snippet: mCherry and c-Fos immunofluorescence following C21 home-cage challenge. A , B , Experimental design and timeline. Mice underwent surgery for AAV injection and were allowed 2 weeks for recovery. Mice underwent behavioral testing and were then given a 3 d washout period. Mice were then injected with C21 (1.5 mg/kg) in their home cage and were euthanized 90 min later to evaluate the immediate early gene c-Fos. C , The percentage of colocalization of c-Fos + and mCherry + cells following C21 challenge was significantly lower in C57BL/6J mice injected with DIO-mCherry (7 c-Fos + mCherry + /497 mCherry + cells = 1.41%) or DIO-hM3Dq-mCherry (23 c-Fos + mCherry + /1062 mCherry + cells = 2.17%) compared with PV-Cre + mice injected with DIO-hM3Dq-mCherry (267 c-Fos + mCherry/367 mCherry + cells = 72.75%). D–F , Representative images show that C57BL/6J mice lacked the clear elevation of c-Fos (green) in mCherry + cells seen in PV-Cre + mice (yellow; white arrows). **** p

Techniques Used: Immunofluorescence, Mouse Assay, Injection

Amplified expression of DIO-hM3Dq-mCherry in the hippocampi of WT C57BL/6J mice. A , B , Experimental design and timeline. AAV8-hSyn-DIO-hM3Dq-mCherry was injected into the anterior and posterior hippocampus of C57BL/6J mice ( n = 8), and mice were perfused 2–3 weeks later. The viral signal was amplified with rabbit anti-mCherry and goat anti-rabbit 568 antibodies and was visualized on an epifluorescence microscope. C , Representative mCherry immunofluorescence in relatively dorsal (top) and caudal (bottom) sections of the DG. Amplified mCherry expression appeared primarily within hilar cells and a sparse number of GCs (yellow arrows). D , Quantification of mCherry + cells revealed that expression was restricted to the injected hippocampus. Female (clear circles) and male (dotted circles) data points are identified, but no sex differences were found. *** p
Figure Legend Snippet: Amplified expression of DIO-hM3Dq-mCherry in the hippocampi of WT C57BL/6J mice. A , B , Experimental design and timeline. AAV8-hSyn-DIO-hM3Dq-mCherry was injected into the anterior and posterior hippocampus of C57BL/6J mice ( n = 8), and mice were perfused 2–3 weeks later. The viral signal was amplified with rabbit anti-mCherry and goat anti-rabbit 568 antibodies and was visualized on an epifluorescence microscope. C , Representative mCherry immunofluorescence in relatively dorsal (top) and caudal (bottom) sections of the DG. Amplified mCherry expression appeared primarily within hilar cells and a sparse number of GCs (yellow arrows). D , Quantification of mCherry + cells revealed that expression was restricted to the injected hippocampus. Female (clear circles) and male (dotted circles) data points are identified, but no sex differences were found. *** p

Techniques Used: Amplification, Expressing, Mouse Assay, Injection, Microscopy, Immunofluorescence

39) Product Images from "Cytotoxic lymphocytes target HIV-1 Gag through granzyme M-mediated cleavage"

Article Title: Cytotoxic lymphocytes target HIV-1 Gag through granzyme M-mediated cleavage

Journal: bioRxiv

doi: 10.1101/2021.02.24.432686

GrM degrades various HIV-1 subtype Gag proteins. HEK293FT cells were transfected with plasmids encoding for C-terminal mCherry-tagged HIV-1C Gag PYQEi (A-B) , HIV-1C Gag WT (C-D) or HIV-1B Gag WT (E-F) and lysates (10 µg) were incubated with increasing concentrations of GrM or GrM-SA (500 nM) for 4 h at 37°C. Samples were subjected to immunoblotting using an anti-mCherry antibody (A, C, E) or anti-p55 Gag antibody (B, D, F) to detect full length Gag-mCherry and degradation products. (G) Band intensities of full-length Gag-mCherry from all four Gag variants as detected with the anti-mCherry antibody in figures 1 and 2 and additional experiments were quantified and plotted with Gag incubated with 0 nM GrM set at 100%. Data points represent the mean ±SEM from two individual experiments.
Figure Legend Snippet: GrM degrades various HIV-1 subtype Gag proteins. HEK293FT cells were transfected with plasmids encoding for C-terminal mCherry-tagged HIV-1C Gag PYQEi (A-B) , HIV-1C Gag WT (C-D) or HIV-1B Gag WT (E-F) and lysates (10 µg) were incubated with increasing concentrations of GrM or GrM-SA (500 nM) for 4 h at 37°C. Samples were subjected to immunoblotting using an anti-mCherry antibody (A, C, E) or anti-p55 Gag antibody (B, D, F) to detect full length Gag-mCherry and degradation products. (G) Band intensities of full-length Gag-mCherry from all four Gag variants as detected with the anti-mCherry antibody in figures 1 and 2 and additional experiments were quantified and plotted with Gag incubated with 0 nM GrM set at 100%. Data points represent the mean ±SEM from two individual experiments.

Techniques Used: Transfection, Incubation

Cytotoxic lymphoma cell line KHYG-1 target Gag PYKEi in living target cells through GrM. (A) HeLa cells were transfected with both mCherry-tagged HIV-1C Gag PYKEi and pEGFP-N1 in a 3:1 plasmid ratio and at 24 h post-transfection these cells were challenged with increasing effector:target (E:T) ratios of KHYG-1 cells for 8 h at 37°C. Lysates were subjected to immunoblotting using an anti-mCherry, anti-p55 Gag or anti-GFP antibody. (B) HeLa cells were transfected with both mCherry-tagged HIV-1C Gag PYKEi and pEGFP-N1 in a 3:1 plasmid ratio and seeded in the presence of zVAD-FMK (100 µM) or only DMSO. Then, 24 h post-transfection, these cells were challenged with KHYG-1 cells (effector:target ratio of 2:1) in the presence of zVAD-FMK (100 µM) or only DMSO for indicated time points at 37°C. Lysates were subjected to immunoblotting using an anti-mCherry, anti-p55 Gag or anti-GFP antibody. (C) Co-cultures were performed as in (b) except KHYG-1 cells were pretreated with GrM inhibitor peptide AcKVPL-CMK (100 µM) or left untreated for 1 h at 37°C before challenging mCherry-tagged Gag PYKEi expressing HeLa cell with KHYG-1 cells for 4 h at 37°C. (D) Band intensities of the ~53 kDa Gag degradation product as detected with the anti-p55 Gag antibody in (c) and two additional experiments were quantified, and values were normalized for GFP band intensities. Data points are plotted as mean ±SEM arbitrary units (AU) from triplicate samples. (*p
Figure Legend Snippet: Cytotoxic lymphoma cell line KHYG-1 target Gag PYKEi in living target cells through GrM. (A) HeLa cells were transfected with both mCherry-tagged HIV-1C Gag PYKEi and pEGFP-N1 in a 3:1 plasmid ratio and at 24 h post-transfection these cells were challenged with increasing effector:target (E:T) ratios of KHYG-1 cells for 8 h at 37°C. Lysates were subjected to immunoblotting using an anti-mCherry, anti-p55 Gag or anti-GFP antibody. (B) HeLa cells were transfected with both mCherry-tagged HIV-1C Gag PYKEi and pEGFP-N1 in a 3:1 plasmid ratio and seeded in the presence of zVAD-FMK (100 µM) or only DMSO. Then, 24 h post-transfection, these cells were challenged with KHYG-1 cells (effector:target ratio of 2:1) in the presence of zVAD-FMK (100 µM) or only DMSO for indicated time points at 37°C. Lysates were subjected to immunoblotting using an anti-mCherry, anti-p55 Gag or anti-GFP antibody. (C) Co-cultures were performed as in (b) except KHYG-1 cells were pretreated with GrM inhibitor peptide AcKVPL-CMK (100 µM) or left untreated for 1 h at 37°C before challenging mCherry-tagged Gag PYKEi expressing HeLa cell with KHYG-1 cells for 4 h at 37°C. (D) Band intensities of the ~53 kDa Gag degradation product as detected with the anti-p55 Gag antibody in (c) and two additional experiments were quantified, and values were normalized for GFP band intensities. Data points are plotted as mean ±SEM arbitrary units (AU) from triplicate samples. (*p

Techniques Used: Transfection, Plasmid Preparation, Expressing

GrM cleaves HIV-1C Gag PYKEi after Leu 483 . (A-B) Based on the HIV-1C Gag PYKEi protein sequence and the GrM acid consensus GrM substrate motif KEPL, we predicted that GrM can cleave Gag PYKEi after the leucine residue at position 483 (Leu 483 ). To test this, we mutated Leu 483 into an alanine (KEPL/A mutant). Then, HEK293FT cells were transfected with mCherry-tagged HIV-1C Gag PYKEi or the KEPL/A mutant. Lysates (10 µg) were incubated with increasing concentrations of GrM or GrM-SA (100 nM) for 4 h at 37°C and immunoblotted using an anti-mCherry antibody (A) or anti-p55 Gag antibody (B). (C) Schematic overview of GrM cleavage site within Gag PYKEi as well as other tested mutants that were not GrM cleavage sites (MA, p17 matrix protein; CA, p24 capsid protein; SP1, spacer peptide 1; NC, p7 nucleocapsid protein; SP2, spacer peptide 2; P6, p6 late domain).
Figure Legend Snippet: GrM cleaves HIV-1C Gag PYKEi after Leu 483 . (A-B) Based on the HIV-1C Gag PYKEi protein sequence and the GrM acid consensus GrM substrate motif KEPL, we predicted that GrM can cleave Gag PYKEi after the leucine residue at position 483 (Leu 483 ). To test this, we mutated Leu 483 into an alanine (KEPL/A mutant). Then, HEK293FT cells were transfected with mCherry-tagged HIV-1C Gag PYKEi or the KEPL/A mutant. Lysates (10 µg) were incubated with increasing concentrations of GrM or GrM-SA (100 nM) for 4 h at 37°C and immunoblotted using an anti-mCherry antibody (A) or anti-p55 Gag antibody (B). (C) Schematic overview of GrM cleavage site within Gag PYKEi as well as other tested mutants that were not GrM cleavage sites (MA, p17 matrix protein; CA, p24 capsid protein; SP1, spacer peptide 1; NC, p7 nucleocapsid protein; SP2, spacer peptide 2; P6, p6 late domain).

Techniques Used: Sequencing, Mutagenesis, Transfection, Incubation

IL-2-activated PBMCs target Gag PYKEi in living target cells. (A) HeLa cells were transfected with both mCherry-tagged HIV-1C Gag PYKEi and pEGFP-N1 in a 3:1 plasmid ratio and at 24 h post-transfection these cells were challenged with increasing effector:target (E:T) ratios of IL-2-activated PBMCs for 4 h at 37°C. Lysates were subjected to immunoblotting using an anti-mCherry, anti-p55 Gag or anti-GFP antibody. (B) HeLa cells were transfected with both mCherry-tagged HIV-1C Gag PYKEi and pEGFP-N1 in a 3:1 plasmid ratio and seeded in the presence of zVAD-FMK (100 µM) or only DMSO. Then, 24 h post-transfection, these cells were challenged with increasing E:T ratios of IL-2-activated PBMCs in the presence of zVAD-FMK (100 µM) or only DMSO for 4 h at 37°C. Lysates were subjected to immunoblotting using an anti-mCherry, anti-p55 Gag or anti-GFP antibody.
Figure Legend Snippet: IL-2-activated PBMCs target Gag PYKEi in living target cells. (A) HeLa cells were transfected with both mCherry-tagged HIV-1C Gag PYKEi and pEGFP-N1 in a 3:1 plasmid ratio and at 24 h post-transfection these cells were challenged with increasing effector:target (E:T) ratios of IL-2-activated PBMCs for 4 h at 37°C. Lysates were subjected to immunoblotting using an anti-mCherry, anti-p55 Gag or anti-GFP antibody. (B) HeLa cells were transfected with both mCherry-tagged HIV-1C Gag PYKEi and pEGFP-N1 in a 3:1 plasmid ratio and seeded in the presence of zVAD-FMK (100 µM) or only DMSO. Then, 24 h post-transfection, these cells were challenged with increasing E:T ratios of IL-2-activated PBMCs in the presence of zVAD-FMK (100 µM) or only DMSO for 4 h at 37°C. Lysates were subjected to immunoblotting using an anti-mCherry, anti-p55 Gag or anti-GFP antibody.

Techniques Used: Transfection, Plasmid Preparation

GrM degrades HIV-1 Gag PYKEi protein. (A-B) HEK293FT cells were transfected with plasmids encoding for C-terminal mCherry-tagged HIV-1C Gag PYKEi and lysates (10 µg) were incubated with increasing concentrations of GrM or GrM-SA (500 nM) for 4 h at 37°C. Samples were subjected to immunoblotting using an anti-mCherry antibody (A) or anti-p55 Gag antibody (B) to detect full length Gag-mCherry and degradation products. (C-D) Lysates (10 µg) were incubated with 5 nM of GrM for the indicated time points or GrM-SA for 4 h at 37°C and immunoblotted using an anti-mCherry antibody (C) or anti-p55 Gag antibody (D) . Of note, the C-terminal mCherry tag is partially degraded by other cellular proteases as observed by the smaller Gag-mCherry product around 65 kDa (Gag-mCherry*). Data depicted is representable for at least two individual experiments.
Figure Legend Snippet: GrM degrades HIV-1 Gag PYKEi protein. (A-B) HEK293FT cells were transfected with plasmids encoding for C-terminal mCherry-tagged HIV-1C Gag PYKEi and lysates (10 µg) were incubated with increasing concentrations of GrM or GrM-SA (500 nM) for 4 h at 37°C. Samples were subjected to immunoblotting using an anti-mCherry antibody (A) or anti-p55 Gag antibody (B) to detect full length Gag-mCherry and degradation products. (C-D) Lysates (10 µg) were incubated with 5 nM of GrM for the indicated time points or GrM-SA for 4 h at 37°C and immunoblotted using an anti-mCherry antibody (C) or anti-p55 Gag antibody (D) . Of note, the C-terminal mCherry tag is partially degraded by other cellular proteases as observed by the smaller Gag-mCherry product around 65 kDa (Gag-mCherry*). Data depicted is representable for at least two individual experiments.

Techniques Used: Transfection, Incubation

40) Product Images from "Time-dependent enhancement in ventral tegmental area dopamine neuron activity drives pain-facilitated fentanyl intake in males"

Article Title: Time-dependent enhancement in ventral tegmental area dopamine neuron activity drives pain-facilitated fentanyl intake in males

Journal: bioRxiv

doi: 10.1101/2022.08.19.504549

Protracted increases in VTA DA cell calcium dynamics are necessary for pain-dependent increases in fentanyl consumption in males. A, Schematic depicting experimental timeline. Male TH-Cre+ rats were injected in the VTA with cre-dependent jGCaMP and inhibitory DREADDS (Gi) or control virus (mCh). Two weeks later, rats received hind-paw injections of CFA before fentanyl self-administration. The last three days of week 2, rats were habituated to the injection procedure with injections of saline (SAL, i.p.). During week 3, rats expressing Gi-DREADDs received either SAL (GI-SAL) or CNO (Gi-CNO) and rats expressing mCh received CNO (mCh-CNO) before the self-administration session. B, Representative images of Gi-DREADD (mCherry) expression colocalized with tyrosine hydroxylase (TH) and fiber placement in the VTA of male TH-Cre+ rats. C, Representative traces of tonic calcium transient events throughout the 2-hr self-administration session. D, Percent change in tonic activity (frequency) after treatment with CNO (1 mg/kg, i.p.) or SAL (1 mL/kg, i.p.) (one-way ANOVA, F (2,15) =6.495, P=0.0093, Tukey’s post-hoc test, *P
Figure Legend Snippet: Protracted increases in VTA DA cell calcium dynamics are necessary for pain-dependent increases in fentanyl consumption in males. A, Schematic depicting experimental timeline. Male TH-Cre+ rats were injected in the VTA with cre-dependent jGCaMP and inhibitory DREADDS (Gi) or control virus (mCh). Two weeks later, rats received hind-paw injections of CFA before fentanyl self-administration. The last three days of week 2, rats were habituated to the injection procedure with injections of saline (SAL, i.p.). During week 3, rats expressing Gi-DREADDs received either SAL (GI-SAL) or CNO (Gi-CNO) and rats expressing mCh received CNO (mCh-CNO) before the self-administration session. B, Representative images of Gi-DREADD (mCherry) expression colocalized with tyrosine hydroxylase (TH) and fiber placement in the VTA of male TH-Cre+ rats. C, Representative traces of tonic calcium transient events throughout the 2-hr self-administration session. D, Percent change in tonic activity (frequency) after treatment with CNO (1 mg/kg, i.p.) or SAL (1 mL/kg, i.p.) (one-way ANOVA, F (2,15) =6.495, P=0.0093, Tukey’s post-hoc test, *P

Techniques Used: Injection, Expressing, Activity Assay

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    Abcam anti mcherry antibody
    OsNF-YC11 and OsNF-YC12 interact with OsNF-YB1 in vivo and mediate the nuclear localization of OsNF-YB1. (A) Observation of OsNF-YB1 subcellular localization showed a dual cytosolic–nuclear localization in root cells and nucleus-specific localization in aleurone layer cells. Root of young seedlings and aleurone layer cells of 10 DAF seeds expressing pUbi:OsNF-YB1-GFP were observed. Bars: 50 µm (upper) or 20 µm (bottom). (B) OsNF-YB1-GFP protein showed a dual cytosolic–nuclear localization in the presence of red fluorescent protein (RFP) or OsNF-YC2-RFP, and was translocated to nucleus of rice protoplast cells in the presence of OsNF-YC11-RFP or OsNF-YC12-RFP. Bars: 5 µm. (C, D) Co-immunoprecipitation analysis revealed the interaction of OsNF-YB1-GFP and <t>OsNF-YC11-mCherry</t> (C), and OsNF-YB1-GFP and OsNF-YC12-mCherry (D) in tobacco cells. Total protein extracts (Input) or immunoprecipitated (IP) fractions using an anti-GFP antibody were analyzed using anti-GFP or anti-mCherry antibodies.
    Anti Mcherry Antibody, supplied by Abcam, used in various techniques. Bioz Stars score: 98/100, based on 9 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    OsNF-YC11 and OsNF-YC12 interact with OsNF-YB1 in vivo and mediate the nuclear localization of OsNF-YB1. (A) Observation of OsNF-YB1 subcellular localization showed a dual cytosolic–nuclear localization in root cells and nucleus-specific localization in aleurone layer cells. Root of young seedlings and aleurone layer cells of 10 DAF seeds expressing pUbi:OsNF-YB1-GFP were observed. Bars: 50 µm (upper) or 20 µm (bottom). (B) OsNF-YB1-GFP protein showed a dual cytosolic–nuclear localization in the presence of red fluorescent protein (RFP) or OsNF-YC2-RFP, and was translocated to nucleus of rice protoplast cells in the presence of OsNF-YC11-RFP or OsNF-YC12-RFP. Bars: 5 µm. (C, D) Co-immunoprecipitation analysis revealed the interaction of OsNF-YB1-GFP and OsNF-YC11-mCherry (C), and OsNF-YB1-GFP and OsNF-YC12-mCherry (D) in tobacco cells. Total protein extracts (Input) or immunoprecipitated (IP) fractions using an anti-GFP antibody were analyzed using anti-GFP or anti-mCherry antibodies.

    Journal: Journal of Experimental Botany

    Article Title: Rice aleurone layer specific OsNF-YB1 regulates grain filling and endosperm development by interacting with an ERF transcription factor

    doi: 10.1093/jxb/erw409

    Figure Lengend Snippet: OsNF-YC11 and OsNF-YC12 interact with OsNF-YB1 in vivo and mediate the nuclear localization of OsNF-YB1. (A) Observation of OsNF-YB1 subcellular localization showed a dual cytosolic–nuclear localization in root cells and nucleus-specific localization in aleurone layer cells. Root of young seedlings and aleurone layer cells of 10 DAF seeds expressing pUbi:OsNF-YB1-GFP were observed. Bars: 50 µm (upper) or 20 µm (bottom). (B) OsNF-YB1-GFP protein showed a dual cytosolic–nuclear localization in the presence of red fluorescent protein (RFP) or OsNF-YC2-RFP, and was translocated to nucleus of rice protoplast cells in the presence of OsNF-YC11-RFP or OsNF-YC12-RFP. Bars: 5 µm. (C, D) Co-immunoprecipitation analysis revealed the interaction of OsNF-YB1-GFP and OsNF-YC11-mCherry (C), and OsNF-YB1-GFP and OsNF-YC12-mCherry (D) in tobacco cells. Total protein extracts (Input) or immunoprecipitated (IP) fractions using an anti-GFP antibody were analyzed using anti-GFP or anti-mCherry antibodies.

    Article Snippet: The bound proteins were eluted with 2× SDS sample buffer, and subjected to immunoblot analysis using monoclonal anti-GFP antibody (sc-9996; Santa) and anti-mCherry antibody (ab125096; Abcam).

    Techniques: In Vivo, Expressing, Immunoprecipitation

    Generation of SKIP-mCherry knock-in (KI) mice. ( a ) Construct of SKIP-mCherry KI mice. mCherry-poly(A)-loxp-Neo-loxp was inserted into exon 1 of wild-type SKIP gene, and later, loxp-Neo was deleted to generate the mutant allele. In the mutant protein sequence, SKIP expression was deleted by mCherry expression. mRNA expression of SKIP and mCherry in isolated islets from homo SKIP-mCherry KI (SKIP −/− ) mice and wild type (SKIP +/+ ) mice detected by RT-PCR ( b ) and qRT-PCR ( c ); data are expressed as average ± standard error of the mean (SEM). **p

    Journal: Scientific Reports

    Article Title: Sphingosine kinase 1-interacting protein is a novel regulator of glucose-stimulated insulin secretion

    doi: 10.1038/s41598-017-00900-7

    Figure Lengend Snippet: Generation of SKIP-mCherry knock-in (KI) mice. ( a ) Construct of SKIP-mCherry KI mice. mCherry-poly(A)-loxp-Neo-loxp was inserted into exon 1 of wild-type SKIP gene, and later, loxp-Neo was deleted to generate the mutant allele. In the mutant protein sequence, SKIP expression was deleted by mCherry expression. mRNA expression of SKIP and mCherry in isolated islets from homo SKIP-mCherry KI (SKIP −/− ) mice and wild type (SKIP +/+ ) mice detected by RT-PCR ( b ) and qRT-PCR ( c ); data are expressed as average ± standard error of the mean (SEM). **p

    Article Snippet: Antibodies The following antibodies were used for western blot and immunohistochemistry: guinea pig polyclonal to insulin (Abcam, ab7842); rabbit monoclonal [D16G10] to glucagon (Cell signaling, #8233); mouse monoclonal to GAPDH (Santa cruz, sc-32233); mouse monoclonal [1C51] to mCherry (Abcam, ab125096).

    Techniques: Knock-In, Mouse Assay, Construct, Mutagenesis, Sequencing, Expressing, Isolation, Reverse Transcription Polymerase Chain Reaction, Quantitative RT-PCR

    Localization of SKIP in the islets. ( a ) Immunohistochemical images in the pancreas from SKIP −/− mice and SKIP +/+ mice; green, anti-insulin; blue: anti-glucagon; and red, anti-mCherry. ( b ) 3D imaging by incubator two-photon microscopy in isolated islets from SKIP −/− mice. ( c ) Incubator two-photon excitation microscopy images of living β-cells from SKIP −/− (SKIP-mCherry KI) mice and MIP-GFP mice; left column, bright field; second column, mCherry; third column, GFP; right column, merged images. 12-week-old mice were used for the experiments, n = 3.

    Journal: Scientific Reports

    Article Title: Sphingosine kinase 1-interacting protein is a novel regulator of glucose-stimulated insulin secretion

    doi: 10.1038/s41598-017-00900-7

    Figure Lengend Snippet: Localization of SKIP in the islets. ( a ) Immunohistochemical images in the pancreas from SKIP −/− mice and SKIP +/+ mice; green, anti-insulin; blue: anti-glucagon; and red, anti-mCherry. ( b ) 3D imaging by incubator two-photon microscopy in isolated islets from SKIP −/− mice. ( c ) Incubator two-photon excitation microscopy images of living β-cells from SKIP −/− (SKIP-mCherry KI) mice and MIP-GFP mice; left column, bright field; second column, mCherry; third column, GFP; right column, merged images. 12-week-old mice were used for the experiments, n = 3.

    Article Snippet: Antibodies The following antibodies were used for western blot and immunohistochemistry: guinea pig polyclonal to insulin (Abcam, ab7842); rabbit monoclonal [D16G10] to glucagon (Cell signaling, #8233); mouse monoclonal to GAPDH (Santa cruz, sc-32233); mouse monoclonal [1C51] to mCherry (Abcam, ab125096).

    Techniques: Immunohistochemistry, Mouse Assay, Imaging, Microscopy, Isolation

    Glycolytic enzymes are present on various vesicles in axons and dendrites. ( a ) Axonal localization of synaptophysin-immunopositive vesicles (red channel) with glycolytic enzymes and SNAP25 (green channel). ( b ) Axonal localization of glycolytic enzymes (green channel) with VAMP2-mCherry-containing vesicles (red channel). ( c , d ) Axonal localization of glycolytic enzymes or SNAP25 (green channel) with secretory vesicles such as chromogranin A-immunopositive vesicles (red channel) ( c ) or BDNF-mCherry-containing vesicles (red channel) ( d ). ( e ) Vesicles expressing APP-mCherry (red channel) show co-localization with glycolytic enzymes (green channel). ( f ) The nuclear neuronal protein Ctip2 (red channel) does not co-localize with synaptophysin of PK (green channel). Nuclear staining of Ctip2 was used as positive control. ( g ) The Transferrin receptor (TfR, red channel) co-localizes with PGK (green channel) in dendrites of cortical neurons. Co-staining was analysed by Airyscan microscopy. Scale bar, 5 μm.

    Journal: Nature Communications

    Article Title: Self-propelling vesicles define glycolysis as the minimal energy machinery for neuronal transport

    doi: 10.1038/ncomms13233

    Figure Lengend Snippet: Glycolytic enzymes are present on various vesicles in axons and dendrites. ( a ) Axonal localization of synaptophysin-immunopositive vesicles (red channel) with glycolytic enzymes and SNAP25 (green channel). ( b ) Axonal localization of glycolytic enzymes (green channel) with VAMP2-mCherry-containing vesicles (red channel). ( c , d ) Axonal localization of glycolytic enzymes or SNAP25 (green channel) with secretory vesicles such as chromogranin A-immunopositive vesicles (red channel) ( c ) or BDNF-mCherry-containing vesicles (red channel) ( d ). ( e ) Vesicles expressing APP-mCherry (red channel) show co-localization with glycolytic enzymes (green channel). ( f ) The nuclear neuronal protein Ctip2 (red channel) does not co-localize with synaptophysin of PK (green channel). Nuclear staining of Ctip2 was used as positive control. ( g ) The Transferrin receptor (TfR, red channel) co-localizes with PGK (green channel) in dendrites of cortical neurons. Co-staining was analysed by Airyscan microscopy. Scale bar, 5 μm.

    Article Snippet: Reagents and antibodies The following antibodies and dilutions were used for western blotting (WB) and immunofluorescence (IF): mouse anti-GFP horseradish peroxidase (Miltenyi Biotec, 130-091-833, 1:5,000 WB), mouse anti-p150glued (BD Transduction Laboratories, 610474, 1:1,000 WB), mouse anti-kinesin heavy chain (Chemicon, MAB1614, 1:500 WB), mouse anti-DIC (Millipore, MAB1618, 1:500 WB), mouse anti-synaptophysin (Sigma-Aldrich, S5768, 1:1,000 WB, 1:500 IF), rabbit anti-synaptophysin (Abcam, ab14692, 1:500 IF), mouse anti-α-tubulin (Sigma-Aldrich, T9026, 1:1,000 WB), rabbit anti-BDNF (Chemicon, AB1534, 1:1,000 WB), rabbit anti-p50 dynamitin (Millipore, AB5869P, 1:250 WB), mouse anti-SNAP25 (Abcam, ab24737,1:2,000 WB and IF), rabbit anti-HK I (Cell Signaling, mAb2024, 1:1,000 WB, 1:100 IF), mouse anti PGI (Santa Cruz Biotechnology, sc-30392, 1:500 WB), rabbit anti-PFK (Cell Signaling, #5412P, 1: 1,000 WB, 1:100 IF), goat anti-ALDO (Santa Cruz Biotechnology 1:200 WB, sc-12059, 1:50 IF), rabbit anti-GAPDH (Santa Cruz, sc-32233, 1:1,000 WB, 1:100 IF), mouse anti-PGK1 (Abcam, ab90787, 1:1,000 WB), goat anti-PGK (Santa Cruz, sc-23805, 1:1,000 WB, 1:100 IF), goat anti-PGM (Santa Cruz Biotechnology, sc-67756, 1:200 WB, 1:100 IF), rabbit anti-ENO (Santa Cruz Biotechnology, sc15343, 1:200 WB, 1:100 IF), rabbit anti-ENO1 (Cell Signaling, #3810, 1:1,000 WB), rabbit anti-ENO2 (Cell Signaling, mAb8171, 1:1,000 WB), rabbit anti-PK (Cell Signaling, mAb3190,1:1,000 WB, 1:100 IF), mouse anti-Chromogranin A (Santa Cruz, sc-393941, 1:500 IF), rabbit anti-Furin (Santa Cruz, sc-20801, 1:1,000 WB), rabbit anti-Glud1 (Abcam, EPR11369, 1:1,000 WB), rat anti-Ctip2 (Abcam, ab18465, 1:500 IF), mouse anti-mCherry (Abcam, ab125096, 1:1,000 IF), mouse anti-Transferrin receptor (Institut Curie, A-M-M#32, 1:500 IF) and rabbit anti-LDH (Cell Signaling, mAb3582, 1:1,000 WB).

    Techniques: Expressing, Staining, Positive Control, Microscopy

    FAT relies on glycolytic enzymes. ( a ) Silencing of the glycolytic enzymes from the pay-off phase reduces the velocity of BDNF-mCherry vesicles (left panel). Mean and s.e.m. for anterograde and retrograde velocities of control and si-GAPDH (anterograde velocity, t =6.355, P =4.3 × 10 −10 , control: n =321, si-GAPDH: n =235; retrograde velocity, t =5.4, P =9.8 × 10 −8 , control: n =254, si-GAPDH: n =239), control and si-PGK (anterograde velocity, t =11.88, P =1.9 × 10 −31 , control: n =973, si-PGK: n =915; retrograde velocity, t =9.6, 1.8 × 10 −21 , control: n =818, si-PGK: n =829); control and si-PGM (anterograde velocity, t =6.289, P =4.2 × 10 −10 , control: n =890, si-PGM: n =1,126; retrograde velocity, t =5.940, P =3.6 × 10 −9 , control: n =802, si-PGM: n =981); control and si-ENO1+2 (anterograde velocity, t =6.852, P =1.5 × 10 −11 , control: n =455, si-ENO1+2: n =325; retrograde velocity, t =6.498, P =1.6 × 10 −10 , control n =446, si- ENO1+2=335), and control and si-PK1 (anterograde velocity, t =9.974, P =2.9 × 10 −22 , control: n =441, si-PK1: n =437; retrograde velocity, control: t =11.58, P =7.3 × 10 −24 , n =576, si-PK1: n =618). Representative kymographs showing the trajectories of BDNF-mCherry vesicles of different conditions and analysed trajectories with colour-code red for retrograde, green for anterograde and blue for static vesicles (right panel). Scale bar, 10 μm and 10 s. ( b ) The preparatory phase of glycolysis is required for FAT. Inhibition of HK by 2-DG reduces FAT that is rescued by addition of glucose. Mean and s.e.m. for anterograde and retrograde velocities of control, 2-DG and glucose (anterograde velocity, F(2,784)=27.96, P =1.9 × 10 −12 , control: n =248, 2-DG: n =151, glucose: n =388; retrograde velocity, F(2,783)=35.42, P =1.9 × 10 −15 , control: n =242, 2-DG: n =165, glucose: n =379); ( c ) PK activation by PEP rescues transport defect induced by GAPDH silencing. Anterograde and retrograde velocities are represented as mean+s.e.m. (anterograde velocity, F(2,293)=40.02, P =4.3 × 10 −16 , control: n =115, si-GAPDH: n =93, si-GAPDH+PEP: n =87; retrograde velocity, F(2, 262)=34.64, P =4.5 × 10 −14 , control: n =112, si-GAPDH: n =97, si-GAPDH+PEP: n =85). *** P

    Journal: Nature Communications

    Article Title: Self-propelling vesicles define glycolysis as the minimal energy machinery for neuronal transport

    doi: 10.1038/ncomms13233

    Figure Lengend Snippet: FAT relies on glycolytic enzymes. ( a ) Silencing of the glycolytic enzymes from the pay-off phase reduces the velocity of BDNF-mCherry vesicles (left panel). Mean and s.e.m. for anterograde and retrograde velocities of control and si-GAPDH (anterograde velocity, t =6.355, P =4.3 × 10 −10 , control: n =321, si-GAPDH: n =235; retrograde velocity, t =5.4, P =9.8 × 10 −8 , control: n =254, si-GAPDH: n =239), control and si-PGK (anterograde velocity, t =11.88, P =1.9 × 10 −31 , control: n =973, si-PGK: n =915; retrograde velocity, t =9.6, 1.8 × 10 −21 , control: n =818, si-PGK: n =829); control and si-PGM (anterograde velocity, t =6.289, P =4.2 × 10 −10 , control: n =890, si-PGM: n =1,126; retrograde velocity, t =5.940, P =3.6 × 10 −9 , control: n =802, si-PGM: n =981); control and si-ENO1+2 (anterograde velocity, t =6.852, P =1.5 × 10 −11 , control: n =455, si-ENO1+2: n =325; retrograde velocity, t =6.498, P =1.6 × 10 −10 , control n =446, si- ENO1+2=335), and control and si-PK1 (anterograde velocity, t =9.974, P =2.9 × 10 −22 , control: n =441, si-PK1: n =437; retrograde velocity, control: t =11.58, P =7.3 × 10 −24 , n =576, si-PK1: n =618). Representative kymographs showing the trajectories of BDNF-mCherry vesicles of different conditions and analysed trajectories with colour-code red for retrograde, green for anterograde and blue for static vesicles (right panel). Scale bar, 10 μm and 10 s. ( b ) The preparatory phase of glycolysis is required for FAT. Inhibition of HK by 2-DG reduces FAT that is rescued by addition of glucose. Mean and s.e.m. for anterograde and retrograde velocities of control, 2-DG and glucose (anterograde velocity, F(2,784)=27.96, P =1.9 × 10 −12 , control: n =248, 2-DG: n =151, glucose: n =388; retrograde velocity, F(2,783)=35.42, P =1.9 × 10 −15 , control: n =242, 2-DG: n =165, glucose: n =379); ( c ) PK activation by PEP rescues transport defect induced by GAPDH silencing. Anterograde and retrograde velocities are represented as mean+s.e.m. (anterograde velocity, F(2,293)=40.02, P =4.3 × 10 −16 , control: n =115, si-GAPDH: n =93, si-GAPDH+PEP: n =87; retrograde velocity, F(2, 262)=34.64, P =4.5 × 10 −14 , control: n =112, si-GAPDH: n =97, si-GAPDH+PEP: n =85). *** P

    Article Snippet: Reagents and antibodies The following antibodies and dilutions were used for western blotting (WB) and immunofluorescence (IF): mouse anti-GFP horseradish peroxidase (Miltenyi Biotec, 130-091-833, 1:5,000 WB), mouse anti-p150glued (BD Transduction Laboratories, 610474, 1:1,000 WB), mouse anti-kinesin heavy chain (Chemicon, MAB1614, 1:500 WB), mouse anti-DIC (Millipore, MAB1618, 1:500 WB), mouse anti-synaptophysin (Sigma-Aldrich, S5768, 1:1,000 WB, 1:500 IF), rabbit anti-synaptophysin (Abcam, ab14692, 1:500 IF), mouse anti-α-tubulin (Sigma-Aldrich, T9026, 1:1,000 WB), rabbit anti-BDNF (Chemicon, AB1534, 1:1,000 WB), rabbit anti-p50 dynamitin (Millipore, AB5869P, 1:250 WB), mouse anti-SNAP25 (Abcam, ab24737,1:2,000 WB and IF), rabbit anti-HK I (Cell Signaling, mAb2024, 1:1,000 WB, 1:100 IF), mouse anti PGI (Santa Cruz Biotechnology, sc-30392, 1:500 WB), rabbit anti-PFK (Cell Signaling, #5412P, 1: 1,000 WB, 1:100 IF), goat anti-ALDO (Santa Cruz Biotechnology 1:200 WB, sc-12059, 1:50 IF), rabbit anti-GAPDH (Santa Cruz, sc-32233, 1:1,000 WB, 1:100 IF), mouse anti-PGK1 (Abcam, ab90787, 1:1,000 WB), goat anti-PGK (Santa Cruz, sc-23805, 1:1,000 WB, 1:100 IF), goat anti-PGM (Santa Cruz Biotechnology, sc-67756, 1:200 WB, 1:100 IF), rabbit anti-ENO (Santa Cruz Biotechnology, sc15343, 1:200 WB, 1:100 IF), rabbit anti-ENO1 (Cell Signaling, #3810, 1:1,000 WB), rabbit anti-ENO2 (Cell Signaling, mAb8171, 1:1,000 WB), rabbit anti-PK (Cell Signaling, mAb3190,1:1,000 WB, 1:100 IF), mouse anti-Chromogranin A (Santa Cruz, sc-393941, 1:500 IF), rabbit anti-Furin (Santa Cruz, sc-20801, 1:1,000 WB), rabbit anti-Glud1 (Abcam, EPR11369, 1:1,000 WB), rat anti-Ctip2 (Abcam, ab18465, 1:500 IF), mouse anti-mCherry (Abcam, ab125096, 1:1,000 IF), mouse anti-Transferrin receptor (Institut Curie, A-M-M#32, 1:500 IF) and rabbit anti-LDH (Cell Signaling, mAb3582, 1:1,000 WB).

    Techniques: Inhibition, Activation Assay

    The interaction of ARF-PB1 and AID mediates rescue of endogenous protein levels. ( A ). Positions on the X -axis are relative to ARF16 residue numbers. ( B ) The amino acids D994 and D998 of ARF16 and K114 of IAA17 are shown in the left panel. The amino acid side chains corresponding to D183 and D187 of IAA17 and K944 of ARF16 are highlighted in the right ) into each chain of an A. thaliana ). These mutations in ARF16, which disrupt the electrostatic binding interface, fail to rescue chronic ZNF143 ( C ) and TEAD4 ( D ) degradation. ( E ) These mutations disrupt this coimmunoprecipitation of mCherry-AID with eGFP-tagged ARF16-PB1. Consistent with lower stability of the ARF16-MT in C and D , the mutant GFP-ARF16-PB1 plasmid was transfected at a concentration three times higher than the wild type to achieve comparable expression of each protein. ( F ) ARF16-PB1 is detected upon mCherry-AID immunoprecipitation; however, we were unable to detect TIR1 associating with AID.

    Journal: Genes & Development

    Article Title: An improved auxin-inducible degron system preserves native protein levels and enables rapid and specific protein depletion

    doi: 10.1101/gad.328237.119

    Figure Lengend Snippet: The interaction of ARF-PB1 and AID mediates rescue of endogenous protein levels. ( A ). Positions on the X -axis are relative to ARF16 residue numbers. ( B ) The amino acids D994 and D998 of ARF16 and K114 of IAA17 are shown in the left panel. The amino acid side chains corresponding to D183 and D187 of IAA17 and K944 of ARF16 are highlighted in the right ) into each chain of an A. thaliana ). These mutations in ARF16, which disrupt the electrostatic binding interface, fail to rescue chronic ZNF143 ( C ) and TEAD4 ( D ) degradation. ( E ) These mutations disrupt this coimmunoprecipitation of mCherry-AID with eGFP-tagged ARF16-PB1. Consistent with lower stability of the ARF16-MT in C and D , the mutant GFP-ARF16-PB1 plasmid was transfected at a concentration three times higher than the wild type to achieve comparable expression of each protein. ( F ) ARF16-PB1 is detected upon mCherry-AID immunoprecipitation; however, we were unable to detect TIR1 associating with AID.

    Article Snippet: We used the following antibodies for the Western blots: anti-GFP (gift from Daniel Foltz, Northwestern University), anti-mCherry (rabbit, 1:5000; Abcam, ab183628), ZNF143 (1:5000; H00007702-MO1, Abnova), TEAD4 (1:1000; Santa Cruz Biotechnology, sc-101184), p53 (1:1000; Santa Cruz Biotechnology, DO1), anti-TIR1 (1: 10,000; gift from Masato Kanemaki, Osaka University), β -Actin (1:5000; Sigma, A1978), β -Tubulin (AA2; gift from Todd Stukenberg, University of Virginia), and CENP-I (rabbit; gift from Todd Stukenberg, University of Virginia).

    Techniques: Binding Assay, Mutagenesis, Plasmid Preparation, Transfection, Concentration Assay, Expressing, Immunoprecipitation