Structured Review

Carl Zeiss green immunostaining
Callose degradation is retarded in osdex1 . A to D, TEM of extracellular materials from the wild type and osdex1 at stage 9 (A and B), and stage 10 (C and D). Black arrows show the site of callose deposition. E to L, Immunolabeling of wild-type (E–H) and osdex1 (I–L) anther sections from stage 7 to stage 10 observed by epifluorescence microscopy. M to P, Negative controls of immunolabeling. In (E) to (P), the green channel shows <t>immunostaining</t> with callose antibody; blue counterstaining shows 1,4- and 1,3;1,4-glucan polymers stained with 0.01% calcofluor white; and red staining shows background autofluorescence. Bars = 2 μm (A), 5 μm (B–D), and 15 μm (E–P).
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Images

1) Product Images from "A Rice Ca2+ Binding Protein Is Required for Tapetum Function and Pollen Formation 1 Binding Protein Is Required for Tapetum Function and Pollen Formation 1 [OPEN]"

Article Title: A Rice Ca2+ Binding Protein Is Required for Tapetum Function and Pollen Formation 1 Binding Protein Is Required for Tapetum Function and Pollen Formation 1 [OPEN]

Journal: Plant Physiology

doi: 10.1104/pp.16.01261

Callose degradation is retarded in osdex1 . A to D, TEM of extracellular materials from the wild type and osdex1 at stage 9 (A and B), and stage 10 (C and D). Black arrows show the site of callose deposition. E to L, Immunolabeling of wild-type (E–H) and osdex1 (I–L) anther sections from stage 7 to stage 10 observed by epifluorescence microscopy. M to P, Negative controls of immunolabeling. In (E) to (P), the green channel shows immunostaining with callose antibody; blue counterstaining shows 1,4- and 1,3;1,4-glucan polymers stained with 0.01% calcofluor white; and red staining shows background autofluorescence. Bars = 2 μm (A), 5 μm (B–D), and 15 μm (E–P).
Figure Legend Snippet: Callose degradation is retarded in osdex1 . A to D, TEM of extracellular materials from the wild type and osdex1 at stage 9 (A and B), and stage 10 (C and D). Black arrows show the site of callose deposition. E to L, Immunolabeling of wild-type (E–H) and osdex1 (I–L) anther sections from stage 7 to stage 10 observed by epifluorescence microscopy. M to P, Negative controls of immunolabeling. In (E) to (P), the green channel shows immunostaining with callose antibody; blue counterstaining shows 1,4- and 1,3;1,4-glucan polymers stained with 0.01% calcofluor white; and red staining shows background autofluorescence. Bars = 2 μm (A), 5 μm (B–D), and 15 μm (E–P).

Techniques Used: Transmission Electron Microscopy, Immunolabeling, Epifluorescence Microscopy, Immunostaining, Staining

2) Product Images from "Complementary RNA amplification methods enhance microarray identification of transcripts expressed in the C. elegans nervous system"

Article Title: Complementary RNA amplification methods enhance microarray identification of transcripts expressed in the C. elegans nervous system

Journal: BMC Genomics

doi: 10.1186/1471-2164-9-84

Promoter-GFP reporter genes confirm neural expression of transcripts from pan-neural enriched data sets . Transgenic animals expressing GFP reporters for representative transcripts exclusively enriched in either the IVT derived data set ( A-D ) or the WT-Pico-amplified sample ( E-L ). A . F32B6.11 ::GFP is expressed throughout the C. elegans nervous system including neurons associated with the Nerve ring in the head, motor neurons throughout the Ventral Nerve Cord (VNC) and in tail ganglia. D . F49H12.4 ::GFP is selectively expressed in PVD nociceptive neuron and in two additional neurons in the tail region. Note the highly branched PVD dendritic architecture (arrowheads). E . Differential Interference Contrast (DIC) image of midbody region of 2 nd stage larva ( F.) expressing F47B8.3 ::GFP in GABAergic motor neurons (DD5, VD10, VD11) in the ventral nerve cord. P9 and P10 denote landmark hypodermal blast cells. G-H . ZC155.2 ::GFP and C50F4.4 ::GFP are expressed in VNC motor neurons (e.g. VA10, VB11, etc). Anterior to left, Ventral down. VNC (Ventral Nerve Cord).
Figure Legend Snippet: Promoter-GFP reporter genes confirm neural expression of transcripts from pan-neural enriched data sets . Transgenic animals expressing GFP reporters for representative transcripts exclusively enriched in either the IVT derived data set ( A-D ) or the WT-Pico-amplified sample ( E-L ). A . F32B6.11 ::GFP is expressed throughout the C. elegans nervous system including neurons associated with the Nerve ring in the head, motor neurons throughout the Ventral Nerve Cord (VNC) and in tail ganglia. D . F49H12.4 ::GFP is selectively expressed in PVD nociceptive neuron and in two additional neurons in the tail region. Note the highly branched PVD dendritic architecture (arrowheads). E . Differential Interference Contrast (DIC) image of midbody region of 2 nd stage larva ( F.) expressing F47B8.3 ::GFP in GABAergic motor neurons (DD5, VD10, VD11) in the ventral nerve cord. P9 and P10 denote landmark hypodermal blast cells. G-H . ZC155.2 ::GFP and C50F4.4 ::GFP are expressed in VNC motor neurons (e.g. VA10, VB11, etc). Anterior to left, Ventral down. VNC (Ventral Nerve Cord).

Techniques Used: Expressing, Transgenic Assay, Derivative Assay, Amplification

3) Product Images from "Choice suppression is achieved through opponent but not independent function of the striatal indirect pathway in mice"

Article Title: Choice suppression is achieved through opponent but not independent function of the striatal indirect pathway in mice

Journal: bioRxiv

doi: 10.1101/675850

iSPN chemogenetic inhibition does not significantly affect Test Phase reward accumulation compared to D2-mCherry control mice. Linear regression lines fit to first 10 choices of Test Phase with 95% confidence bands plotted. D2-mCherry and D2-hM4Di regression slope estimates overlapped at the 95% confidence interval.
Figure Legend Snippet: iSPN chemogenetic inhibition does not significantly affect Test Phase reward accumulation compared to D2-mCherry control mice. Linear regression lines fit to first 10 choices of Test Phase with 95% confidence bands plotted. D2-mCherry and D2-hM4Di regression slope estimates overlapped at the 95% confidence interval.

Techniques Used: Inhibition, Mouse Assay

Chemogenetic manipulation data show dSPN inhibition impairs suppression of nonrewarded choices. (a) Top panel: schematic illustrating injection site and viral spread D1-Cre DIO-mCherry (N=7) and DIO-hM4Di (N=15) mice. Opacity indicates the number of mice in each cohort that expressed virus in a given location. Bottom panel: summary of behavior. (b) There was no significant difference between groups in Acquisition Phase choices to criterion (p=0.99 Mann-Whitney U test). (c) There was a significant effect of odor identity (F(1.1, 22.3) = 26.6, **p
Figure Legend Snippet: Chemogenetic manipulation data show dSPN inhibition impairs suppression of nonrewarded choices. (a) Top panel: schematic illustrating injection site and viral spread D1-Cre DIO-mCherry (N=7) and DIO-hM4Di (N=15) mice. Opacity indicates the number of mice in each cohort that expressed virus in a given location. Bottom panel: summary of behavior. (b) There was no significant difference between groups in Acquisition Phase choices to criterion (p=0.99 Mann-Whitney U test). (c) There was a significant effect of odor identity (F(1.1, 22.3) = 26.6, **p

Techniques Used: Inhibition, Injection, Mouse Assay, MANN-WHITNEY

iSPN activation increases choice stochasticity ( a ) Acquisition and Test Phase trial history data from D2-Cre DREADD mice and controls were modeled using an RL model, and best fit parameters were inferred using hierarchical Bayesian model fitting. ( b ) Test Phase alpha and β parameters did not significantly differ among manipulation groups (p > 0.05). ( c ) Test Phase β was significantly lower in the D2-hM3Dq group compared to mCherry control (*p
Figure Legend Snippet: iSPN activation increases choice stochasticity ( a ) Acquisition and Test Phase trial history data from D2-Cre DREADD mice and controls were modeled using an RL model, and best fit parameters were inferred using hierarchical Bayesian model fitting. ( b ) Test Phase alpha and β parameters did not significantly differ among manipulation groups (p > 0.05). ( c ) Test Phase β was significantly lower in the D2-hM3Dq group compared to mCherry control (*p

Techniques Used: Activation Assay, Mouse Assay

Chemogenetic manipulation of DMS direct and indirect pathway neurons reduced entries and increased single trial choices. Outside of the task context, chemogenetic manipulation spared rotarod performance but did affect open field locomotion in a manner uncorrelated with task choice effects. ( a ) Test Phase quadrant entries. ( b ) D2-Cre mice expressing activating DREADD (D2-hM3Dq) or D1-Cre mice expressing inhibitory DREADD (D1-hM4Di) made fewer entries during Test Phase on CNO compared to mCherry control mice on CNO (*p
Figure Legend Snippet: Chemogenetic manipulation of DMS direct and indirect pathway neurons reduced entries and increased single trial choices. Outside of the task context, chemogenetic manipulation spared rotarod performance but did affect open field locomotion in a manner uncorrelated with task choice effects. ( a ) Test Phase quadrant entries. ( b ) D2-Cre mice expressing activating DREADD (D2-hM3Dq) or D1-Cre mice expressing inhibitory DREADD (D1-hM4Di) made fewer entries during Test Phase on CNO compared to mCherry control mice on CNO (*p

Techniques Used: Mouse Assay, Expressing

iSPN chemogenetic activation not inhibition impairs suppression of nonrewarded choices. ( a ) Top panel: schematic adapted from ( Franklin and Paxinos, 1997 ) illustrating injection site and viral spread D2-Cre DIO-mCherry (N=21), DIO-hM4Di (N=12), and DIO-hM3Dq (N= 11) mice. Opacity indicates the number of mice in each cohort that expressed virus in a given location. Bottom panel: summary of behavior; during pre-training mice are habituated to the arena and cheerio rewards (habituation) and learn to dig in unscented wood shavings to retrieve buried cheerio reward (shaping); during Acquisition, mice learn that only Odor 1 is rewarded (criterion = 8/10 correct consecutive choices); 24 hours later during Test, mice are tested for their recall of Acquisition learning (criterion = 8/10 correct consecutive choices). Mice received i.p. injection of Saline or 1 mg/kg CNO (clozapine-N-oxide) 30 minutes prior to testing. ( b ) Acquisition (Saline) choices to criterion did not differ across groups (F(2,19.4)=0.64, p=0.54; Welch’s ANOVA). (c) There was a significant effect of odor identity (F(1.41, 57.9)= 75.6, **p
Figure Legend Snippet: iSPN chemogenetic activation not inhibition impairs suppression of nonrewarded choices. ( a ) Top panel: schematic adapted from ( Franklin and Paxinos, 1997 ) illustrating injection site and viral spread D2-Cre DIO-mCherry (N=21), DIO-hM4Di (N=12), and DIO-hM3Dq (N= 11) mice. Opacity indicates the number of mice in each cohort that expressed virus in a given location. Bottom panel: summary of behavior; during pre-training mice are habituated to the arena and cheerio rewards (habituation) and learn to dig in unscented wood shavings to retrieve buried cheerio reward (shaping); during Acquisition, mice learn that only Odor 1 is rewarded (criterion = 8/10 correct consecutive choices); 24 hours later during Test, mice are tested for their recall of Acquisition learning (criterion = 8/10 correct consecutive choices). Mice received i.p. injection of Saline or 1 mg/kg CNO (clozapine-N-oxide) 30 minutes prior to testing. ( b ) Acquisition (Saline) choices to criterion did not differ across groups (F(2,19.4)=0.64, p=0.54; Welch’s ANOVA). (c) There was a significant effect of odor identity (F(1.41, 57.9)= 75.6, **p

Techniques Used: Activation Assay, Inhibition, Injection, Mouse Assay

Selective modulation of dorsomedial striatum (DMS) SPNs using designer receptor exclusively activated by designer drugs (DREADDs). ( a) DMS of D2-Cre were transduced with a 2:1 mixture of Cre-dependent hM4Di-mCherry and ChR2-EYFP, leading to expression in the indirect pathway. Scale bars indicate 1 mm. (b) Acute 300 μM sagittal slices containing GPe were prepared and GPe neurons were targeted for patch-clamp recording. (c) Left panel: cell-attached recording configuration; right panel: brief light stimulation (10 ms) causes long-lasting suppression of postsynaptic spiking in example tonically firing GPe neuron. Asterisk indicates raster that corresponds to raw trace above. (d) Peristimulus spike histogram indicates that 10 ms light stimulation significantly reduces spontaneous firing rate for 160 ms ( > 3 z-scores below pre-stimulus mean firing rate; n= 6 cells). (e) Left panel: whole-cell recording configuration; right panel: example recording of eIPSC before and after CNO (10μM) wash on. (f) Normalized eIPSC amplitude before and after CNO wash on (mean ± SEM plotted for 6 cells). Pre-CNO vs. post-CNO normalized eIPSC amplitude (D=0.9839, ***p
Figure Legend Snippet: Selective modulation of dorsomedial striatum (DMS) SPNs using designer receptor exclusively activated by designer drugs (DREADDs). ( a) DMS of D2-Cre were transduced with a 2:1 mixture of Cre-dependent hM4Di-mCherry and ChR2-EYFP, leading to expression in the indirect pathway. Scale bars indicate 1 mm. (b) Acute 300 μM sagittal slices containing GPe were prepared and GPe neurons were targeted for patch-clamp recording. (c) Left panel: cell-attached recording configuration; right panel: brief light stimulation (10 ms) causes long-lasting suppression of postsynaptic spiking in example tonically firing GPe neuron. Asterisk indicates raster that corresponds to raw trace above. (d) Peristimulus spike histogram indicates that 10 ms light stimulation significantly reduces spontaneous firing rate for 160 ms ( > 3 z-scores below pre-stimulus mean firing rate; n= 6 cells). (e) Left panel: whole-cell recording configuration; right panel: example recording of eIPSC before and after CNO (10μM) wash on. (f) Normalized eIPSC amplitude before and after CNO wash on (mean ± SEM plotted for 6 cells). Pre-CNO vs. post-CNO normalized eIPSC amplitude (D=0.9839, ***p

Techniques Used: Transduction, Expressing, Patch Clamp

4) Product Images from "Formin and capping protein together embrace the actin filament in a ménage à trois"

Article Title: Formin and capping protein together embrace the actin filament in a ménage à trois

Journal: Nature Communications

doi: 10.1038/ncomms9730

The BFC state splits into CP-capped (BC) and formin mDia1-bound (BF) states. ( a ) Schematic representation of the experimental set-up #2. Fluorescent actin filaments are initiated from formins anchored on the coverslip. The filaments are then sequentially exposed to flows containing PA (non-fluorescent) and CP as indicated. ( b ) Kymographs of two formin-anchored (indicated by the yellow dot) filaments switching from the rapidly elongating state (BF) to the pausing state (BFC) on binding CP, followed by the transition to either BF (top) or BC (bottom). Rapidly elongating filaments were initiated by exposing formins to the fluorescent actin and profilin. Filaments were then exposed to a solution containing PA with non-fluorescent actin and 100 nM CP for 30 s. On removal of CP from the flow and introduction of non-fluorescent PA, filaments either resume fast elongation (BFC→BF) or detach (BFC→BC). Elongation by formin in non-fluorescent actin gives the appearance of ‘fluorescent segment' moving further away. ( c ) Fraction of BFC filaments (black symbols; n =76 filaments) undergoing dissociation into either BC (filaments released in the flow, blue squares) or BF (filaments resuming fast growth, red circles). For comparison, the time course of BF produced from BFC in set-up #1 ( Fig. 1 ) is plotted (open red circles). The solid lines are the exponential fits. The data were fitted with an exponential process (continuous line) consistent with a rate constant k obs = k ′ −F + k ′ −C .
Figure Legend Snippet: The BFC state splits into CP-capped (BC) and formin mDia1-bound (BF) states. ( a ) Schematic representation of the experimental set-up #2. Fluorescent actin filaments are initiated from formins anchored on the coverslip. The filaments are then sequentially exposed to flows containing PA (non-fluorescent) and CP as indicated. ( b ) Kymographs of two formin-anchored (indicated by the yellow dot) filaments switching from the rapidly elongating state (BF) to the pausing state (BFC) on binding CP, followed by the transition to either BF (top) or BC (bottom). Rapidly elongating filaments were initiated by exposing formins to the fluorescent actin and profilin. Filaments were then exposed to a solution containing PA with non-fluorescent actin and 100 nM CP for 30 s. On removal of CP from the flow and introduction of non-fluorescent PA, filaments either resume fast elongation (BFC→BF) or detach (BFC→BC). Elongation by formin in non-fluorescent actin gives the appearance of ‘fluorescent segment' moving further away. ( c ) Fraction of BFC filaments (black symbols; n =76 filaments) undergoing dissociation into either BC (filaments released in the flow, blue squares) or BF (filaments resuming fast growth, red circles). For comparison, the time course of BF produced from BFC in set-up #1 ( Fig. 1 ) is plotted (open red circles). The solid lines are the exponential fits. The data were fitted with an exponential process (continuous line) consistent with a rate constant k obs = k ′ −F + k ′ −C .

Techniques Used: Binding Assay, Flow Cytometry, Produced

CP associates to mDia1 formin-bound barbed ends in a capped ternary BFC complex that releases either formin or CP. ( a ) Schematic representation of experimental set-up #1. Barbed-end growth is initiated from spectrin–actin seeds bound on the coverslip surface. The filaments are then sequentially exposed to flows containing PA, formin, CP as indicated. ( b ) Kymograph of a filament showing evidence for the pathway BF+C→BFC→BF+C with unlabelled formin and CP. In the kymograph, an actin filament exposed to 20 nM formin (F) for 10 s exhibits fast elongation in the presence of PA. On exposure to 100 nM CP (CP+PA) for 20 s, filament growth is arrested. On exposure to PA, CP dissociates leaving behind fast-elongating BF. ( c ) Similar kymograph as in b but with fluorescently labelled formin F*. Filaments growing in PA (red) were exposed to 20 nM labelled formin F* (green) for 10 s after which filaments were re-exposed to PA. The higher green background appears to stay for slightly longer (∼30 s) even after formin is removed from the flow due to non-specific interaction of formin with the surface. Formin can be seen bound to the barbed end in the paused state in the BF*C complex as a result of exposure to PA+CP. Fast processive elongation resumes when CP falls off (BF*C→BF*+C) and the same formin continues to processively track the barbed end ( Supplementary Movie 1 ). ( d ) Fraction of rapidly elongating BF filaments converting to arrested BFC state on binding CP at the following concentrations (nM): 200 nM (black symbols, n =36 filaments), 100 nM (blue symbols, n =27 filaments) and 50 nM (red symbols, n =29 filaments). Symbols represent the experimental data and the solid lines are the exponential fits providing k ′ obs values. Inset: plot of k ′ obs+C versus [CP] giving k ′ +C =0.21±0.01 μM −1 ·s −1 (see Supplementary Fig. 3 for histograms. Error bars: s.e.m.).
Figure Legend Snippet: CP associates to mDia1 formin-bound barbed ends in a capped ternary BFC complex that releases either formin or CP. ( a ) Schematic representation of experimental set-up #1. Barbed-end growth is initiated from spectrin–actin seeds bound on the coverslip surface. The filaments are then sequentially exposed to flows containing PA, formin, CP as indicated. ( b ) Kymograph of a filament showing evidence for the pathway BF+C→BFC→BF+C with unlabelled formin and CP. In the kymograph, an actin filament exposed to 20 nM formin (F) for 10 s exhibits fast elongation in the presence of PA. On exposure to 100 nM CP (CP+PA) for 20 s, filament growth is arrested. On exposure to PA, CP dissociates leaving behind fast-elongating BF. ( c ) Similar kymograph as in b but with fluorescently labelled formin F*. Filaments growing in PA (red) were exposed to 20 nM labelled formin F* (green) for 10 s after which filaments were re-exposed to PA. The higher green background appears to stay for slightly longer (∼30 s) even after formin is removed from the flow due to non-specific interaction of formin with the surface. Formin can be seen bound to the barbed end in the paused state in the BF*C complex as a result of exposure to PA+CP. Fast processive elongation resumes when CP falls off (BF*C→BF*+C) and the same formin continues to processively track the barbed end ( Supplementary Movie 1 ). ( d ) Fraction of rapidly elongating BF filaments converting to arrested BFC state on binding CP at the following concentrations (nM): 200 nM (black symbols, n =36 filaments), 100 nM (blue symbols, n =27 filaments) and 50 nM (red symbols, n =29 filaments). Symbols represent the experimental data and the solid lines are the exponential fits providing k ′ obs values. Inset: plot of k ′ obs+C versus [CP] giving k ′ +C =0.21±0.01 μM −1 ·s −1 (see Supplementary Fig. 3 for histograms. Error bars: s.e.m.).

Techniques Used: Flow Cytometry, Binding Assay

Formin mDia1 binds to CP-capped filaments and rapidly uncaps via a transient BCF state. ( a ) Pyrene actin polymerization assay demonstrates uncapping of capped barbed ends (BC), by formin. Capped filaments (5 μM F-actin, 2% pyrenyl labelled and 5 nM CP) were diluted 50-fold in F-buffer containing 2 μM G-actin (2% pyrenyl labelled) and 6 μM profilin and the following additions: none (black), 2 nM formin (blue) and 4 μM CIN85 (magenta). Red curve is a filament nucleation control (2 μM actin, 6 μM profilin and 2 nM formin) in the absence of CP-capped filaments. Note that a small percentage of non-capped filaments are responsible for the non-zero initial rate in the black (free barbed ends) and blue (formin-bound barbed ends) curves. Dead time is about 20 s. ( b ) Kymograph of a capped filament undergoing uncapping and fast processive growth on exposure to formin mDia1. Filament was elongated from anchored spectrin–actin seeds in the presence of PA, then exposed to 20 nM CP and PA for 1 min (B+C→BC). The capped filament is later exposed to 40 nM formin in the absence of PA for 40 s (BC+F→BCF). Once formin was removed from the flow and PA was introduced, fast elongation was observed (BCF→BF+C). ( c ) Fraction of filaments (in experiment described in b ) that resume rapid elongation (BF state) during exposure to PA only, versus time, from an initial population of capped filaments exposed to 10 nM (black, n =91 filaments), 20 nM (red, n =50 filaments) and 40 nM (blue, n =69 filaments) formin for 30 s. Symbols represent the experimental data and the solid lines are the exponential fits. Only three representative CDFs are shown here for the ease of reading, see Supplementary Fig. 9 for details. ( d ) The maximum fraction of BF filaments (plateau values of curves such as shown in c ) as a function of formin concentration [F] times the exposure duration ( T expo ). The solid line is an exponential fit corresponding to equation (3) . Inset: k obs = k ′ −C + k ′ −F is independent of the experimental condition. Horizontal lines represent the average (blue) plus or minus the s.d. (grey). Error bars: s.e.m.
Figure Legend Snippet: Formin mDia1 binds to CP-capped filaments and rapidly uncaps via a transient BCF state. ( a ) Pyrene actin polymerization assay demonstrates uncapping of capped barbed ends (BC), by formin. Capped filaments (5 μM F-actin, 2% pyrenyl labelled and 5 nM CP) were diluted 50-fold in F-buffer containing 2 μM G-actin (2% pyrenyl labelled) and 6 μM profilin and the following additions: none (black), 2 nM formin (blue) and 4 μM CIN85 (magenta). Red curve is a filament nucleation control (2 μM actin, 6 μM profilin and 2 nM formin) in the absence of CP-capped filaments. Note that a small percentage of non-capped filaments are responsible for the non-zero initial rate in the black (free barbed ends) and blue (formin-bound barbed ends) curves. Dead time is about 20 s. ( b ) Kymograph of a capped filament undergoing uncapping and fast processive growth on exposure to formin mDia1. Filament was elongated from anchored spectrin–actin seeds in the presence of PA, then exposed to 20 nM CP and PA for 1 min (B+C→BC). The capped filament is later exposed to 40 nM formin in the absence of PA for 40 s (BC+F→BCF). Once formin was removed from the flow and PA was introduced, fast elongation was observed (BCF→BF+C). ( c ) Fraction of filaments (in experiment described in b ) that resume rapid elongation (BF state) during exposure to PA only, versus time, from an initial population of capped filaments exposed to 10 nM (black, n =91 filaments), 20 nM (red, n =50 filaments) and 40 nM (blue, n =69 filaments) formin for 30 s. Symbols represent the experimental data and the solid lines are the exponential fits. Only three representative CDFs are shown here for the ease of reading, see Supplementary Fig. 9 for details. ( d ) The maximum fraction of BF filaments (plateau values of curves such as shown in c ) as a function of formin concentration [F] times the exposure duration ( T expo ). The solid line is an exponential fit corresponding to equation (3) . Inset: k obs = k ′ −C + k ′ −F is independent of the experimental condition. Horizontal lines represent the average (blue) plus or minus the s.d. (grey). Error bars: s.e.m.

Techniques Used: Polymerization Assay, Flow Cytometry, Concentration Assay

Formin FMNL2 also binds to CP-capped filaments and uncaps via a transient BCF state. ( a ) Kymograph of a capped filament undergoing uncapping and fast processive growth on exposure to formin FMNL2. Filaments elongating from spectrin–actin seeds (set-up #1, Fig. 1a ) were first exposed to 20 nM CP and PA for a couple of minutes (B+C→BC). Paused filaments were then exposed to 250 nM FMNL2 for 30 s (BC+F→BCF). Once formin was removed from the flow and PA was introduced, fast elongation was observed (BCF→BF+C). Note that, as expected, the FMNL2 elongation rate seen here is much slower compared with that of formin mDia1 as seen in Fig. 3b . ( b ) Fraction of CP-capped paused filaments that resume rapid elongation (BF state) during exposure to PA only, versus time, from an initial population of capped filaments exposed to 250 nM (black, n =56 filaments), 500 nM (red, n =55 filaments) and 750 nM (blue, n =43 filaments) FMNL2 for 30 s. Symbols represent the experimental data and the solid lines are the exponential fits. Only three representative CDFs are shown here for the ease of reading, see Supplementary Fig. 10 for details. ( c ) The maximum fraction of BF filaments (plateau values of curves such as the ones shown in) as a function the product of formin FMNL2 concentration [F] and exposure duration ( T expo ). The solid line is an exponential fit corresponding to equation (3) . Inset: k obs = k ′ −C + k ′ −F is independent of the experimental condition. Horizontal line represents the average (Error bars: s.e.m.).
Figure Legend Snippet: Formin FMNL2 also binds to CP-capped filaments and uncaps via a transient BCF state. ( a ) Kymograph of a capped filament undergoing uncapping and fast processive growth on exposure to formin FMNL2. Filaments elongating from spectrin–actin seeds (set-up #1, Fig. 1a ) were first exposed to 20 nM CP and PA for a couple of minutes (B+C→BC). Paused filaments were then exposed to 250 nM FMNL2 for 30 s (BC+F→BCF). Once formin was removed from the flow and PA was introduced, fast elongation was observed (BCF→BF+C). Note that, as expected, the FMNL2 elongation rate seen here is much slower compared with that of formin mDia1 as seen in Fig. 3b . ( b ) Fraction of CP-capped paused filaments that resume rapid elongation (BF state) during exposure to PA only, versus time, from an initial population of capped filaments exposed to 250 nM (black, n =56 filaments), 500 nM (red, n =55 filaments) and 750 nM (blue, n =43 filaments) FMNL2 for 30 s. Symbols represent the experimental data and the solid lines are the exponential fits. Only three representative CDFs are shown here for the ease of reading, see Supplementary Fig. 10 for details. ( c ) The maximum fraction of BF filaments (plateau values of curves such as the ones shown in) as a function the product of formin FMNL2 concentration [F] and exposure duration ( T expo ). The solid line is an exponential fit corresponding to equation (3) . Inset: k obs = k ′ −C + k ′ −F is independent of the experimental condition. Horizontal line represents the average (Error bars: s.e.m.).

Techniques Used: Flow Cytometry, Concentration Assay

Structural clashes between CP and formin at the barbed end must cause partial dissociation of each protein in the BFC state. ( a ) Steric clashes between CP and mDia1-FH2 in the BFC complex. The 167°-twisted F-actin barbed end is depicted as surface representation (4A7N), while the α/β heterodimeric CP 30 and the dimeric mDia1-FH2 domain (3O4X) are illustrated in ribbon diagrams. The FH2 domain hemidimers (FH2 1 and FH2 2 ) are shown in the previously defined ‘open' state 4 32 and bind with an amphipathic α-helix (αD (ref. 32 ), orange, knob region) to the hydrophobic target-binding cleft (TBC) of actin. CP interacts with its amphipathic β-tentacle (βT, yellow) with B1, while the basic α-tentacle (αT) binds to B1 and B2. ( b , c ) Zoom-in of the clashes. The α-tentacle of CP is able to interact with B1 and B2 of F-actin, while there is a steric clash between CPβ and the post region of FH2 1 (clash 1). FH2 1 competes with CP for binding to B1-TBC (clash 2). Since CPβ and αD of FH2 2 are in close proximity, there might be a minor steric hindrance for simultaneous binding to actin B2 (clash 3). ( d ) Cartoon depicting complex formation and dissociation of BFC, based on the present work and the structural model presented in Supplementary Fig. 13 . Left panel: association of PA to the barbed end is prohibited in BC state and permitted in ‘open' BF state. Right panel: formin binds to BC by association of FH2 2 to B1 and B2 (top) followed by displacement of β-tentacle by FH2 1 (bottom). Similarly CP can associate with BF without inserting the β-tentacle in B1-TBC (bottom). For the detailed model, see Supplementary Fig. 13 .
Figure Legend Snippet: Structural clashes between CP and formin at the barbed end must cause partial dissociation of each protein in the BFC state. ( a ) Steric clashes between CP and mDia1-FH2 in the BFC complex. The 167°-twisted F-actin barbed end is depicted as surface representation (4A7N), while the α/β heterodimeric CP 30 and the dimeric mDia1-FH2 domain (3O4X) are illustrated in ribbon diagrams. The FH2 domain hemidimers (FH2 1 and FH2 2 ) are shown in the previously defined ‘open' state 4 32 and bind with an amphipathic α-helix (αD (ref. 32 ), orange, knob region) to the hydrophobic target-binding cleft (TBC) of actin. CP interacts with its amphipathic β-tentacle (βT, yellow) with B1, while the basic α-tentacle (αT) binds to B1 and B2. ( b , c ) Zoom-in of the clashes. The α-tentacle of CP is able to interact with B1 and B2 of F-actin, while there is a steric clash between CPβ and the post region of FH2 1 (clash 1). FH2 1 competes with CP for binding to B1-TBC (clash 2). Since CPβ and αD of FH2 2 are in close proximity, there might be a minor steric hindrance for simultaneous binding to actin B2 (clash 3). ( d ) Cartoon depicting complex formation and dissociation of BFC, based on the present work and the structural model presented in Supplementary Fig. 13 . Left panel: association of PA to the barbed end is prohibited in BC state and permitted in ‘open' BF state. Right panel: formin binds to BC by association of FH2 2 to B1 and B2 (top) followed by displacement of β-tentacle by FH2 1 (bottom). Similarly CP can associate with BF without inserting the β-tentacle in B1-TBC (bottom). For the detailed model, see Supplementary Fig. 13 .

Techniques Used: Binding Assay

5) Product Images from "Comprehensive Analysis of RNA-Protein Interactions by High Throughput Sequencing-RNA Affinity Profiling"

Article Title: Comprehensive Analysis of RNA-Protein Interactions by High Throughput Sequencing-RNA Affinity Profiling

Journal: Nature methods

doi: 10.1038/nmeth.2970

RNA-protein interactions can be assayed by HiTS-RAP on an Illumina GAIIx instrument ( a ) HiTS-RAP schematic. Sequencing is done following the standard Illumina workflow. The strand synthesized during sequencing is then stripped away, a primer is annealed and the second strand is regenerated with Klenow enzyme. Tus is then bound to the ter site, and DNAs on the flowcell are transcribed. T7 RNAP initiates at its promoter, transcribes through the sequence of interest, and halts just upstream of the Tus bound ter site. The RNA transcript is stably linked to its DNA template through the polymerase. Fluorescently labeled protein is then bound to the RNA and imaged. ( b ) Images from a HiTS-RAP run with GFP and SRB-2 aptamers. ‘All clusters’ (left panels) are labeled during sequencing and shown as a maximum intensity projection of the four channels. After transcription halting and EGFP-mOrange binding, the flowcell is imaged at 625 nM EGFP-mOrange. GFPapt clusters are labeled by mOrange while SRB-2 aptamer clusters are not. Scale bars: 6.75 μM, 1.125 μM in inset ( c ) Binding curves for the GFP aptamer ( n = 2,665,064), and mutants C58U ( n = 3,833), C76U ( n = 4,758), and G8U_U56C ( n = 29), and the SRB-2 aptamer ( n = 1,588,404). G8U_U56C and SRB-2 aptamer are scored as not binding. Data are from one lane of the sequencer (SRB-2 aptamer is from a separate lane). Intensities are the average of all clusters of each sequence in the lane, normalized by dividing by their average sequencing intensity and subtracting their average intensity at no EGFP-mOrange. Error bars represent standard error. Error of fitted K d s are the square root of variances returned by the fitting algorithm. ( d ) EMSA of RNAs in part c. K d s are determined from a single fit to two replicate EMSA experiments for each sequence, ± standard deviation fitted by IGOR. The G8U_U56C gel was also scanned to visualize EGFP.
Figure Legend Snippet: RNA-protein interactions can be assayed by HiTS-RAP on an Illumina GAIIx instrument ( a ) HiTS-RAP schematic. Sequencing is done following the standard Illumina workflow. The strand synthesized during sequencing is then stripped away, a primer is annealed and the second strand is regenerated with Klenow enzyme. Tus is then bound to the ter site, and DNAs on the flowcell are transcribed. T7 RNAP initiates at its promoter, transcribes through the sequence of interest, and halts just upstream of the Tus bound ter site. The RNA transcript is stably linked to its DNA template through the polymerase. Fluorescently labeled protein is then bound to the RNA and imaged. ( b ) Images from a HiTS-RAP run with GFP and SRB-2 aptamers. ‘All clusters’ (left panels) are labeled during sequencing and shown as a maximum intensity projection of the four channels. After transcription halting and EGFP-mOrange binding, the flowcell is imaged at 625 nM EGFP-mOrange. GFPapt clusters are labeled by mOrange while SRB-2 aptamer clusters are not. Scale bars: 6.75 μM, 1.125 μM in inset ( c ) Binding curves for the GFP aptamer ( n = 2,665,064), and mutants C58U ( n = 3,833), C76U ( n = 4,758), and G8U_U56C ( n = 29), and the SRB-2 aptamer ( n = 1,588,404). G8U_U56C and SRB-2 aptamer are scored as not binding. Data are from one lane of the sequencer (SRB-2 aptamer is from a separate lane). Intensities are the average of all clusters of each sequence in the lane, normalized by dividing by their average sequencing intensity and subtracting their average intensity at no EGFP-mOrange. Error bars represent standard error. Error of fitted K d s are the square root of variances returned by the fitting algorithm. ( d ) EMSA of RNAs in part c. K d s are determined from a single fit to two replicate EMSA experiments for each sequence, ± standard deviation fitted by IGOR. The G8U_U56C gel was also scanned to visualize EGFP.

Techniques Used: Sequencing, Synthesized, Stable Transfection, Labeling, Sulforhodamine B Assay, Binding Assay, Standard Deviation

T7 RNA polymerase halting with Tus gives stable complexes containing DNA and functional RNA ( a. ) EMSA was carried out with radiolabeled DNA that encodes the GFP aptamer and has a binding site for Tus. Naked DNA runs with high mobility. Binding Tus to the ter DNA element (red segment) retards DNA mobility. After transcription, nearly every DNA participates in a complex of intermediate mobility, containing Tus, T7 RNA polymerase (orange), and RNA (green). This band is very sharp, indicating that each DNA-Tus-T7RNAP-RNA complex is of homogeneous composition. b. 454 Life Sciences polystyrene beads covered in covalently linked DNA templates for transcription. After transcription halting, beads were incubated with EGFP and then washed. EGFP bound to beads presenting halted GFP aptamer RNA, but not to beads presenting SRB-2 aptamer RNA. Scale bar is 20 μm.
Figure Legend Snippet: T7 RNA polymerase halting with Tus gives stable complexes containing DNA and functional RNA ( a. ) EMSA was carried out with radiolabeled DNA that encodes the GFP aptamer and has a binding site for Tus. Naked DNA runs with high mobility. Binding Tus to the ter DNA element (red segment) retards DNA mobility. After transcription, nearly every DNA participates in a complex of intermediate mobility, containing Tus, T7 RNA polymerase (orange), and RNA (green). This band is very sharp, indicating that each DNA-Tus-T7RNAP-RNA complex is of homogeneous composition. b. 454 Life Sciences polystyrene beads covered in covalently linked DNA templates for transcription. After transcription halting, beads were incubated with EGFP and then washed. EGFP bound to beads presenting halted GFP aptamer RNA, but not to beads presenting SRB-2 aptamer RNA. Scale bar is 20 μm.

Techniques Used: Functional Assay, Binding Assay, Incubation, Sulforhodamine B Assay

6) Product Images from "SRRF: Universal live-cell super-resolution microscopy"

Article Title: SRRF: Universal live-cell super-resolution microscopy

Journal: The International Journal of Biochemistry & Cell Biology

doi: 10.1016/j.biocel.2018.05.014

SRRF IMAGING USING DIFFERENT MICROSCOPES. a) Left: SRRF image of Alexa Fluor-647-labelled phalloidin in fixed Cos7 cells imaged using TIRF with intense laser illumination. Scale bar = 10 μm. Right: enlarged view of the boxed region with the non-super-resolved TIRF image shown. Scale bar = 2 μm. b) Greyscale images: individual SRRF time-points (each frame represents 1 s of imaging) of Cos7 cells expressing Utrophin-GFP imaged using confocal microscopy (scale bars = 5 μm). Enlarged views of the boxed region are displayed below as a split between the diffraction-limited confocal image and the SRRF image (scale bars = 1 μm). Coloured image: temporally colour-coded projection of 200 SRRF reconstructions at 1 Hz from 200 s of continuous imaging (scale bar = 5 μm). c) Greyscale images: individual SRRF time-points (1 s imaging per reconstructed SRRF frame) of Utrophin-GFP images using widefield laser-based microscopy (scale bars = 5 μm) and enlarged insets showing split between corresponding diffraction-limited images below (scale bars = 2 μm). Coloured image: temporally colour-coded projection on 200 SRRF reconstructions at 1 Hz from 200 s of continuous imaging (scale bar = 5 μm). d) Temporally colour-coded projections of SRRF reconstructions of 30 min. of continuous LED-illuminated widefield Utrophin-GFP imaging. Left: projection of all 590 SRRF reconstructions at 0.33 Hz. Right: same dataset, selected SRRF frames at 5 min intervals. Scale bars = 10 μm. e) Individual SRRF frames (3 s imaging per time-point) from the projected dataset in the right-hand panel of d) (scale bars = 10 μm), with insets below showing enlarged boxed region split with diffraction-limited LED widefield (scale bars = 5 μm). f) Long-term widefield LED timelapse imaging of Utrophin-GFP with SRRF images acquired every 10 min. Greyscale images: individual SRRF frames (3 s imaging per time-point, scale bars = 10 μm) with enlarged insets below showing the diffraction-limited equivalent (scale bars = 2 μm). Coloured image: temporally colour-coded projection of 10 SRRF reconstructions from imaging once every 10 min (scale bar = 10 μm). g) Resolutions as measured using the ‘FRC Map’ tool in NanoJ-SQUIRREL. For the fixed cell data, SRRF (mean FRC) is the average resolution across the whole image, with SRRF (min. FRC) representing the best local resolution in the image. For the live cell data, mean FRC is the resolution averaged across all images in a time series, and min. FRC is the average value for the best individual frame within the series. All values are in nm, errors ± SD.
Figure Legend Snippet: SRRF IMAGING USING DIFFERENT MICROSCOPES. a) Left: SRRF image of Alexa Fluor-647-labelled phalloidin in fixed Cos7 cells imaged using TIRF with intense laser illumination. Scale bar = 10 μm. Right: enlarged view of the boxed region with the non-super-resolved TIRF image shown. Scale bar = 2 μm. b) Greyscale images: individual SRRF time-points (each frame represents 1 s of imaging) of Cos7 cells expressing Utrophin-GFP imaged using confocal microscopy (scale bars = 5 μm). Enlarged views of the boxed region are displayed below as a split between the diffraction-limited confocal image and the SRRF image (scale bars = 1 μm). Coloured image: temporally colour-coded projection of 200 SRRF reconstructions at 1 Hz from 200 s of continuous imaging (scale bar = 5 μm). c) Greyscale images: individual SRRF time-points (1 s imaging per reconstructed SRRF frame) of Utrophin-GFP images using widefield laser-based microscopy (scale bars = 5 μm) and enlarged insets showing split between corresponding diffraction-limited images below (scale bars = 2 μm). Coloured image: temporally colour-coded projection on 200 SRRF reconstructions at 1 Hz from 200 s of continuous imaging (scale bar = 5 μm). d) Temporally colour-coded projections of SRRF reconstructions of 30 min. of continuous LED-illuminated widefield Utrophin-GFP imaging. Left: projection of all 590 SRRF reconstructions at 0.33 Hz. Right: same dataset, selected SRRF frames at 5 min intervals. Scale bars = 10 μm. e) Individual SRRF frames (3 s imaging per time-point) from the projected dataset in the right-hand panel of d) (scale bars = 10 μm), with insets below showing enlarged boxed region split with diffraction-limited LED widefield (scale bars = 5 μm). f) Long-term widefield LED timelapse imaging of Utrophin-GFP with SRRF images acquired every 10 min. Greyscale images: individual SRRF frames (3 s imaging per time-point, scale bars = 10 μm) with enlarged insets below showing the diffraction-limited equivalent (scale bars = 2 μm). Coloured image: temporally colour-coded projection of 10 SRRF reconstructions from imaging once every 10 min (scale bar = 10 μm). g) Resolutions as measured using the ‘FRC Map’ tool in NanoJ-SQUIRREL. For the fixed cell data, SRRF (mean FRC) is the average resolution across the whole image, with SRRF (min. FRC) representing the best local resolution in the image. For the live cell data, mean FRC is the resolution averaged across all images in a time series, and min. FRC is the average value for the best individual frame within the series. All values are in nm, errors ± SD.

Techniques Used: Imaging, Expressing, Confocal Microscopy, Microscopy

7) Product Images from "The Anticancer Drug 3-Bromopyruvate Induces DNA Damage Potentially Through Reactive Oxygen Species in Yeast and in Human Cancer Cells"

Article Title: The Anticancer Drug 3-Bromopyruvate Induces DNA Damage Potentially Through Reactive Oxygen Species in Yeast and in Human Cancer Cells

Journal: Cells

doi: 10.3390/cells9051161

Homologous recombination is crucial for tolerance to 3-BP: ( A,B ) Homologous recombination, nonhomologous end-joining and base excision repair pathways are required for maintaining viability of yeast cells in the presence of 3-BP. Serial dilutions of indicated strains were plated on solid media in the presence or absence of 3-BP at 30 °C and photographed after 3 days. ( C – E ) 3-BP treatment triggers formation of homologous recombination repair centers. Logarithmically growing cultures of cells bearing Mre11-YFP ( A ), Rfa1-YFP ( B ) and Rad52-YFP ( C ) were treated with 3-BP or MMS for 1.5 h. Next, cells were washed and processed for microscopic analysis. Error bars represent mean value ± standard deviations of mean ( n = 3).
Figure Legend Snippet: Homologous recombination is crucial for tolerance to 3-BP: ( A,B ) Homologous recombination, nonhomologous end-joining and base excision repair pathways are required for maintaining viability of yeast cells in the presence of 3-BP. Serial dilutions of indicated strains were plated on solid media in the presence or absence of 3-BP at 30 °C and photographed after 3 days. ( C – E ) 3-BP treatment triggers formation of homologous recombination repair centers. Logarithmically growing cultures of cells bearing Mre11-YFP ( A ), Rfa1-YFP ( B ) and Rad52-YFP ( C ) were treated with 3-BP or MMS for 1.5 h. Next, cells were washed and processed for microscopic analysis. Error bars represent mean value ± standard deviations of mean ( n = 3).

Techniques Used: Homologous Recombination

8) Product Images from "Dose Response Effects of Dermally applied Diethanolamine on Neurogenesis in Fetal Mouse Hippocampus and Potential Exposure of Humans"

Article Title: Dose Response Effects of Dermally applied Diethanolamine on Neurogenesis in Fetal Mouse Hippocampus and Potential Exposure of Humans

Journal:

doi: 10.1093/toxsci/kfn227

Immunohistochemical analysis of cell proliferation and caspase-3 activation in fetal E17 mouse hippocampus. Pregnant mice were treated dermally with 80 mg/kg/day DEA (or vehicle control) from E7 until they were killed on E17. Coronal sections were prepared
Figure Legend Snippet: Immunohistochemical analysis of cell proliferation and caspase-3 activation in fetal E17 mouse hippocampus. Pregnant mice were treated dermally with 80 mg/kg/day DEA (or vehicle control) from E7 until they were killed on E17. Coronal sections were prepared

Techniques Used: Immunohistochemistry, Activation Assay, Mouse Assay

Maternal DEA treatment increases apoptosis in fetal mouse hippocampus on E17. Pregnant mice were treated with different doses of DEA, and fetal hippocampal sections were assessed as presented in . These were analyzed using activated cleaved caspase-3
Figure Legend Snippet: Maternal DEA treatment increases apoptosis in fetal mouse hippocampus on E17. Pregnant mice were treated with different doses of DEA, and fetal hippocampal sections were assessed as presented in . These were analyzed using activated cleaved caspase-3

Techniques Used: Mouse Assay

9) Product Images from "Vascular Associations and Dynamic Process Motility in Perivascular Myeloid Cells of the Mouse Choroid: Implications for Function and Senescent Change"

Article Title: Vascular Associations and Dynamic Process Motility in Perivascular Myeloid Cells of the Mouse Choroid: Implications for Function and Senescent Change

Journal: Investigative Ophthalmology & Visual Science

doi: 10.1167/iovs.13-13522

Distribution and morphologies of GFP-positive resident myeloid cells are shown in the mouse choroid. ( A ) Choroidoscleral flat-mount from a 3-month-old adult CX3CR1 +/GFP mouse that had been perfused intravascularly with lipophilic dye, DiI ( red ) is shown. Choroidal myeloid cells ( green ) were found distributed throughout the choroidal layer from central to peripheral areas. Scale bar : 500 μm. ( B ) Higher magnification view of the inset in ( A ) demonstrates that resident myeloid cells showed a generally perivascular distribution. ( C – H ) Resident myeloid cells were visualized by immunohistochemical staining for myeloid markers in CX3CR1 +/GFP mice. Green fluorescent protein–expressing myeloid cells ([ C ], green ) stained positively for MHCII ([ D ], red ) and Iba1 ([ E ], blue ). A merged imaged of all three markers ( F ) revealed that choroidal myeloid cells were composed of cells with dendritiform morphologies ( upper inset ), with a minority of rounded cells with minimal or no processes ( lower inset ). Most GFP + cells were also immunopositive for CD163 (ED2; [ G ]) and CD169 (ED3; [ H ]). ( I ) Green fluorescent protein + cells with dendritiform morphologies were significantly more prevalent than those with rounded morphologies. ( J – N ) Myeloid cells with dendritiform morphologies were typically concurrently positive for CX3CR1-GFP ( J ), MHCII ( K ), and Iba1 ( L ) markers ( upper panels ), as quantified in cell counts ( M ). Rounded myeloid cells ([ J – L ], lower panels ), however, were significantly more varied in their immunopositivity for the three myeloid markers, indicating a more diverse composition ( N ). Cells were counted from 27 high-magnification fields in nine choroidal flat mounts ( n = 5 animals). Scale bar : 50 μm.
Figure Legend Snippet: Distribution and morphologies of GFP-positive resident myeloid cells are shown in the mouse choroid. ( A ) Choroidoscleral flat-mount from a 3-month-old adult CX3CR1 +/GFP mouse that had been perfused intravascularly with lipophilic dye, DiI ( red ) is shown. Choroidal myeloid cells ( green ) were found distributed throughout the choroidal layer from central to peripheral areas. Scale bar : 500 μm. ( B ) Higher magnification view of the inset in ( A ) demonstrates that resident myeloid cells showed a generally perivascular distribution. ( C – H ) Resident myeloid cells were visualized by immunohistochemical staining for myeloid markers in CX3CR1 +/GFP mice. Green fluorescent protein–expressing myeloid cells ([ C ], green ) stained positively for MHCII ([ D ], red ) and Iba1 ([ E ], blue ). A merged imaged of all three markers ( F ) revealed that choroidal myeloid cells were composed of cells with dendritiform morphologies ( upper inset ), with a minority of rounded cells with minimal or no processes ( lower inset ). Most GFP + cells were also immunopositive for CD163 (ED2; [ G ]) and CD169 (ED3; [ H ]). ( I ) Green fluorescent protein + cells with dendritiform morphologies were significantly more prevalent than those with rounded morphologies. ( J – N ) Myeloid cells with dendritiform morphologies were typically concurrently positive for CX3CR1-GFP ( J ), MHCII ( K ), and Iba1 ( L ) markers ( upper panels ), as quantified in cell counts ( M ). Rounded myeloid cells ([ J – L ], lower panels ), however, were significantly more varied in their immunopositivity for the three myeloid markers, indicating a more diverse composition ( N ). Cells were counted from 27 high-magnification fields in nine choroidal flat mounts ( n = 5 animals). Scale bar : 50 μm.

Techniques Used: Immunohistochemistry, Staining, Mouse Assay, Expressing

Dendritiform resident myeloid cells are nonmigratory but demonstrate rapid dynamic process motility. ( A ) GFP + resident myeloid cells in ex vivo choroidoscleral explants from a CX3CR1 +/GFP mouse were monitored using time-lapse confocal live imaging. Images of choriocapillaris myeloid cells taken at time 0 ( green , top ) and 100 minutes ( red , middle ) were highly overlapping when superimposed ( bottom ), demonstrating an absence of significant cellular migration or soma translocation within this time interval. ( B ) Resident myeloid cells demonstrated dynamic motility in their dendritiform processes. Comparison of a confocal image of a choroidal artery-associated myeloid cell (L, arterial lumen), captured at a single time point ( top ), to that of a summed image of 100 individual images (captured every 10 seconds over 1000 seconds) ( bottom ) demonstrates the coverage of perivascular space by motile processes. Note that the elongated morphology and parallel alignment to the vessel wall are maintained despite marked process movement. ( C ) Comparison of a confocal image of a choriocapillaris-associated myeloid cell captured at a single time point ( top ) to that of a summed image of 30 images (captured at 42-second intervals over 1000 seconds) ( bottom ) illustrates the dynamism of the processes and their ability to occupy the extravascular space on the surface of the choriocapillaris. ( D ) Time-lapse images (sequential images taken 2 minutes apart) from a recording of a spindle-shaped myeloid cell associated with a primary arteriole demonstrates varying motility in its processes; processes that were directed outward into the extravascular space ( process 1 ) demonstrated more prominent dynamism than juxtavascular axial processes closely associated with the vessel wall ( processes 2 and 3 ) that were relatively more stable. ( E ) Time-lapse images (sequential images taken 2 minutes apart) from a recording of a stellate-shaped myeloid cell associated with the choriocapillaris ( C ) demonstrate dynamism in all processes (1–4).
Figure Legend Snippet: Dendritiform resident myeloid cells are nonmigratory but demonstrate rapid dynamic process motility. ( A ) GFP + resident myeloid cells in ex vivo choroidoscleral explants from a CX3CR1 +/GFP mouse were monitored using time-lapse confocal live imaging. Images of choriocapillaris myeloid cells taken at time 0 ( green , top ) and 100 minutes ( red , middle ) were highly overlapping when superimposed ( bottom ), demonstrating an absence of significant cellular migration or soma translocation within this time interval. ( B ) Resident myeloid cells demonstrated dynamic motility in their dendritiform processes. Comparison of a confocal image of a choroidal artery-associated myeloid cell (L, arterial lumen), captured at a single time point ( top ), to that of a summed image of 100 individual images (captured every 10 seconds over 1000 seconds) ( bottom ) demonstrates the coverage of perivascular space by motile processes. Note that the elongated morphology and parallel alignment to the vessel wall are maintained despite marked process movement. ( C ) Comparison of a confocal image of a choriocapillaris-associated myeloid cell captured at a single time point ( top ) to that of a summed image of 30 images (captured at 42-second intervals over 1000 seconds) ( bottom ) illustrates the dynamism of the processes and their ability to occupy the extravascular space on the surface of the choriocapillaris. ( D ) Time-lapse images (sequential images taken 2 minutes apart) from a recording of a spindle-shaped myeloid cell associated with a primary arteriole demonstrates varying motility in its processes; processes that were directed outward into the extravascular space ( process 1 ) demonstrated more prominent dynamism than juxtavascular axial processes closely associated with the vessel wall ( processes 2 and 3 ) that were relatively more stable. ( E ) Time-lapse images (sequential images taken 2 minutes apart) from a recording of a stellate-shaped myeloid cell associated with the choriocapillaris ( C ) demonstrate dynamism in all processes (1–4).

Techniques Used: Ex Vivo, Imaging, Migration, Translocation Assay

10) Product Images from "Tau mutant A152T, a risk factor for FTD/PSP, induces neuronal dysfunction and reduced lifespan independently of aggregation in a C. elegans Tauopathy model"

Article Title: Tau mutant A152T, a risk factor for FTD/PSP, induces neuronal dysfunction and reduced lifespan independently of aggregation in a C. elegans Tauopathy model

Journal: Molecular Neurodegeneration

doi: 10.1186/s13024-016-0096-1

Mutant Tau AT worms show aberrant localization of presynaptic components in mechanosensory neurons. a Schematic representation of presynaptic cargo distribution in a normal healthy mechanosensory neuron and a neuron in an aged animal. b Day-1 old worms visualized after crossing them into vdEx262: [ Pmec-4::mCherry::rab-3 ] transgene that expresses mCherry fused to synaptic vesicle associated RAB-3 in mechanosensory neurons. Tau wt -lo and Tau wt -hi show a similar distribution as non-tg reporter strain. Tau AT -lo worms show accumulation of mCherry::RAB-3 in the end neuron (yellow arrowhead), cell body (CB, white arrowhead) and posterior neurite (white arrow). By contrast, the mid-neuron of Tau AT -lo shows less puncta (6 ± 3 measured per 40 μm length) than non-tg (15 ± 5) and Tau wt -lo (17 ± 6) worms. On day 3, mislocalization of mCherry::RAB-3 worsens in Tau AT -lo worms, whereas Tau wt -lo and Tau wt -hi start accumulating mCherry:RAB-3 puncta in the distal axon and posterior neurite (see Additional file 12 )
Figure Legend Snippet: Mutant Tau AT worms show aberrant localization of presynaptic components in mechanosensory neurons. a Schematic representation of presynaptic cargo distribution in a normal healthy mechanosensory neuron and a neuron in an aged animal. b Day-1 old worms visualized after crossing them into vdEx262: [ Pmec-4::mCherry::rab-3 ] transgene that expresses mCherry fused to synaptic vesicle associated RAB-3 in mechanosensory neurons. Tau wt -lo and Tau wt -hi show a similar distribution as non-tg reporter strain. Tau AT -lo worms show accumulation of mCherry::RAB-3 in the end neuron (yellow arrowhead), cell body (CB, white arrowhead) and posterior neurite (white arrow). By contrast, the mid-neuron of Tau AT -lo shows less puncta (6 ± 3 measured per 40 μm length) than non-tg (15 ± 5) and Tau wt -lo (17 ± 6) worms. On day 3, mislocalization of mCherry::RAB-3 worsens in Tau AT -lo worms, whereas Tau wt -lo and Tau wt -hi start accumulating mCherry:RAB-3 puncta in the distal axon and posterior neurite (see Additional file 12 )

Techniques Used: Mutagenesis

Mutant Tau AT induces morphological changes in mechanosensory neurons early in the adulthood and reduces life-span. a Cartoon depicting a normal healthy neuron and a neuron showing morphological changes associated with aging like sprouting, non-specific branching and bending. b Mutant Tau AT worms show neuronal abnormalities reminiscent of aging neurons. Pmec-4::gfp reporter that expresses GFP in mechanosensory neurons, was crossed into tau-transgenic worms and neurons visualized for morphological abnormalities at the days indicated. Note the soma outgrowth (yellow arrowhead) and bending of neuronal process (white arrow) in Tau AT -lo at day 1. At this age, Tau wt (both -lo and -hi) show normal morphology and do not differ from non-tg. Only the incidence of an infrequent posterior extension (white arrow heads) increases in the Tau wt worms (to 30 % compared with 10 % in non-tg at day 1), whereas in Tau AT -lo this posterior extension occurs in almost 100 % of the animals. c The severity of the phenotype increases with age. Soma outgrowths visible in Tau AT -lo animals at day 1 grow and undergo further branching with age (yellow double arrowhead). The volume marker GFP accumulates in beaded structures in the posterior extension in Tau AT -lo worms and small outgrowths emanate from posterior extension (white double arrowhead). These beaded structures could represent starting points of new branches, and were previously shown to be associated with mitochondria [ 24 ]. d Quantification of animals with gross non-specific neuronal abnormalities (bends and branches) at two time points, day 1 and day 3. Error bars denote mean ± SEM, n ≥ 20. ** P ≤ 0.01, *** P ≤ 0.001. Paired t-test with unequal variance was used for comparison. e Representative survival curves of tau-transgenic animals, non-tg serves as control. Mantel-Cox log-rank test was performed to determine the statistical significance for the worm life-span. (for P -values see Table 2 )
Figure Legend Snippet: Mutant Tau AT induces morphological changes in mechanosensory neurons early in the adulthood and reduces life-span. a Cartoon depicting a normal healthy neuron and a neuron showing morphological changes associated with aging like sprouting, non-specific branching and bending. b Mutant Tau AT worms show neuronal abnormalities reminiscent of aging neurons. Pmec-4::gfp reporter that expresses GFP in mechanosensory neurons, was crossed into tau-transgenic worms and neurons visualized for morphological abnormalities at the days indicated. Note the soma outgrowth (yellow arrowhead) and bending of neuronal process (white arrow) in Tau AT -lo at day 1. At this age, Tau wt (both -lo and -hi) show normal morphology and do not differ from non-tg. Only the incidence of an infrequent posterior extension (white arrow heads) increases in the Tau wt worms (to 30 % compared with 10 % in non-tg at day 1), whereas in Tau AT -lo this posterior extension occurs in almost 100 % of the animals. c The severity of the phenotype increases with age. Soma outgrowths visible in Tau AT -lo animals at day 1 grow and undergo further branching with age (yellow double arrowhead). The volume marker GFP accumulates in beaded structures in the posterior extension in Tau AT -lo worms and small outgrowths emanate from posterior extension (white double arrowhead). These beaded structures could represent starting points of new branches, and were previously shown to be associated with mitochondria [ 24 ]. d Quantification of animals with gross non-specific neuronal abnormalities (bends and branches) at two time points, day 1 and day 3. Error bars denote mean ± SEM, n ≥ 20. ** P ≤ 0.01, *** P ≤ 0.001. Paired t-test with unequal variance was used for comparison. e Representative survival curves of tau-transgenic animals, non-tg serves as control. Mantel-Cox log-rank test was performed to determine the statistical significance for the worm life-span. (for P -values see Table 2 )

Techniques Used: Mutagenesis, Transgenic Assay, Marker

11) Product Images from "Identification of Secretion Determinants of the Bordetella pertussis BrkA Autotransporter"

Article Title: Identification of Secretion Determinants of the Bordetella pertussis BrkA Autotransporter

Journal: Journal of Bacteriology

doi: 10.1128/JB.185.2.489-495.2003

BrkA expression in E. coli strain UT5600. (A) BrkA domain organization: signal peptide (SP [residues 1 to 42]), passenger or α-domain (residues 43 to 731), and β-domain (residues 732 to 1010). (B) Western immunoblot of E. coli UT5600 whole-cell lysates resolved by SDS-PAGE (11% polyacrylamide), probed with anti BrkA antiserum, and detected with goat anti-rabbit antiserum conjugated to horseradish peroxidase. Lanes: 1 and 2, pDO6935 (wild-type copy of brk A gene); 3 and 4, pBluescript (vector control). Specific BrkA bands are indicated. U, unprocessed 103-kDa precursor protein; *, 73-kDa processed passenger moiety. Cells were processed in the presence (+) or absence (−) of trypsin as described in Materials and Methods. (C) Surface expression of BrkA in E. coli UT5600 detected via indirect immunofluorescence. The top panels show phase-contrast images, and the bottom panels show epifluorescence images.
Figure Legend Snippet: BrkA expression in E. coli strain UT5600. (A) BrkA domain organization: signal peptide (SP [residues 1 to 42]), passenger or α-domain (residues 43 to 731), and β-domain (residues 732 to 1010). (B) Western immunoblot of E. coli UT5600 whole-cell lysates resolved by SDS-PAGE (11% polyacrylamide), probed with anti BrkA antiserum, and detected with goat anti-rabbit antiserum conjugated to horseradish peroxidase. Lanes: 1 and 2, pDO6935 (wild-type copy of brk A gene); 3 and 4, pBluescript (vector control). Specific BrkA bands are indicated. U, unprocessed 103-kDa precursor protein; *, 73-kDa processed passenger moiety. Cells were processed in the presence (+) or absence (−) of trypsin as described in Materials and Methods. (C) Surface expression of BrkA in E. coli UT5600 detected via indirect immunofluorescence. The top panels show phase-contrast images, and the bottom panels show epifluorescence images.

Techniques Used: Expressing, Western Blot, SDS Page, Plasmid Preparation, Immunofluorescence

12) Product Images from "Severe neuromuscular denervation of clinically relevant muscles in a mouse model of spinal muscular atrophy"

Article Title: Severe neuromuscular denervation of clinically relevant muscles in a mouse model of spinal muscular atrophy

Journal: Human Molecular Genetics

doi: 10.1093/hmg/ddr453

NMJs are formed but not maintained in the vulnerable appendicular muscle, FDB-2/3, in SMNΔ7 mice. ( A ) Sample immunomicrographs of the control and SMNΔ7 FDB-2 muscles at P1, P4 and P7. NMJs were immunostained for nerve terminals with anti-synaptophysin (in green) and endplates with α-bungarotoxin (in red). Insets are magnified views of sample NMJs. ( B ) Quantification of fully innervated endplates in FDB-2/3 muscles of control and SMNΔ7 mice during postnatal development (E19.5–P14). ( C ) Quantification of partially or fully denervated endplates in SMNΔ7 FDB-2/3 muscles during E19.5–P14. ( D ) Quantification of the number of presynaptic inputs (labeled by anti-neurofilament) at the vulnerable FDB-2 muscle and the resistant FDB-4 muscles in the control and SMNΔ7 mice at P1. All quantitative data are mean ± SEM. * P
Figure Legend Snippet: NMJs are formed but not maintained in the vulnerable appendicular muscle, FDB-2/3, in SMNΔ7 mice. ( A ) Sample immunomicrographs of the control and SMNΔ7 FDB-2 muscles at P1, P4 and P7. NMJs were immunostained for nerve terminals with anti-synaptophysin (in green) and endplates with α-bungarotoxin (in red). Insets are magnified views of sample NMJs. ( B ) Quantification of fully innervated endplates in FDB-2/3 muscles of control and SMNΔ7 mice during postnatal development (E19.5–P14). ( C ) Quantification of partially or fully denervated endplates in SMNΔ7 FDB-2/3 muscles during E19.5–P14. ( D ) Quantification of the number of presynaptic inputs (labeled by anti-neurofilament) at the vulnerable FDB-2 muscle and the resistant FDB-4 muscles in the control and SMNΔ7 mice at P1. All quantitative data are mean ± SEM. * P

Techniques Used: Mouse Assay, Labeling

Severe denervation of proximal and distal muscles in end-stage SMNΔ7 mice. ( A ) Axial muscles [serratus posterior superior (SPS), SPI, trapezius and splenius] and appendicular muscles (FDB-4 and -2) of YFP-SMA mice were immunostained for nerve terminals with anti-synaptophysin [syn] (in green) and motor endplates with α-bungarotoxin (in red). ( B ) Selected high-magnification images of NMJs in SPI muscles of control and SMNΔ7 mice at P14. Arrows indicate dispersing AChR clusters at a denervated endplate in SMNΔ7 mice.
Figure Legend Snippet: Severe denervation of proximal and distal muscles in end-stage SMNΔ7 mice. ( A ) Axial muscles [serratus posterior superior (SPS), SPI, trapezius and splenius] and appendicular muscles (FDB-4 and -2) of YFP-SMA mice were immunostained for nerve terminals with anti-synaptophysin [syn] (in green) and motor endplates with α-bungarotoxin (in red). ( B ) Selected high-magnification images of NMJs in SPI muscles of control and SMNΔ7 mice at P14. Arrows indicate dispersing AChR clusters at a denervated endplate in SMNΔ7 mice.

Techniques Used: Mouse Assay

Quantitative analyses of innervation in a wide range of axial and appendicular muscles in end-staged (P12–14) SMNΔ7 mice and age-matched control mice. Axial and appendicular muscles were immunostained for nerve terminals with anti-synaptophysin and motor endplates with α-bungarotoxin. Innervation percentage was expressed as the number of fully innervated endplates over the total number of analyzed endplates ( > 200 NMJs) in each animal. The average percentage was obtained from three to five pairs of animals. Data are expressed as mean ± SEM. * P
Figure Legend Snippet: Quantitative analyses of innervation in a wide range of axial and appendicular muscles in end-staged (P12–14) SMNΔ7 mice and age-matched control mice. Axial and appendicular muscles were immunostained for nerve terminals with anti-synaptophysin and motor endplates with α-bungarotoxin. Innervation percentage was expressed as the number of fully innervated endplates over the total number of analyzed endplates ( > 200 NMJs) in each animal. The average percentage was obtained from three to five pairs of animals. Data are expressed as mean ± SEM. * P

Techniques Used: Mouse Assay

13) Product Images from "Choline Availability During Embryonic Development Alters Progenitor Cell Mitosis in Developing Mouse Hippocampus 1Choline Availability During Embryonic Development Alters Progenitor Cell Mitosis in Developing Mouse Hippocampus 1 , 2"

Article Title: Choline Availability During Embryonic Development Alters Progenitor Cell Mitosis in Developing Mouse Hippocampus 1Choline Availability During Embryonic Development Alters Progenitor Cell Mitosis in Developing Mouse Hippocampus 1 , 2

Journal: The Journal of nutrition

doi:

Maternal dietary choline deficiency in timed-pregnant mice fed choline-supplemented (CS), control (CT) or choline-deficient (CD) AIN-76 diet from embryonic day 12 to 17 (E12–17) increases apoptosis in day E17 mouse hippocampus. Coronal sections were prepared from the brains of day E17 fetuses from each diet group and were analyzed for apoptosis using a combination of classical apoptotic morphology, active caspase-3 immunoreactivity and TUNEL (terminal deoxynucleotidyl transferase (TdT)-mediated dUTP-digoxigenin anti-digoxigenin fluorescein conjugate antibody nick end-labeling). Consecutive serial sections were used for TUNEL and active (cleaved) caspase-3 staining as described in Materials and Methods. Panel A : The graph shows apoptotic cell counts using morphological criteria (means ± SEM, n = 10–12 pups/group from at least 5 dams), and TUNEL-cleaved caspase-3 immunostaining (means ± SEM, n = 6 pups/group from 3 different dams). Means without common letters differ, P
Figure Legend Snippet: Maternal dietary choline deficiency in timed-pregnant mice fed choline-supplemented (CS), control (CT) or choline-deficient (CD) AIN-76 diet from embryonic day 12 to 17 (E12–17) increases apoptosis in day E17 mouse hippocampus. Coronal sections were prepared from the brains of day E17 fetuses from each diet group and were analyzed for apoptosis using a combination of classical apoptotic morphology, active caspase-3 immunoreactivity and TUNEL (terminal deoxynucleotidyl transferase (TdT)-mediated dUTP-digoxigenin anti-digoxigenin fluorescein conjugate antibody nick end-labeling). Consecutive serial sections were used for TUNEL and active (cleaved) caspase-3 staining as described in Materials and Methods. Panel A : The graph shows apoptotic cell counts using morphological criteria (means ± SEM, n = 10–12 pups/group from at least 5 dams), and TUNEL-cleaved caspase-3 immunostaining (means ± SEM, n = 6 pups/group from 3 different dams). Means without common letters differ, P

Techniques Used: Mouse Assay, TUNEL Assay, End Labeling, Staining, Immunostaining

14) Product Images from "The Bud14p-Glc7p complex functions as a cortical regulator of dynein in budding yeast"

Article Title: The Bud14p-Glc7p complex functions as a cortical regulator of dynein in budding yeast

Journal: The EMBO Journal

doi: 10.1038/sj.emboj.7600783

The localization of Bud14p to sites of polarized growth requires an intact actin cytoskeleton and the cortical kelch-domain-containing proteins Kel1p and Kel2p. ( A ) The localization of YFP-Bud14p (pMK113) expressed from the ADH1 promoter (upper panel) and CFP-Tub1p (middle panel) was determined by epifluorescence microscopy in wt (yMK78) cells. ( B ) The level of Bud14p-myc expressed from its endogenous genomic locus (yMK164) was analyzed by immunoblotting after cell cycle synchronization by α-factor arrest/release. Cell cycle progression was monitored at the times indicated (in min) by immunoblotting for Clb2p (middle panels). Actin served as a loading control (lower panels). An untagged wt strain (K699) controls for the specificity of Bud14p-myc detection. ( C ) The localization of YFP-Bud14p (pMK113) and CFP-Tub1p was determined in wt (yMK78) cells in the presence (+) or absence (DMSO) of the actin depolymerization drug LAT-A, or the MT-depolymerization drug Nocodazole. Arrows mark the localization of YFP-Bud14p at the bud cortex and bud neck, respectively. ( D, E ) The localization of GFP-Bud14p (pMK60) expressed from its endogenous promoter was determined in wt (BY4741), kel1 Δ (YO2852), kel2 Δ (YO6996) and kel1 Δ kel2 Δ (yMK91) cells. Arrows mark GFP-Bud14p at bud tips, the mother-bud neck, and shmoo tips in cells exposed to α-factor (panels D and E). Asterisks denote loss of GFP-Bud14p at bud or shmoo tips in kel1 Δ and kel1 Δ kel2 Δ cells, while crosses indicate loss of GFP-Bud14p staining at the mother-bud neck. Percentages (%) of cells that have lost polarized GFP-Bud14p localization at shmoo tips ( n =200) are shown in (E).
Figure Legend Snippet: The localization of Bud14p to sites of polarized growth requires an intact actin cytoskeleton and the cortical kelch-domain-containing proteins Kel1p and Kel2p. ( A ) The localization of YFP-Bud14p (pMK113) expressed from the ADH1 promoter (upper panel) and CFP-Tub1p (middle panel) was determined by epifluorescence microscopy in wt (yMK78) cells. ( B ) The level of Bud14p-myc expressed from its endogenous genomic locus (yMK164) was analyzed by immunoblotting after cell cycle synchronization by α-factor arrest/release. Cell cycle progression was monitored at the times indicated (in min) by immunoblotting for Clb2p (middle panels). Actin served as a loading control (lower panels). An untagged wt strain (K699) controls for the specificity of Bud14p-myc detection. ( C ) The localization of YFP-Bud14p (pMK113) and CFP-Tub1p was determined in wt (yMK78) cells in the presence (+) or absence (DMSO) of the actin depolymerization drug LAT-A, or the MT-depolymerization drug Nocodazole. Arrows mark the localization of YFP-Bud14p at the bud cortex and bud neck, respectively. ( D, E ) The localization of GFP-Bud14p (pMK60) expressed from its endogenous promoter was determined in wt (BY4741), kel1 Δ (YO2852), kel2 Δ (YO6996) and kel1 Δ kel2 Δ (yMK91) cells. Arrows mark GFP-Bud14p at bud tips, the mother-bud neck, and shmoo tips in cells exposed to α-factor (panels D and E). Asterisks denote loss of GFP-Bud14p at bud or shmoo tips in kel1 Δ and kel1 Δ kel2 Δ cells, while crosses indicate loss of GFP-Bud14p staining at the mother-bud neck. Percentages (%) of cells that have lost polarized GFP-Bud14p localization at shmoo tips ( n =200) are shown in (E).

Techniques Used: Epifluorescence Microscopy, Staining

15) Product Images from "Caenorhabditis elegans Muscleblind homolog mbl-1 functions in neurons to regulate synapse formation"

Article Title: Caenorhabditis elegans Muscleblind homolog mbl-1 functions in neurons to regulate synapse formation

Journal: Neural Development

doi: 10.1186/1749-8104-7-7

Localization of post-synaptic receptor ACR-16 and body wall muscle morphology is normal in mbl-1 . (A-F) Postsynaptic receptor ACR-16::GFP co-localizes with presynaptic mCherry::RAB-3 in wild type (A-C) and mbl-1(tm1563) (D-F) mutants in the dorsal nerve cord. ACR-16 is shown in green and RAB-3 is shown in red. (G, H) Dorsal body wall muscles labeled with myr-mCherry extend muscle arms towards the dorsal nerve cord where DA9 synapses are labeled with SNB-1::YFP. Muscles and SNB-1 are pseudocolored green and red, respectively. Two representative muscle arms are indicated with a white triangle for each genotype. (I, J) Lateral body wall muscles labeled with membrane-tethered yellow fluorescent protein (YFP; green) extend muscle arms towards the dorsal nerve cord labeled with mCherry (red). Four muscle arms, each indicated by a white triangle, are observed per muscle for both wild type and mbl-1(tm1563) .
Figure Legend Snippet: Localization of post-synaptic receptor ACR-16 and body wall muscle morphology is normal in mbl-1 . (A-F) Postsynaptic receptor ACR-16::GFP co-localizes with presynaptic mCherry::RAB-3 in wild type (A-C) and mbl-1(tm1563) (D-F) mutants in the dorsal nerve cord. ACR-16 is shown in green and RAB-3 is shown in red. (G, H) Dorsal body wall muscles labeled with myr-mCherry extend muscle arms towards the dorsal nerve cord where DA9 synapses are labeled with SNB-1::YFP. Muscles and SNB-1 are pseudocolored green and red, respectively. Two representative muscle arms are indicated with a white triangle for each genotype. (I, J) Lateral body wall muscles labeled with membrane-tethered yellow fluorescent protein (YFP; green) extend muscle arms towards the dorsal nerve cord labeled with mCherry (red). Four muscle arms, each indicated by a white triangle, are observed per muscle for both wild type and mbl-1(tm1563) .

Techniques Used: Labeling

Quantification of rescue of DA9 synaptic defect. (A) Quantification of the rescue of the DA9 synapse defect in mbl-1(tm1563) mutants with fosmid WRM0616bE04 tagged with GFP::SL2::mCherry ( wyEx4512 and wyEx4513 ). Error is standard error of the mean. ** P
Figure Legend Snippet: Quantification of rescue of DA9 synaptic defect. (A) Quantification of the rescue of the DA9 synapse defect in mbl-1(tm1563) mutants with fosmid WRM0616bE04 tagged with GFP::SL2::mCherry ( wyEx4512 and wyEx4513 ). Error is standard error of the mean. ** P

Techniques Used:

Localization of pre-synaptic proteins is disrupted in mbl-1 mutants. (A) Schematic of DA9 and wild-type localization of RAB-3. (B-G) Co-localization of RAB-3 and UNC-10 in wild type (B-D) and mbl-1(tm1563) (E-G). (wyEx3709) . (H-M) RAB-3 and the active zone protein SYD-2 in wild type (H-J) and mbl-1(tm1563) (K-M). Transgene used is wyEx2055 . Scale bar represents 10 μm.
Figure Legend Snippet: Localization of pre-synaptic proteins is disrupted in mbl-1 mutants. (A) Schematic of DA9 and wild-type localization of RAB-3. (B-G) Co-localization of RAB-3 and UNC-10 in wild type (B-D) and mbl-1(tm1563) (E-G). (wyEx3709) . (H-M) RAB-3 and the active zone protein SYD-2 in wild type (H-J) and mbl-1(tm1563) (K-M). Transgene used is wyEx2055 . Scale bar represents 10 μm.

Techniques Used:

Measurement of synapse length. (A-D) Representative images of DA9 synapses labeled with SNB-1::YFP in animals that are approximately 15 hours old and at the L2 stage (A, B) and approximately 40 hours old and at the L4 stage (C, D). Transgene used is wyIs92 . (E-F) Images of GFP::RAB-3 in early-stage adult animals. Trasgene used is wyIs85 . (G) Quantification of synapse length at three stages for wild-type (blue) and mbl-1(tm1563) (red) animals.
Figure Legend Snippet: Measurement of synapse length. (A-D) Representative images of DA9 synapses labeled with SNB-1::YFP in animals that are approximately 15 hours old and at the L2 stage (A, B) and approximately 40 hours old and at the L4 stage (C, D). Transgene used is wyIs92 . (E-F) Images of GFP::RAB-3 in early-stage adult animals. Trasgene used is wyIs85 . (G) Quantification of synapse length at three stages for wild-type (blue) and mbl-1(tm1563) (red) animals.

Techniques Used: Labeling

MBL-1 is required cell autonomously in DA9 and is expressed in many dorsal cord neurons. (A) Genomic region of the X chromosome including mbl-1. mbl-1(tm1563) deletion is indicated above a schematic of genomic region. All cDNA isoforms were reported by Sasagawa et al. [ 31 ]. Bottom: schematic of genomic rescuing constructs. Black boxes represent exons and thin black lines represent introns. Thin gray lines indicate splicing patterns. (B) Quantification of the number of DA9 synapses present in the dorsal cord. Two independent transgenic lines were scored for each rescuing construct. Error bars represent standard error of the mean. ***P
Figure Legend Snippet: MBL-1 is required cell autonomously in DA9 and is expressed in many dorsal cord neurons. (A) Genomic region of the X chromosome including mbl-1. mbl-1(tm1563) deletion is indicated above a schematic of genomic region. All cDNA isoforms were reported by Sasagawa et al. [ 31 ]. Bottom: schematic of genomic rescuing constructs. Black boxes represent exons and thin black lines represent introns. Thin gray lines indicate splicing patterns. (B) Quantification of the number of DA9 synapses present in the dorsal cord. Two independent transgenic lines were scored for each rescuing construct. Error bars represent standard error of the mean. ***P

Techniques Used: Construct, Transgenic Assay

mbl-1 disrupts synaptic vesicle localization in the motorneuron DA9. (A-C) Localization of synaptic vesicle-associated GFP::RAB-3 in adult wild type ( wyIs85 ) (A), mbl-1(wy560) (B) and mbl-1(tm1563) (C) mutants. Animals are oriented such that anterior is to the left and dorsal is up. Synaptic region (square bracket), dendrite (curly bracket) and DA9 cell body (asterisk) are indicated. Scale bar represents 10 μm. (D) Quantification of the number of axonal RAB-3 puncta observed by epifluorescence microscopy. Error bars represent standard error of the mean. *** P
Figure Legend Snippet: mbl-1 disrupts synaptic vesicle localization in the motorneuron DA9. (A-C) Localization of synaptic vesicle-associated GFP::RAB-3 in adult wild type ( wyIs85 ) (A), mbl-1(wy560) (B) and mbl-1(tm1563) (C) mutants. Animals are oriented such that anterior is to the left and dorsal is up. Synaptic region (square bracket), dendrite (curly bracket) and DA9 cell body (asterisk) are indicated. Scale bar represents 10 μm. (D) Quantification of the number of axonal RAB-3 puncta observed by epifluorescence microscopy. Error bars represent standard error of the mean. *** P

Techniques Used: Epifluorescence Microscopy

16) Product Images from "Cyanate and Urea are Substrates for Nitrification by Thaumarchaeota in the Marine Environment"

Article Title: Cyanate and Urea are Substrates for Nitrification by Thaumarchaeota in the Marine Environment

Journal: Nature microbiology

doi: 10.1038/s41564-018-0316-2

Thaumarchaeota single cell ammonium, urea and cyanate uptake determined by nanoSIMS at Station 2, 14m depth. a) Representative CARD-FISH image of Thaumarchaeota (green; counterstained by DAPI, blue) with a specific probe (Thaum726). b) corresponding nanoSIMS image of 15 N/( 14 N+ 15 N) enrichment after addition of 15 N-cyanate. Thaumarchaeota are marked by white outlines. Scale bar is 1 μm. In total, 9, 6 and 8 fields of view were analyzed by nanoSIMS for the 15 N-cyanate, 15 N-ammonium and 15 N-urea treatment. c) 15 N/( 14 N+ 15 N) enrichment of Thaumarchaeota (green) and non-targeted cells (grey) after incubation with 15 N-ammonium (left), 15 N-urea (middle, without added 14 N-ammonium) or 15 N-cyanate (right, without added 14 N-ammonium). Note the different scales for 15 N-ammonium and 15 N-urea and 15 N-cyanate, respectively. Number of cells analyzed per category is indicated above each boxplot. Boxplots depict the 25 – 75 % quantile range, with the center line depicting the median (50% quantile); whiskers encompass data points within 1.5 × the interquartile range. NA is the natural abundance 15 N/( 14 N+ 15 N) value (0.0037). Four non- Thaumarchaeota cell values in the 15 N-urea treatment are not depicted and have 15 N/( 14 N+ 15 N) values of 0.326, 0.095, 0.118 and 0.139, these cells were included in all calculations. More ammonium was assimilated than urea and cyanate by the Thaumarchaeota, and the Thaumarchaeota assimilated significantly more 15 N compared to surrounding cells in all treatments (one-sided Mann-Whitney U Test, U = 3348.5, p = 6.19×10 -14 ; U = 873, p = 0.001; U = 3409, p = 2.91×10 -12 for ammonium, urea and cyanate, respectively).
Figure Legend Snippet: Thaumarchaeota single cell ammonium, urea and cyanate uptake determined by nanoSIMS at Station 2, 14m depth. a) Representative CARD-FISH image of Thaumarchaeota (green; counterstained by DAPI, blue) with a specific probe (Thaum726). b) corresponding nanoSIMS image of 15 N/( 14 N+ 15 N) enrichment after addition of 15 N-cyanate. Thaumarchaeota are marked by white outlines. Scale bar is 1 μm. In total, 9, 6 and 8 fields of view were analyzed by nanoSIMS for the 15 N-cyanate, 15 N-ammonium and 15 N-urea treatment. c) 15 N/( 14 N+ 15 N) enrichment of Thaumarchaeota (green) and non-targeted cells (grey) after incubation with 15 N-ammonium (left), 15 N-urea (middle, without added 14 N-ammonium) or 15 N-cyanate (right, without added 14 N-ammonium). Note the different scales for 15 N-ammonium and 15 N-urea and 15 N-cyanate, respectively. Number of cells analyzed per category is indicated above each boxplot. Boxplots depict the 25 – 75 % quantile range, with the center line depicting the median (50% quantile); whiskers encompass data points within 1.5 × the interquartile range. NA is the natural abundance 15 N/( 14 N+ 15 N) value (0.0037). Four non- Thaumarchaeota cell values in the 15 N-urea treatment are not depicted and have 15 N/( 14 N+ 15 N) values of 0.326, 0.095, 0.118 and 0.139, these cells were included in all calculations. More ammonium was assimilated than urea and cyanate by the Thaumarchaeota, and the Thaumarchaeota assimilated significantly more 15 N compared to surrounding cells in all treatments (one-sided Mann-Whitney U Test, U = 3348.5, p = 6.19×10 -14 ; U = 873, p = 0.001; U = 3409, p = 2.91×10 -12 for ammonium, urea and cyanate, respectively).

Techniques Used: Fluorescence In Situ Hybridization, Incubation, MANN-WHITNEY

17) Product Images from "Arabidopsis thaliana POLYOL/MONOSACCHARIDE TRANSPORTERS 1 and 2: fructose and xylitol/H+ symporters in pollen and young xylem cells"

Article Title: Arabidopsis thaliana POLYOL/MONOSACCHARIDE TRANSPORTERS 1 and 2: fructose and xylitol/H+ symporters in pollen and young xylem cells

Journal: Journal of Experimental Botany

doi: 10.1093/jxb/erp322

Reporter gene analyses of p AtPMT2 / GFP and p AtPMT2 / GUS plants. (A) Inflorescence of a p AtPMT2 / GUS plant with strong GUS staining in the mature anthers. (B) Higher magnification of an anther with very strong GUS staining in fully developed pollen grains (arrows) and in germinated and ungerminated pollen on agar medium (insert). Staining of cells in the anther surface results from the diffusion of excess stain out of the pollen grains and can even reach the sepals and petals of stained flowers (see A). (C) GFP-fluorescence (epifluorescence) in pollen grains on an opened anther from a p AtPMT2 / GFP plants. No fluorescence is seen in WT anthers (insert). (D) Strong GUS staining in source leaf hydathodes and very weak GUS staining in minor veins (arrows). (E) Cross-section of a flower stalk with GUS staining in young xylem cells (ca, cambium; ph, phloem; xy, xylem). A bar indicates the region where the single-celled row of cambial cells is located. The cambial cells themselves cannot be identified. Bars are 2 mm (A, D), 200 μm (B, insert of C), 20 μm (insert of B), 100 μm (C), 25 μm (E).
Figure Legend Snippet: Reporter gene analyses of p AtPMT2 / GFP and p AtPMT2 / GUS plants. (A) Inflorescence of a p AtPMT2 / GUS plant with strong GUS staining in the mature anthers. (B) Higher magnification of an anther with very strong GUS staining in fully developed pollen grains (arrows) and in germinated and ungerminated pollen on agar medium (insert). Staining of cells in the anther surface results from the diffusion of excess stain out of the pollen grains and can even reach the sepals and petals of stained flowers (see A). (C) GFP-fluorescence (epifluorescence) in pollen grains on an opened anther from a p AtPMT2 / GFP plants. No fluorescence is seen in WT anthers (insert). (D) Strong GUS staining in source leaf hydathodes and very weak GUS staining in minor veins (arrows). (E) Cross-section of a flower stalk with GUS staining in young xylem cells (ca, cambium; ph, phloem; xy, xylem). A bar indicates the region where the single-celled row of cambial cells is located. The cambial cells themselves cannot be identified. Bars are 2 mm (A, D), 200 μm (B, insert of C), 20 μm (insert of B), 100 μm (C), 25 μm (E).

Techniques Used: Staining, Diffusion-based Assay, Fluorescence

18) Product Images from "Deciphering Protein Dynamics of the Siderophore Pyoverdine Pathway in Pseudomonas aeruginosa"

Article Title: Deciphering Protein Dynamics of the Siderophore Pyoverdine Pathway in Pseudomonas aeruginosa

Journal: PLoS ONE

doi: 10.1371/journal.pone.0079111

A. Scheme depicting the PVD pathway involving biosynthesis, iron uptake and gene expression. For details and explanations, refer to the “Introduction” section. The results obtained in this work on protein dynamics are indicated as follows: the stars in red, purple and blue indicate the proteins with rapid, moderate and slow dynamics, respectively. B. Fluorescence microscopy analysis of fluorescent fusions with, from left to right and up to down, OpmQ, FpvF, PvdQ, TonB, PvdT, PvdA and PvdS. Cells were grown twice in minimal medium, washed in minimal medium and spotted onto slides coated with agarose made up in minimal medium. Brightfield images, when available, are presented on the left. Due to low fluorescent signals, epifluorescence images of pvdS-yfp , mcherry-pvdT and mcherry-opmQ were recorded using a high sensitivity camera. Images of fpvF-mcherry in both epifluorescence (left panel) and TIRF (right panel) are shown. Epifluorescence images of pvdA-yfp , mcherry-pvdQ and pvdQ-mcherry are presented. For tonB-mcherry, from left to right, brightfield, epifluorescence and TIRF mode images are presented (scale bar 2 µm). For the fluorescence miscrocopy pictures of PAO1 strain harboring a plasmid encoding a cytoplasmic mCHERRY fluorescent protein expressed under the control of the pvdA promoter (PAO1(pMMB- mcherry )) see in Supplemental Materials (Figure 5-SM).
Figure Legend Snippet: A. Scheme depicting the PVD pathway involving biosynthesis, iron uptake and gene expression. For details and explanations, refer to the “Introduction” section. The results obtained in this work on protein dynamics are indicated as follows: the stars in red, purple and blue indicate the proteins with rapid, moderate and slow dynamics, respectively. B. Fluorescence microscopy analysis of fluorescent fusions with, from left to right and up to down, OpmQ, FpvF, PvdQ, TonB, PvdT, PvdA and PvdS. Cells were grown twice in minimal medium, washed in minimal medium and spotted onto slides coated with agarose made up in minimal medium. Brightfield images, when available, are presented on the left. Due to low fluorescent signals, epifluorescence images of pvdS-yfp , mcherry-pvdT and mcherry-opmQ were recorded using a high sensitivity camera. Images of fpvF-mcherry in both epifluorescence (left panel) and TIRF (right panel) are shown. Epifluorescence images of pvdA-yfp , mcherry-pvdQ and pvdQ-mcherry are presented. For tonB-mcherry, from left to right, brightfield, epifluorescence and TIRF mode images are presented (scale bar 2 µm). For the fluorescence miscrocopy pictures of PAO1 strain harboring a plasmid encoding a cytoplasmic mCHERRY fluorescent protein expressed under the control of the pvdA promoter (PAO1(pMMB- mcherry )) see in Supplemental Materials (Figure 5-SM).

Techniques Used: Expressing, Fluorescence, Microscopy, Plasmid Preparation

19) Product Images from "An essential role for the nuclear protein Akirin2 in mouse limb interdigital tissue regression"

Article Title: An essential role for the nuclear protein Akirin2 in mouse limb interdigital tissue regression

Journal: Scientific Reports

doi: 10.1038/s41598-018-30801-2

Fgf8 expression is aberrantly retained in Akirin2 knockout limb epithelium. Whole mount in situ hybridisation using Fgf8 riboprobes comparing control and Akirin2 Emx-KO limb at E10.5 ( a ), E11.5 ( c – f ), E12.5 ( g – n ) and E13 ( o – v ). Fgf8 signal is retained in the Akirin2 Emx-KO limb distal epithelia in the regions that display syndactyly from E12.5 onwards ( g – v ): digits 2–3 in forelimb and digits 2–4 in hindlimb (black arrowheads). Insets at E12.5 ( k – n ) and E13 ( s – v ) show higher magnification images of the distal limb. Panel (b) is a schematic showing the KO phenotype of perduring Fgf8 expression (blue). Key: A, anterior; D, dorsal; Di, distal; P, posterior; Pr, proximal; V, ventral. Scale bar: 500 μm in each row.
Figure Legend Snippet: Fgf8 expression is aberrantly retained in Akirin2 knockout limb epithelium. Whole mount in situ hybridisation using Fgf8 riboprobes comparing control and Akirin2 Emx-KO limb at E10.5 ( a ), E11.5 ( c – f ), E12.5 ( g – n ) and E13 ( o – v ). Fgf8 signal is retained in the Akirin2 Emx-KO limb distal epithelia in the regions that display syndactyly from E12.5 onwards ( g – v ): digits 2–3 in forelimb and digits 2–4 in hindlimb (black arrowheads). Insets at E12.5 ( k – n ) and E13 ( s – v ) show higher magnification images of the distal limb. Panel (b) is a schematic showing the KO phenotype of perduring Fgf8 expression (blue). Key: A, anterior; D, dorsal; Di, distal; P, posterior; Pr, proximal; V, ventral. Scale bar: 500 μm in each row.

Techniques Used: Expressing, Knock-Out, In Situ, Hybridization

20) Product Images from "Nodule-Specific Regulation of Phosphatidylinositol Transfer Protein Expression in Lotus japonicus"

Article Title: Nodule-Specific Regulation of Phosphatidylinositol Transfer Protein Expression in Lotus japonicus

Journal: The Plant Cell

doi:

The Conserved Nlj16 Domain Mediates the Plasma Membrane Targeting of the LjPLP-IV Protein. (A) to (E) Subcellular localization of the mGFP5–Nlj16 fusion proteins in onion epidermal cells. Fluorescent images of onion cells expressing mGFP5 alone (A) , the mGFP5–Nlj16 fusion ( [B] and [D] ), and the mGFP5–Nlj16ΔCC fusion (C) are shown. (E) is a bright-field image of the cell shown in (D) . The onion cell shown in (D) and (E) has undergone plasmolysis, and mGFP5 fluorescence can be seen on the periphery of the protoplast. P, protoplast; V, vacuole; CW, cell wall. (F) Alignment of the C-terminal Nlj16-like domains of LjPLP-II, LjPLP-III, LjPLP-IV, and Arabidopsis AtPLP. Similar residues are boxed, and identical amino acids are boxed and shaded. Dashes represent gaps in the sequences.
Figure Legend Snippet: The Conserved Nlj16 Domain Mediates the Plasma Membrane Targeting of the LjPLP-IV Protein. (A) to (E) Subcellular localization of the mGFP5–Nlj16 fusion proteins in onion epidermal cells. Fluorescent images of onion cells expressing mGFP5 alone (A) , the mGFP5–Nlj16 fusion ( [B] and [D] ), and the mGFP5–Nlj16ΔCC fusion (C) are shown. (E) is a bright-field image of the cell shown in (D) . The onion cell shown in (D) and (E) has undergone plasmolysis, and mGFP5 fluorescence can be seen on the periphery of the protoplast. P, protoplast; V, vacuole; CW, cell wall. (F) Alignment of the C-terminal Nlj16-like domains of LjPLP-II, LjPLP-III, LjPLP-IV, and Arabidopsis AtPLP. Similar residues are boxed, and identical amino acids are boxed and shaded. Dashes represent gaps in the sequences.

Techniques Used: Expressing, Fluorescence

21) Product Images from "Cyanate and Urea are Substrates for Nitrification by Thaumarchaeota in the Marine Environment"

Article Title: Cyanate and Urea are Substrates for Nitrification by Thaumarchaeota in the Marine Environment

Journal: Nature microbiology

doi: 10.1038/s41564-018-0316-2

Thaumarchaeota single cell ammonium, urea and cyanate uptake determined by nanoSIMS at Station 2, 14m depth. a) Representative CARD-FISH image of Thaumarchaeota (green; counterstained by DAPI, blue) with a specific probe (Thaum726). b) corresponding nanoSIMS image of 15 N/( 14 N+ 15 N) enrichment after addition of 15 N-cyanate. Thaumarchaeota are marked by white outlines. Scale bar is 1 μm. In total, 9, 6 and 8 fields of view were analyzed by nanoSIMS for the 15 N-cyanate, 15 N-ammonium and 15 N-urea treatment. c) 15 N/( 14 N+ 15 N) enrichment of Thaumarchaeota (green) and non-targeted cells (grey) after incubation with 15 N-ammonium (left), 15 N-urea (middle, without added 14 N-ammonium) or 15 N-cyanate (right, without added 14 N-ammonium). Note the different scales for 15 N-ammonium and 15 N-urea and 15 N-cyanate, respectively. Number of cells analyzed per category is indicated above each boxplot. Boxplots depict the 25 – 75 % quantile range, with the center line depicting the median (50% quantile); whiskers encompass data points within 1.5 × the interquartile range. NA is the natural abundance 15 N/( 14 N+ 15 N) value (0.0037). Four non- Thaumarchaeota cell values in the 15 N-urea treatment are not depicted and have 15 N/( 14 N+ 15 N) values of 0.326, 0.095, 0.118 and 0.139, these cells were included in all calculations. More ammonium was assimilated than urea and cyanate by the Thaumarchaeota, and the Thaumarchaeota assimilated significantly more 15 N compared to surrounding cells in all treatments (one-sided Mann-Whitney U Test, U = 3348.5, p = 6.19×10 -14 ; U = 873, p = 0.001; U = 3409, p = 2.91×10 -12 for ammonium, urea and cyanate, respectively).
Figure Legend Snippet: Thaumarchaeota single cell ammonium, urea and cyanate uptake determined by nanoSIMS at Station 2, 14m depth. a) Representative CARD-FISH image of Thaumarchaeota (green; counterstained by DAPI, blue) with a specific probe (Thaum726). b) corresponding nanoSIMS image of 15 N/( 14 N+ 15 N) enrichment after addition of 15 N-cyanate. Thaumarchaeota are marked by white outlines. Scale bar is 1 μm. In total, 9, 6 and 8 fields of view were analyzed by nanoSIMS for the 15 N-cyanate, 15 N-ammonium and 15 N-urea treatment. c) 15 N/( 14 N+ 15 N) enrichment of Thaumarchaeota (green) and non-targeted cells (grey) after incubation with 15 N-ammonium (left), 15 N-urea (middle, without added 14 N-ammonium) or 15 N-cyanate (right, without added 14 N-ammonium). Note the different scales for 15 N-ammonium and 15 N-urea and 15 N-cyanate, respectively. Number of cells analyzed per category is indicated above each boxplot. Boxplots depict the 25 – 75 % quantile range, with the center line depicting the median (50% quantile); whiskers encompass data points within 1.5 × the interquartile range. NA is the natural abundance 15 N/( 14 N+ 15 N) value (0.0037). Four non- Thaumarchaeota cell values in the 15 N-urea treatment are not depicted and have 15 N/( 14 N+ 15 N) values of 0.326, 0.095, 0.118 and 0.139, these cells were included in all calculations. More ammonium was assimilated than urea and cyanate by the Thaumarchaeota, and the Thaumarchaeota assimilated significantly more 15 N compared to surrounding cells in all treatments (one-sided Mann-Whitney U Test, U = 3348.5, p = 6.19×10 -14 ; U = 873, p = 0.001; U = 3409, p = 2.91×10 -12 for ammonium, urea and cyanate, respectively).

Techniques Used: Fluorescence In Situ Hybridization, Incubation, MANN-WHITNEY

22) Product Images from "Nr4a1-eGFP Is a Marker of Striosome-Matrix Architecture, Development and Activity in the Extended Striatum"

Article Title: Nr4a1-eGFP Is a Marker of Striosome-Matrix Architecture, Development and Activity in the Extended Striatum

Journal: PLoS ONE

doi: 10.1371/journal.pone.0016619

Developmental expression of Nr4a1 in striatonigral projections compared to Drd1, TH and mu-OR in horizontal sections. eGFP expression (A1, B1, C1) overlaps with Drd1 immunoreactivity (A2, A3, A5 merged with the DAPI channel) and TH immunoreactivity (B2, B3, B5 merged with the DAPI channel) Mu-OR immunoreactivity (C2) colocalized with Nr4a1-eGFP is shown in C3 and in C5 merged with the DAPI channel. DAPI staining is less intense in the striosomes (A4, B4, C4), indicating that the striosome-like distribution is not due to increased cell density. The scale bar in H (200 µm) applies to all images. Panels A and B were taken with a Zeiss Axiovert microscope. Panels in C were taken with a Zeiss Lumar stereomicroscope.
Figure Legend Snippet: Developmental expression of Nr4a1 in striatonigral projections compared to Drd1, TH and mu-OR in horizontal sections. eGFP expression (A1, B1, C1) overlaps with Drd1 immunoreactivity (A2, A3, A5 merged with the DAPI channel) and TH immunoreactivity (B2, B3, B5 merged with the DAPI channel) Mu-OR immunoreactivity (C2) colocalized with Nr4a1-eGFP is shown in C3 and in C5 merged with the DAPI channel. DAPI staining is less intense in the striosomes (A4, B4, C4), indicating that the striosome-like distribution is not due to increased cell density. The scale bar in H (200 µm) applies to all images. Panels A and B were taken with a Zeiss Axiovert microscope. Panels in C were taken with a Zeiss Lumar stereomicroscope.

Techniques Used: Expressing, Staining, Microscopy

23) Product Images from "Meconium Impairs Pulmonary Surfactant by a Combined Action of Cholesterol and Bile Acids"

Article Title: Meconium Impairs Pulmonary Surfactant by a Combined Action of Cholesterol and Bile Acids

Journal: Biophysical Journal

doi: 10.1016/j.bpj.2010.12.3715

Effect of meconium on the structure of surfactant films. ( A ) Epifluorescence microscopy images from interfacial films of native surfactant containing a trace of the fluorescent probe BODIPY-PC, in the absence ( left pictures ) or in the presence ( right
Figure Legend Snippet: Effect of meconium on the structure of surfactant films. ( A ) Epifluorescence microscopy images from interfacial films of native surfactant containing a trace of the fluorescent probe BODIPY-PC, in the absence ( left pictures ) or in the presence ( right

Techniques Used: Epifluorescence Microscopy

24) Product Images from "Isolation and initial propagation of guinea pig adenovirus (GPAdV) inCavia porcellus cell lines"

Article Title: Isolation and initial propagation of guinea pig adenovirus (GPAdV) inCavia porcellus cell lines

Journal: F1000Research

doi: 10.12688/f1000research.20135.2

Characterization of guinea pig tracheal epithelial cell line GPTEC-T. A . Chromosome numbers in metaphases of GPTEC-T cells at passage 23. A total of 105 metaphases were examined by light microscopy. B . Detection of T antigen coding sequence by PCR for quality control at DSMZ, Germany. Total DNA was extracted from GPTEC-T and various other cell lines included in the test as controls, and used as a template. M: Molecular marker Generuler 1kB plus (Thermo Fisher Scientific). C . Indirect immunofluorescence staining of GPTEC-T cells for ZO-1 and SV40 T antigen. Cells were stained with primary antibodies anti ZO-1 and anti SV40 T antigen and appropriate secondary antibodies as described in the Methods section. Nuclei were stained with Hoechst. Images were acquired with a Zeiss Axioskop epifluorescence microscope at 40X magnification. Merged image of ZO-1 staining at the plasma membrane (green) and nuclear staining for T antigen (red) and Hoechst (blue).
Figure Legend Snippet: Characterization of guinea pig tracheal epithelial cell line GPTEC-T. A . Chromosome numbers in metaphases of GPTEC-T cells at passage 23. A total of 105 metaphases were examined by light microscopy. B . Detection of T antigen coding sequence by PCR for quality control at DSMZ, Germany. Total DNA was extracted from GPTEC-T and various other cell lines included in the test as controls, and used as a template. M: Molecular marker Generuler 1kB plus (Thermo Fisher Scientific). C . Indirect immunofluorescence staining of GPTEC-T cells for ZO-1 and SV40 T antigen. Cells were stained with primary antibodies anti ZO-1 and anti SV40 T antigen and appropriate secondary antibodies as described in the Methods section. Nuclei were stained with Hoechst. Images were acquired with a Zeiss Axioskop epifluorescence microscope at 40X magnification. Merged image of ZO-1 staining at the plasma membrane (green) and nuclear staining for T antigen (red) and Hoechst (blue).

Techniques Used: Light Microscopy, Sequencing, Polymerase Chain Reaction, Marker, Immunofluorescence, Staining, Microscopy

25) Product Images from "Fluorescence fluctuation spectroscopy reveals differential SUN protein oligomerization in living cells"

Article Title: Fluorescence fluctuation spectroscopy reveals differential SUN protein oligomerization in living cells

Journal: Molecular Biology of the Cell

doi: 10.1091/mbc.E17-04-0233

SUN1 and SUN2 oligomerization in the cytoplasm. (A) Constructs used in this figure. (B) Representative epifluorescence images of U2OS cells expressing the indicated constructs. Scale bar: 5 μm. (C–G) Plots of b vs. N for the indicated constructs. The data in C were fitted to a trimeric binding model (solid blue line), which is shown in D and E (dashed blue line) with K MD = 8000 (60 μM) ± 4000 and K DT = 0.3 (0.002 μM) ± 0.2. The data in F were fitted to a monomer/trimer/hexamer binding model (solid blue line) with K MT = 100 (0.7 μM) ± 60 and a trimer–hexamer dissociation coefficient K TH = 1500 (10 μM) ± 400, which is then shown in G (dashed blue line). Estimated binding curves (dashed red lines) for the data obtained in the NE for the indicated constructs are presented in C and F by converting N from the NE to its cytoplasmic value.
Figure Legend Snippet: SUN1 and SUN2 oligomerization in the cytoplasm. (A) Constructs used in this figure. (B) Representative epifluorescence images of U2OS cells expressing the indicated constructs. Scale bar: 5 μm. (C–G) Plots of b vs. N for the indicated constructs. The data in C were fitted to a trimeric binding model (solid blue line), which is shown in D and E (dashed blue line) with K MD = 8000 (60 μM) ± 4000 and K DT = 0.3 (0.002 μM) ± 0.2. The data in F were fitted to a monomer/trimer/hexamer binding model (solid blue line) with K MT = 100 (0.7 μM) ± 60 and a trimer–hexamer dissociation coefficient K TH = 1500 (10 μM) ± 400, which is then shown in G (dashed blue line). Estimated binding curves (dashed red lines) for the data obtained in the NE for the indicated constructs are presented in C and F by converting N from the NE to its cytoplasmic value.

Techniques Used: Construct, Expressing, Binding Assay

FFS and brightness analysis in the NE. (A) Identification of the dorsal (NE D ) and ventral (NE V ) NEs in a cell expressing EGFP-tagged NE proteins by z-scan FFS. Fluorescence intensity fluctuations are measured at either NE. (B) Constructs used in this figure. (C) Representative epifluorescence images of U2OS cells expressing the indicated constructs. Scale bar: 5 μm. (D) Brightness analysis of the cells described in C. Each data point represents the average b measured in a single cell.
Figure Legend Snippet: FFS and brightness analysis in the NE. (A) Identification of the dorsal (NE D ) and ventral (NE V ) NEs in a cell expressing EGFP-tagged NE proteins by z-scan FFS. Fluorescence intensity fluctuations are measured at either NE. (B) Constructs used in this figure. (C) Representative epifluorescence images of U2OS cells expressing the indicated constructs. Scale bar: 5 μm. (D) Brightness analysis of the cells described in C. Each data point represents the average b measured in a single cell.

Techniques Used: Expressing, Fluorescence, Construct

SUN1 oligomerization in the NE. (A) Constructs used in this figure. (B) Represen­tative epifluorescence images of U2OS cells expressing the indicated constructs. Scale bar: 5 μm. (C–E) Plots of b vs. N for the indicated constructs. The data in C were fitted to a linear regression (solid red line), which is shown in D and E (dashed red line).
Figure Legend Snippet: SUN1 oligomerization in the NE. (A) Constructs used in this figure. (B) Represen­tative epifluorescence images of U2OS cells expressing the indicated constructs. Scale bar: 5 μm. (C–E) Plots of b vs. N for the indicated constructs. The data in C were fitted to a linear regression (solid red line), which is shown in D and E (dashed red line).

Techniques Used: Construct, Expressing

SUN2 oligomerization in the NE. (A) Constructs used in this figure. (B) Represent­ative epifluorescence images of U2OS cells expressing the indicated constructs. Scale bar: 5 μm. (C–E) Plots of b vs. N for the indicated constructs. The data in C were fitted to a monomer/dimer/trimer binding model (solid red line), which is shown in D and E (dashed red line), with K MD = 4100 (1000 μM) , K DT = 0.06 (0.01 μM) , and a monomer/trimer binding model (solid green line) with K MT = 26 (6 μM) ± 7.
Figure Legend Snippet: SUN2 oligomerization in the NE. (A) Constructs used in this figure. (B) Represent­ative epifluorescence images of U2OS cells expressing the indicated constructs. Scale bar: 5 μm. (C–E) Plots of b vs. N for the indicated constructs. The data in C were fitted to a monomer/dimer/trimer binding model (solid red line), which is shown in D and E (dashed red line), with K MD = 4100 (1000 μM) , K DT = 0.06 (0.01 μM) , and a monomer/trimer binding model (solid green line) with K MT = 26 (6 μM) ± 7.

Techniques Used: Construct, Expressing, Binding Assay

26) Product Images from "Copy Number Variation and Expression Analysis Reveals a Nonorthologous Pinta Gene Family Member Involved in Butterfly Vision"

Article Title: Copy Number Variation and Expression Analysis Reveals a Nonorthologous Pinta Gene Family Member Involved in Butterfly Vision

Journal: Genome Biology and Evolution

doi: 10.1093/gbe/evx230

—Immunohistochemistry of Hme CTD31 in Heliconius melpomene eye and optic lobe. ( A ) Drawing of a longitudinal view of a compound eye and lamina, and longitudinal and transverse sections of a single ommatidium. Green highlights where we find Hme CTD31 expression; L, lamina; c, cornea; cc, crystalline cone; ppc, primary pigment cells; r, rhabdom; R1-9 conventional Lepidoptera numbering of photoreceptor cells; n, cell nucleus; spc, secondary pigment cells; bm, basement membrane; t, trachea; and tc, tracheal cell. ( B ) Brightfield longitudinal section showing pigments in the H. melpomene retina. ( C ) Longitudinal section with Hme CTD31 and LW opsin staining; Hme CTD31 is in green and LW opsin is in magenta. ( D ) Brightfield image of a transverse section of a butterfly eye, pigment is seen in the structures surrounding the ommatidia. ( E ) Transverse view of a butterfly eye stained for LW and Hme CTD31. ( E ′) autofluorescence showing tracheoles surrounding an individual ommatidium. ( E ′′) LW opsin staining showing where the LW photoreceptor cells are. ( E ′′′) CTD31 staining showing where the CRAL-TRIO domain protein Hme CTD31 is expressed. ( E ′′′′) merged image of LWRh, CTD31, and autofluorescence.
Figure Legend Snippet: —Immunohistochemistry of Hme CTD31 in Heliconius melpomene eye and optic lobe. ( A ) Drawing of a longitudinal view of a compound eye and lamina, and longitudinal and transverse sections of a single ommatidium. Green highlights where we find Hme CTD31 expression; L, lamina; c, cornea; cc, crystalline cone; ppc, primary pigment cells; r, rhabdom; R1-9 conventional Lepidoptera numbering of photoreceptor cells; n, cell nucleus; spc, secondary pigment cells; bm, basement membrane; t, trachea; and tc, tracheal cell. ( B ) Brightfield longitudinal section showing pigments in the H. melpomene retina. ( C ) Longitudinal section with Hme CTD31 and LW opsin staining; Hme CTD31 is in green and LW opsin is in magenta. ( D ) Brightfield image of a transverse section of a butterfly eye, pigment is seen in the structures surrounding the ommatidia. ( E ) Transverse view of a butterfly eye stained for LW and Hme CTD31. ( E ′) autofluorescence showing tracheoles surrounding an individual ommatidium. ( E ′′) LW opsin staining showing where the LW photoreceptor cells are. ( E ′′′) CTD31 staining showing where the CRAL-TRIO domain protein Hme CTD31 is expressed. ( E ′′′′) merged image of LWRh, CTD31, and autofluorescence.

Techniques Used: Immunohistochemistry, Expressing, Staining

27) Product Images from "Characterization of Engineered Actin Binding Proteins That Control Filament Assembly and Structure"

Article Title: Characterization of Engineered Actin Binding Proteins That Control Filament Assembly and Structure

Journal: PLoS ONE

doi: 10.1371/journal.pone.0013960

sAB-27 bundles actin filaments. (A) A single TMR-actin filament visualized in epifluorescence in solution. Arrowhead indicates an out of focus free end of F-actin. (B) TMR-actin filaments in solution with sAB-27, visualized in epifluorescence. Multiple filaments are crosslinked in the presence of sAB-27, and these crosslinked assemblies diffuse as a unit in solution. (C) Electron micrograph of single actin filaments. (D) Electron micrograph of actin filaments in the presence of sAB-27 (1 µM), revealing a similar crosslinking of actin filaments. (E) Nucleation assay of actin alone, after 5 min polymerization, visualized with TIRF microscopy. (F) Nucleation assay in the presence of 1 µM sAB-27 after 5 min. No polymerization is evident. Scale bar, 10 µm.
Figure Legend Snippet: sAB-27 bundles actin filaments. (A) A single TMR-actin filament visualized in epifluorescence in solution. Arrowhead indicates an out of focus free end of F-actin. (B) TMR-actin filaments in solution with sAB-27, visualized in epifluorescence. Multiple filaments are crosslinked in the presence of sAB-27, and these crosslinked assemblies diffuse as a unit in solution. (C) Electron micrograph of single actin filaments. (D) Electron micrograph of actin filaments in the presence of sAB-27 (1 µM), revealing a similar crosslinking of actin filaments. (E) Nucleation assay of actin alone, after 5 min polymerization, visualized with TIRF microscopy. (F) Nucleation assay in the presence of 1 µM sAB-27 after 5 min. No polymerization is evident. Scale bar, 10 µm.

Techniques Used: Microscopy

28) Product Images from "Compressed collagen and decellularized tissue – novel components in a pipeline approach for the study of cancer metastasis"

Article Title: Compressed collagen and decellularized tissue – novel components in a pipeline approach for the study of cancer metastasis

Journal: BMC Cancer

doi: 10.1186/s12885-018-4533-0

dCAM provides a collagen-rich 3D substrate for cell culture. Laser scanning confocal spectral unmixing was used to determine the residual components after decellularization of CAM (Nikon A1 Plus at room temperature using a × 60 1.40 Plan Apo ∞/0.17 WD 0.13, NA 1.4 lens). a , combined image, b DAPI only, c CAM background only, d , phalloidin for cellular actin cytoskeleton (scale bar for A = 20 μm). Scanning electron microscopy (SEM) was used to characterize the surface of the decellularized tissue (Quanta FEI), e and f show dCAM surface features including vasculature (yellow arrows) and fibrous extracellular matrix (scale bars: E = 50 μm, F = 5 μm). dCAM used as a growth matrix: g shows a bright field image of dCAM during colonization and h shows MDA-MB-231 GFP+ cells adhering and proliferating over the dCAM (DC, yellow arrows). Images G and H were taken using a Zeiss Axio Vert inverted epifluorescence microscope and × 5 Planar Plan Neofl Ph1 0.15 ∞ /0.17 lens with the DS-Fi2 camera, operating at room temperature, scale bars = 1 mm
Figure Legend Snippet: dCAM provides a collagen-rich 3D substrate for cell culture. Laser scanning confocal spectral unmixing was used to determine the residual components after decellularization of CAM (Nikon A1 Plus at room temperature using a × 60 1.40 Plan Apo ∞/0.17 WD 0.13, NA 1.4 lens). a , combined image, b DAPI only, c CAM background only, d , phalloidin for cellular actin cytoskeleton (scale bar for A = 20 μm). Scanning electron microscopy (SEM) was used to characterize the surface of the decellularized tissue (Quanta FEI), e and f show dCAM surface features including vasculature (yellow arrows) and fibrous extracellular matrix (scale bars: E = 50 μm, F = 5 μm). dCAM used as a growth matrix: g shows a bright field image of dCAM during colonization and h shows MDA-MB-231 GFP+ cells adhering and proliferating over the dCAM (DC, yellow arrows). Images G and H were taken using a Zeiss Axio Vert inverted epifluorescence microscope and × 5 Planar Plan Neofl Ph1 0.15 ∞ /0.17 lens with the DS-Fi2 camera, operating at room temperature, scale bars = 1 mm

Techniques Used: Cell Culture, Chick Chorioallantoic Membrane Assay, Electron Microscopy, Multiple Displacement Amplification, Inverted Epifluorescence

29) Product Images from "Hypoxia-inducible factor cell non-autonomously regulates C. elegans stress responses and behavior via a nuclear receptor"

Article Title: Hypoxia-inducible factor cell non-autonomously regulates C. elegans stress responses and behavior via a nuclear receptor

Journal: eLife

doi: 10.7554/eLife.36828

cyp-36A1 expression in multiple tissues rescues the egg-laying phenotype of cyp-36A1(lf); egl-9(lf) . ( A–H ) Distribution of stages of eggs laid by adult hermaphrodites of the indicated genotypes. All strains contained the agIs219 ( P T24B8.5 ::gfp ) transgene. ( A ) Stages of eggs laid by wild-type animals. ( B ) egl-9(sa307) animals laid later stage eggs than wild type (p
Figure Legend Snippet: cyp-36A1 expression in multiple tissues rescues the egg-laying phenotype of cyp-36A1(lf); egl-9(lf) . ( A–H ) Distribution of stages of eggs laid by adult hermaphrodites of the indicated genotypes. All strains contained the agIs219 ( P T24B8.5 ::gfp ) transgene. ( A ) Stages of eggs laid by wild-type animals. ( B ) egl-9(sa307) animals laid later stage eggs than wild type (p

Techniques Used: Expressing

cyp-36A1 is expressed in many tissues. ( A–D ) Paired fluorescent (left in each panel) and Nomarski (right in each panel) micrographs showing expression of a transcriptional P cyp-36A1 ::gfp reporter ( nIs682 ) in an adult worm. Expression was observed in the head ( A ), midbody ( B and C ), and tail ( D ), including in neurons, body-wall muscle, vulval muscle, intestine, and hypoderm, as indicated. Scale bars, 20 μm.
Figure Legend Snippet: cyp-36A1 is expressed in many tissues. ( A–D ) Paired fluorescent (left in each panel) and Nomarski (right in each panel) micrographs showing expression of a transcriptional P cyp-36A1 ::gfp reporter ( nIs682 ) in an adult worm. Expression was observed in the head ( A ), midbody ( B and C ), and tail ( D ), including in neurons, body-wall muscle, vulval muscle, intestine, and hypoderm, as indicated. Scale bars, 20 μm.

Techniques Used: Expressing

30) Product Images from "The Arabidopsis Phytochrome-Interacting Factor PIF7, Together with PIF3 and PIF4, Regulates Responses to Prolonged Red Light by Modulating phyB Levels [W]"

Article Title: The Arabidopsis Phytochrome-Interacting Factor PIF7, Together with PIF3 and PIF4, Regulates Responses to Prolonged Red Light by Modulating phyB Levels [W]

Journal: The Plant Cell

doi: 10.1105/tpc.107.052142

PIF7 Is a Light-Stable Nuclear Protein That Colocalizes with phyB in Rc-Induced Early Speckles. (A) Immunoblot of protein extracts from T2 segregating 35S:PIF7:CFP seedlings. Seedlings were grown in the dark for 2 d (0), transferred to Rc for a 30-s pulse, followed by 10 min of dark (Rp), or transferred to Rc for 1, 3, 6, 12, or 24 h. PIF7-specific polyclonal antibody (top) and a PIF3-specific polyclonal antibody (middle) were used as probes. As controls, protein extracts from the wild type, pif7-1 , and pif3-3 are included. Tubulin was used as a loading control (bottom). n.s., nonspecific, cross-reacting bands. (B) Immunoblot of protein extracts from 35S:PIF7:CFP seedlings. Seedlings were grown for 4 d in the dark (D) or in Rc at 6.2 μmol·m −2 ·s −1 (R). PIF7-specific polyclonal antibody (top) and a PIF3-specific polyclonal antibody (middle) were used as probes. As controls, protein extracts from the wild type and pif3-3 are included. Tubulin was used as a loading control (bottom). (C) Epifluorescence imaging of YFP (displayed in green) and CFP (displayed in red) fluorescence in nuclei of Arabidopsis transgenic seedlings expressing PIF7:CFP (top panels) or YFP:PIF3 (bottom panels). Seedlings were grown in the dark for 4 d and then either maintained in darkness (Dark), exposed to Rc for 2 min (R2min) provided by passing the microscope light through a red filter (140 μmol·m −2 ·s −1 ), or exposed to Rc for 60 min of 7 μmol·m −2 ·s −1 (R60min). Images were recorded within 2 min of termination of the Rc treatments. Bars = 10 μm. (D) Epifluorescence imaging of YFP (displayed in green) and CFP (displayed in red) fluorescence in nuclei of Arabidopsis transgenic seedlings coexpressing PIF7:CFP (top panels) and phyB:YFP (bottom panels) in the phyB-9 background. Seedlings were grown in the dark for 4 d and then either maintained in darkness (Dark) or exposed to Rc for 2 min (R2min) provided by passing the microscope light through a red filter (140 μmol·m −2 ·s −1 ). Images were recorded within 2 min of termination of the Rc treatments. The overlay of the PIF7:YFP and the phyB:CFP signals after a Rc irradiation is indicated in yellow in the merged image (right panel). Bars = 10 μm.
Figure Legend Snippet: PIF7 Is a Light-Stable Nuclear Protein That Colocalizes with phyB in Rc-Induced Early Speckles. (A) Immunoblot of protein extracts from T2 segregating 35S:PIF7:CFP seedlings. Seedlings were grown in the dark for 2 d (0), transferred to Rc for a 30-s pulse, followed by 10 min of dark (Rp), or transferred to Rc for 1, 3, 6, 12, or 24 h. PIF7-specific polyclonal antibody (top) and a PIF3-specific polyclonal antibody (middle) were used as probes. As controls, protein extracts from the wild type, pif7-1 , and pif3-3 are included. Tubulin was used as a loading control (bottom). n.s., nonspecific, cross-reacting bands. (B) Immunoblot of protein extracts from 35S:PIF7:CFP seedlings. Seedlings were grown for 4 d in the dark (D) or in Rc at 6.2 μmol·m −2 ·s −1 (R). PIF7-specific polyclonal antibody (top) and a PIF3-specific polyclonal antibody (middle) were used as probes. As controls, protein extracts from the wild type and pif3-3 are included. Tubulin was used as a loading control (bottom). (C) Epifluorescence imaging of YFP (displayed in green) and CFP (displayed in red) fluorescence in nuclei of Arabidopsis transgenic seedlings expressing PIF7:CFP (top panels) or YFP:PIF3 (bottom panels). Seedlings were grown in the dark for 4 d and then either maintained in darkness (Dark), exposed to Rc for 2 min (R2min) provided by passing the microscope light through a red filter (140 μmol·m −2 ·s −1 ), or exposed to Rc for 60 min of 7 μmol·m −2 ·s −1 (R60min). Images were recorded within 2 min of termination of the Rc treatments. Bars = 10 μm. (D) Epifluorescence imaging of YFP (displayed in green) and CFP (displayed in red) fluorescence in nuclei of Arabidopsis transgenic seedlings coexpressing PIF7:CFP (top panels) and phyB:YFP (bottom panels) in the phyB-9 background. Seedlings were grown in the dark for 4 d and then either maintained in darkness (Dark) or exposed to Rc for 2 min (R2min) provided by passing the microscope light through a red filter (140 μmol·m −2 ·s −1 ). Images were recorded within 2 min of termination of the Rc treatments. The overlay of the PIF7:YFP and the phyB:CFP signals after a Rc irradiation is indicated in yellow in the merged image (right panel). Bars = 10 μm.

Techniques Used: Imaging, Fluorescence, Transgenic Assay, Expressing, Microscopy, Irradiation

31) Product Images from "Rapid Hair Cell Loss: A Mouse Model for Cochlear Lesions"

Article Title: Rapid Hair Cell Loss: A Mouse Model for Cochlear Lesions

Journal: JARO: Journal of the Association for Research in Otolaryngology

doi: 10.1007/s10162-007-0105-8

Progression of OHC loss in the organ of Corti. A – C Twenty-four hours posttreatment. A Basal turn; B middle turn; C apical turn. Confocal images of whole mount preparations. Phalloidin-FITC ( green ) labels actin assemblies at the reticular lamina and in hair cells. Nuclei counterstained with DAPI (displayed in red channel for greater clarity). A In the basal coil, essentially all OHC are lost except for one remaining ( arrow ), whose nucleus shows condensed chromatin. B In the middle coil, there is scattered loss of OHC, with the nuclei of remaining ones showing apoptotic features: marginated and condensed chromatin and fragmentation. At the arrow , an OHC hair bundle overlies a highly condensed nucleus. Arrowhead indicates fragmented nuclei persisting in the absence of any indication of an OHC. C In the apical coil, OHC are mainly unaffected. D – H Forty-eight hours posttreatment. D Basal coil; E , G middle; F , H apex. D , E Confocal image of whole mount preparation; green , actin labeled with phalloidin-FITC; blue , nuclei counterstained with DAPI; red in D , calretinin labeling. F Wide-field epifluorescence microscopy; green , actin; red , nuclei counterstained with propidium iodide. G , H Scanning electron micrographs (SEM). D In the basal coil, all OHC are lost and “scars” fill their positions, but all IHC (calretinin positive) are still present. E Two remaining OHC; all other OHC are missing. There are no nuclei in the region normally occupied by OHC, but both the apical structures and nuclei of IHC persist. F There are scattered persistent OHC, but nuclear fragments ( arrows ) indicate continuing apoptotic death in the apical coil at this time. G Total OHC loss in the middle turn. F At the apical end of the apical coil, only a few scattered OHC remain, and there is ongoing damage to OHC. Scale bars , 10 μm ( A – E , G , and H ) and 20 μm ( F ).
Figure Legend Snippet: Progression of OHC loss in the organ of Corti. A – C Twenty-four hours posttreatment. A Basal turn; B middle turn; C apical turn. Confocal images of whole mount preparations. Phalloidin-FITC ( green ) labels actin assemblies at the reticular lamina and in hair cells. Nuclei counterstained with DAPI (displayed in red channel for greater clarity). A In the basal coil, essentially all OHC are lost except for one remaining ( arrow ), whose nucleus shows condensed chromatin. B In the middle coil, there is scattered loss of OHC, with the nuclei of remaining ones showing apoptotic features: marginated and condensed chromatin and fragmentation. At the arrow , an OHC hair bundle overlies a highly condensed nucleus. Arrowhead indicates fragmented nuclei persisting in the absence of any indication of an OHC. C In the apical coil, OHC are mainly unaffected. D – H Forty-eight hours posttreatment. D Basal coil; E , G middle; F , H apex. D , E Confocal image of whole mount preparation; green , actin labeled with phalloidin-FITC; blue , nuclei counterstained with DAPI; red in D , calretinin labeling. F Wide-field epifluorescence microscopy; green , actin; red , nuclei counterstained with propidium iodide. G , H Scanning electron micrographs (SEM). D In the basal coil, all OHC are lost and “scars” fill their positions, but all IHC (calretinin positive) are still present. E Two remaining OHC; all other OHC are missing. There are no nuclei in the region normally occupied by OHC, but both the apical structures and nuclei of IHC persist. F There are scattered persistent OHC, but nuclear fragments ( arrows ) indicate continuing apoptotic death in the apical coil at this time. G Total OHC loss in the middle turn. F At the apical end of the apical coil, only a few scattered OHC remain, and there is ongoing damage to OHC. Scale bars , 10 μm ( A – E , G , and H ) and 20 μm ( F ).

Techniques Used: Labeling, Epifluorescence Microscopy, Immunohistochemistry

32) Product Images from "The Trypanosoma brucei subpellicular microtubule array is organized into functionally discrete subdomains defined by microtubule associated proteins"

Article Title: The Trypanosoma brucei subpellicular microtubule array is organized into functionally discrete subdomains defined by microtubule associated proteins

Journal: bioRxiv

doi: 10.1101/2020.11.09.375725

PAVE1 and PAVE2 form a complex in vitro that binds the microtubule lattice. (A) (Left) Coomassie-stained SDS-PAGE gel demonstrating the co-purification of mNeonGreen-PAVE1 and Strep-PAVE2 from E. coli cells. E. coli cells co-expressing [MBP-oligoHis-TEV]-nNeonGreen-PAVE1 and Strep-PAVE2 constructs were harvested, lysed, and clarified supernatant were batch-bound with Ni-NTA resin. The eluate was treated with TEV protease and allowed to interact with Strep-Tactin XT resin. Proteins were eluted using 50 mM biotin. The Strep eluate is 4 μg of protein. (Right) The PAVE complex was diluted into a low salt buffer containing 50 mM NaCl and ultracentrifuged. Equal fractions of both the supernatant and pellet were separated on an SDS-PAGE gel and stained with Coomassie blue. (B) 5, 50, 100, 200, and 500 nM clarified PAVE complex was allowed to interact with Taxol-stabilized bovine microtubules labeled with the Cy5 fluor for 20 min RT. The PAVE complex-microtubule solution was settled onto a blocked flow cell and unbound protein was washed away. The flow cell was imaged using epifluorescence microscopy. (C) (Left) A PEGylated flow cell was blocked and Taxol-stabilized bovine Cy5-microtubules were allowed to attach to the flow cell surface. 5 nM clarified PAVE complex was added to the flow cell chamber and immediately imaged using TIRF microscopy. Images were taken every 10 sec for 20 min. (Middle) Kymograph of PAVE complex on the microtubule outlined in yellow. (Right) Fluorescence intensity line scan of the microtubule outlined in yellow for the first 4 min 10 sec of TIRF imaging. (D) (Left) 5 nM clarified PAVE complex was mixed with Taxol-stabilized bovine Cy5-microtubules for 10 min RT to pre-seed patches. The PAVE complex-microtubule mixture was flowed into a blocked and PEGylated flow cell and allowed to attach to the flow cell surface. Unbound protein was washed away and the flow cell was immediately imaged using TIRF microscopy. Images were taken every 10 sec for 20 min. (Right) Kymograph of PAVE complex on the microtubule outlined in yellow. (E) Control microtubules and microtubules treated with subtilisin A were collected for western blotting. The post-translational modification of glutamylation is no longer present on subtilisin-treated microtubules, as detected with the antibody GT335, and there is a molecular weight shift showing that the C-terminal tails have been cleaved, as detected with anti-a-tubulin. (F) PAVE complex was incubated with control or subtilisin-treated Taxol-stabilized bovine Cy5-microtubules as in (B) and imaged using epifluorescence. There is no difference in PAVE complex binding to subtilisin-treated microtubules cleaved of their C-terminal tails in comparison to control, indicating that the PAVE complex binds directly to the microtubule lattice.
Figure Legend Snippet: PAVE1 and PAVE2 form a complex in vitro that binds the microtubule lattice. (A) (Left) Coomassie-stained SDS-PAGE gel demonstrating the co-purification of mNeonGreen-PAVE1 and Strep-PAVE2 from E. coli cells. E. coli cells co-expressing [MBP-oligoHis-TEV]-nNeonGreen-PAVE1 and Strep-PAVE2 constructs were harvested, lysed, and clarified supernatant were batch-bound with Ni-NTA resin. The eluate was treated with TEV protease and allowed to interact with Strep-Tactin XT resin. Proteins were eluted using 50 mM biotin. The Strep eluate is 4 μg of protein. (Right) The PAVE complex was diluted into a low salt buffer containing 50 mM NaCl and ultracentrifuged. Equal fractions of both the supernatant and pellet were separated on an SDS-PAGE gel and stained with Coomassie blue. (B) 5, 50, 100, 200, and 500 nM clarified PAVE complex was allowed to interact with Taxol-stabilized bovine microtubules labeled with the Cy5 fluor for 20 min RT. The PAVE complex-microtubule solution was settled onto a blocked flow cell and unbound protein was washed away. The flow cell was imaged using epifluorescence microscopy. (C) (Left) A PEGylated flow cell was blocked and Taxol-stabilized bovine Cy5-microtubules were allowed to attach to the flow cell surface. 5 nM clarified PAVE complex was added to the flow cell chamber and immediately imaged using TIRF microscopy. Images were taken every 10 sec for 20 min. (Middle) Kymograph of PAVE complex on the microtubule outlined in yellow. (Right) Fluorescence intensity line scan of the microtubule outlined in yellow for the first 4 min 10 sec of TIRF imaging. (D) (Left) 5 nM clarified PAVE complex was mixed with Taxol-stabilized bovine Cy5-microtubules for 10 min RT to pre-seed patches. The PAVE complex-microtubule mixture was flowed into a blocked and PEGylated flow cell and allowed to attach to the flow cell surface. Unbound protein was washed away and the flow cell was immediately imaged using TIRF microscopy. Images were taken every 10 sec for 20 min. (Right) Kymograph of PAVE complex on the microtubule outlined in yellow. (E) Control microtubules and microtubules treated with subtilisin A were collected for western blotting. The post-translational modification of glutamylation is no longer present on subtilisin-treated microtubules, as detected with the antibody GT335, and there is a molecular weight shift showing that the C-terminal tails have been cleaved, as detected with anti-a-tubulin. (F) PAVE complex was incubated with control or subtilisin-treated Taxol-stabilized bovine Cy5-microtubules as in (B) and imaged using epifluorescence. There is no difference in PAVE complex binding to subtilisin-treated microtubules cleaved of their C-terminal tails in comparison to control, indicating that the PAVE complex binds directly to the microtubule lattice.

Techniques Used: In Vitro, Staining, SDS Page, Copurification, Expressing, Construct, Labeling, Epifluorescence Microscopy, Microscopy, Fluorescence, Imaging, Western Blot, Modification, Molecular Weight, Incubation, Binding Assay

33) Product Images from "GLP-2 Prevents Neuronal and Glial Changes in the Distal Colon of Mice Chronically Treated with Cisplatin"

Article Title: GLP-2 Prevents Neuronal and Glial Changes in the Distal Colon of Mice Chronically Treated with Cisplatin

Journal: International Journal of Molecular Sciences

doi: 10.3390/ijms21228875

ChAT/NeuN ( A – C ) and nNOS/NeuN ( D – F ) double labeling in myenteric ganglia. Controls ( A , D ), cisplatin ( B , E ) and cisplatin + [Gly2]GLP-2 ( C , F ) groups. ChAT and nNOS labeling were detected mainly in the neuronal body and in few nerve fibers; NeuN-IR was always observed in the nucleus. Scale bar = 25 μm. Quantitation of the nitrergic and cholinergic neurons number ( G , H ). Data are expressed as mean ± SEM. One-way ANOVA test, post hoc Newman Keuls’. *** ( p
Figure Legend Snippet: ChAT/NeuN ( A – C ) and nNOS/NeuN ( D – F ) double labeling in myenteric ganglia. Controls ( A , D ), cisplatin ( B , E ) and cisplatin + [Gly2]GLP-2 ( C , F ) groups. ChAT and nNOS labeling were detected mainly in the neuronal body and in few nerve fibers; NeuN-IR was always observed in the nucleus. Scale bar = 25 μm. Quantitation of the nitrergic and cholinergic neurons number ( G , H ). Data are expressed as mean ± SEM. One-way ANOVA test, post hoc Newman Keuls’. *** ( p

Techniques Used: Labeling, Quantitation Assay

34) Product Images from "Kindlin-2 Mediates Mechanical Activation of Cardiac Myofibroblasts"

Article Title: Kindlin-2 Mediates Mechanical Activation of Cardiac Myofibroblasts

Journal: Cells

doi: 10.3390/cells9122702

Kindlin-2 overexpression promotes myofibroblast activation. Human MRC-5 fibroblasts were transiently transfected with kindlin-2 (K-2)-GFP, a kindlin-2-GFP mutant lacking the putative nuclear localization sequence (NLS) (kindlin-2-ΔNLS-GFP) and GFP vector control (ctr). Cells were analyzed after 48 h by ( A , B ) confocal immunofluorescence co-staining for GFP (green), α-smooth muscle actin (α-SMA) (greyscale), kindlin-2 (red), and 4′,6-diamidino-2-phenylindole (DAPI) (blue) and ( C , D ) quantitative immunoblotting against kindlin-2. Scale bar: 25 µm. ( B ) Levels of nuclear and perinuclear (cytosolic) GFP signals were quantified from GFP confocal immunofluorescence images to calculate levels of nuclear GFP and ratios of nuclear/cytosolic GFP only in transfected fibroblasts (green). ( C ) The same was done for kindlin-2 signal, comprising transfected and endogenous kindlin-2. ( D ) Western blot for kindlin-2. ( E ) MRC-5 were additionally transfected with firefly luciferase promoter under full length α-SMA promoter control; signal was first normalized to renilla luciferase and then to GFP control. ( F ) The percentage of α-SMA-positive cells was quantified by manually counting α-SMA stress fiber-positive fibroblasts in the GFP-positive fraction (≥10 cells/image, ≥5 images/experimental condition). Shown are mean values from at least three independent experiments (data points) ±SD (* p
Figure Legend Snippet: Kindlin-2 overexpression promotes myofibroblast activation. Human MRC-5 fibroblasts were transiently transfected with kindlin-2 (K-2)-GFP, a kindlin-2-GFP mutant lacking the putative nuclear localization sequence (NLS) (kindlin-2-ΔNLS-GFP) and GFP vector control (ctr). Cells were analyzed after 48 h by ( A , B ) confocal immunofluorescence co-staining for GFP (green), α-smooth muscle actin (α-SMA) (greyscale), kindlin-2 (red), and 4′,6-diamidino-2-phenylindole (DAPI) (blue) and ( C , D ) quantitative immunoblotting against kindlin-2. Scale bar: 25 µm. ( B ) Levels of nuclear and perinuclear (cytosolic) GFP signals were quantified from GFP confocal immunofluorescence images to calculate levels of nuclear GFP and ratios of nuclear/cytosolic GFP only in transfected fibroblasts (green). ( C ) The same was done for kindlin-2 signal, comprising transfected and endogenous kindlin-2. ( D ) Western blot for kindlin-2. ( E ) MRC-5 were additionally transfected with firefly luciferase promoter under full length α-SMA promoter control; signal was first normalized to renilla luciferase and then to GFP control. ( F ) The percentage of α-SMA-positive cells was quantified by manually counting α-SMA stress fiber-positive fibroblasts in the GFP-positive fraction (≥10 cells/image, ≥5 images/experimental condition). Shown are mean values from at least three independent experiments (data points) ±SD (* p

Techniques Used: Over Expression, Activation Assay, Transfection, Mutagenesis, Sequencing, Plasmid Preparation, Immunofluorescence, Staining, Western Blot, Luciferase

35) Product Images from "Cyanobacterial Ecotypes in Different Optical Microenvironments of a 68?C Hot Spring Mat Community Revealed by 16S-23S rRNA Internal Transcribed Spacer Region Variation †"

Article Title: Cyanobacterial Ecotypes in Different Optical Microenvironments of a 68?C Hot Spring Mat Community Revealed by 16S-23S rRNA Internal Transcribed Spacer Region Variation †

Journal: Applied and Environmental Microbiology

doi: 10.1128/AEM.69.5.2893-2898.2003

Unrooted neighbor-joining tree of thermophilic Synechococcus sequences based on 396 nucleotides of the ITS region adjacent to the 16S rRNA gene. Circles, surface library sequences; squares, subsurface sequences. Identical sequences are positioned together at terminal branches. Bootstrap values over 50% are shown.
Figure Legend Snippet: Unrooted neighbor-joining tree of thermophilic Synechococcus sequences based on 396 nucleotides of the ITS region adjacent to the 16S rRNA gene. Circles, surface library sequences; squares, subsurface sequences. Identical sequences are positioned together at terminal branches. Bootstrap values over 50% are shown.

Techniques Used:

36) Product Images from "The Arabidopsis Rab GTPase RabA4b Localizes to the Tips of Growing Root Hair Cells W⃞"

Article Title: The Arabidopsis Rab GTPase RabA4b Localizes to the Tips of Growing Root Hair Cells W⃞

Journal: The Plant Cell

doi: 10.1105/tpc.021634

Localization of the EYFP-RabA4b Fluorescence in the rhd Mutant Backgrounds. The 35S-EYFP-RabA4b construct was transformed into the mutant root hair lines rhd1-1 ( [A] and [B] ), rhd2-1 ( [C] and [D] ), rhd3-1 ( [E] and [F] ), and rhd4-1 ( [G] and [H] ). Plants were grown in 0.25× MS + 0.3% phytagel and transferred to microscope slides. Root hairs were observed using a Nikon Eclipse E600 microscope with differential interference contrast ( [A] , [C] , [E] , and [G] ) and epifluorescence ( [B] , [D] , [F] , and [H] ) optics. Arrows indicate root hairs with normal tip localization of EYFP-RabA4b. Arrowheads point to abnormal distributions of the EYFP-RabA4b in root hairs.
Figure Legend Snippet: Localization of the EYFP-RabA4b Fluorescence in the rhd Mutant Backgrounds. The 35S-EYFP-RabA4b construct was transformed into the mutant root hair lines rhd1-1 ( [A] and [B] ), rhd2-1 ( [C] and [D] ), rhd3-1 ( [E] and [F] ), and rhd4-1 ( [G] and [H] ). Plants were grown in 0.25× MS + 0.3% phytagel and transferred to microscope slides. Root hairs were observed using a Nikon Eclipse E600 microscope with differential interference contrast ( [A] , [C] , [E] , and [G] ) and epifluorescence ( [B] , [D] , [F] , and [H] ) optics. Arrows indicate root hairs with normal tip localization of EYFP-RabA4b. Arrowheads point to abnormal distributions of the EYFP-RabA4b in root hairs.

Techniques Used: Fluorescence, Mutagenesis, Construct, Transformation Assay, Mass Spectrometry, Microscopy

37) Product Images from "New insights into the targeting of a subset of tail-anchored proteins to the outer mitochondrial membrane"

Article Title: New insights into the targeting of a subset of tail-anchored proteins to the outer mitochondrial membrane

Journal: Frontiers in Plant Science

doi: 10.3389/fpls.2014.00426

Localization of various CTS mutant versions of the mitochondrial-TA protein TraB in BY-2 cells . Shown on the left are schematic illustrations of wild-type and various CTS mutant versions of TraB and their corresponding localization (or lack thereof) to mitochondria in transformed BY-2 cells. The names of the mutant constructs represent the specific amino acids in their modified CTSs. All constructs possess an N-terminal-appended Myc-epitope tag. Shown for each construct is the corresponding C-terminal amino acid sequence, including putative TMD (underlined) and modified (or wild-type) CTS; additional amino acid residues inserted into the TraB CTS (i.e., threonines) are bolded. Mitochondrial localization (indicated as “Yes” or “No”) was assessed based on colocalization (or lack thereof) of the expressed protein and endogenous mitochondrial CoxII. Shown on the right in both are representative immuno-epifluorescence micrographs illustrating the localization of the various constructs shown on the left . Each micrograph is labeled with the name of the expressed Myc-tagged wild-type TraB or CTS mutant version of TraB, or endogenous CoxII. Bar = 10 μm.
Figure Legend Snippet: Localization of various CTS mutant versions of the mitochondrial-TA protein TraB in BY-2 cells . Shown on the left are schematic illustrations of wild-type and various CTS mutant versions of TraB and their corresponding localization (or lack thereof) to mitochondria in transformed BY-2 cells. The names of the mutant constructs represent the specific amino acids in their modified CTSs. All constructs possess an N-terminal-appended Myc-epitope tag. Shown for each construct is the corresponding C-terminal amino acid sequence, including putative TMD (underlined) and modified (or wild-type) CTS; additional amino acid residues inserted into the TraB CTS (i.e., threonines) are bolded. Mitochondrial localization (indicated as “Yes” or “No”) was assessed based on colocalization (or lack thereof) of the expressed protein and endogenous mitochondrial CoxII. Shown on the right in both are representative immuno-epifluorescence micrographs illustrating the localization of the various constructs shown on the left . Each micrograph is labeled with the name of the expressed Myc-tagged wild-type TraB or CTS mutant version of TraB, or endogenous CoxII. Bar = 10 μm.

Techniques Used: Mutagenesis, Transformation Assay, Construct, Modification, Sequencing, Labeling

Localization, topology, membrane insertion, and C-terminal targeting-signal analysis of a novel OMM-TA protein, At1g55450. (A) Representative CLSM micrographs illustrating the localization of N-terminal Myc-tagged At1g55450 or MIRO3 to the OMM in BY-2 cells. Cells were processed for immunofluoescence CLSM as in Figure 1 . Shown in the three panels on the right are images corresponding to a portion of the cell at higher magnification. Solid arrowheads indicate examples of the torus-shaped fluorescent structures containing the transiently-expressed protein delineating the spherical structures attributable to endogenous CoxII. (B) Topological mapping of Myc-At1g55450 in differential-permeabilized BY-2 cells. Cells transiently-transformed with N-terminal Myc-tagged At1g55450 were formaldehyde fixed and permeabilized with either Triton X-100 or digitonin, and then cells were processed for immuno-epifluorescence microscopy, as described in Figure 2 . (C) Insertion of At1g55450 into mitochondrial membranes in vitro . Isolated pea mitochondria were incubated with in vitro synthesized Myc-At1g55450 (lanes 1 and 2) or, for comparative purposes, Myc-TraB (lanes 3 and 4), and then resuspended (+) or not (−) in alkaline Na 2 CO 3 . Equivalent amounts of each alkaline Na 2 CO 3 - or mock-extracted sample were then subjected to SDS-PAGE and phosphoimaging. (D) Representative immuno-epifluorescence micrographs illustrating the localization of a C-terminal mutant or GFP fusion of At1g55450 in BY-2 cells. Each micrograph is labeled with the name of either the transiently-expressed Myc-tagged C-terminal mutant or GFP fusion protein or endogenous CoxII. The name of each construct includes the number of amino acid residues that were either deleted from the C terminus of Myc-tagged At1g55450 (−C26) or fused to the C terminus of GFP (+C26). (E) Representative CLSM micrographs illustrating the localization of the Cherry-At1g55450 fusion protein to mitochondria in living transgenic A. thaliana seedlings co-expressing the mitochondrial marker protein mito-GFP. Labels above the panels indicate the name of the co-expressed protein and labels in panels on the left indicate the seedling tissue type. Note in the top row that not all root cells expressed the Cherry-At1g55450 fusion protein. Bars in (A and B) and (D and E) = 10 μm.
Figure Legend Snippet: Localization, topology, membrane insertion, and C-terminal targeting-signal analysis of a novel OMM-TA protein, At1g55450. (A) Representative CLSM micrographs illustrating the localization of N-terminal Myc-tagged At1g55450 or MIRO3 to the OMM in BY-2 cells. Cells were processed for immunofluoescence CLSM as in Figure 1 . Shown in the three panels on the right are images corresponding to a portion of the cell at higher magnification. Solid arrowheads indicate examples of the torus-shaped fluorescent structures containing the transiently-expressed protein delineating the spherical structures attributable to endogenous CoxII. (B) Topological mapping of Myc-At1g55450 in differential-permeabilized BY-2 cells. Cells transiently-transformed with N-terminal Myc-tagged At1g55450 were formaldehyde fixed and permeabilized with either Triton X-100 or digitonin, and then cells were processed for immuno-epifluorescence microscopy, as described in Figure 2 . (C) Insertion of At1g55450 into mitochondrial membranes in vitro . Isolated pea mitochondria were incubated with in vitro synthesized Myc-At1g55450 (lanes 1 and 2) or, for comparative purposes, Myc-TraB (lanes 3 and 4), and then resuspended (+) or not (−) in alkaline Na 2 CO 3 . Equivalent amounts of each alkaline Na 2 CO 3 - or mock-extracted sample were then subjected to SDS-PAGE and phosphoimaging. (D) Representative immuno-epifluorescence micrographs illustrating the localization of a C-terminal mutant or GFP fusion of At1g55450 in BY-2 cells. Each micrograph is labeled with the name of either the transiently-expressed Myc-tagged C-terminal mutant or GFP fusion protein or endogenous CoxII. The name of each construct includes the number of amino acid residues that were either deleted from the C terminus of Myc-tagged At1g55450 (−C26) or fused to the C terminus of GFP (+C26). (E) Representative CLSM micrographs illustrating the localization of the Cherry-At1g55450 fusion protein to mitochondria in living transgenic A. thaliana seedlings co-expressing the mitochondrial marker protein mito-GFP. Labels above the panels indicate the name of the co-expressed protein and labels in panels on the left indicate the seedling tissue type. Note in the top row that not all root cells expressed the Cherry-At1g55450 fusion protein. Bars in (A and B) and (D and E) = 10 μm.

Techniques Used: Confocal Laser Scanning Microscopy, Transformation Assay, Epifluorescence Microscopy, In Vitro, Isolation, Incubation, Synthesized, SDS Page, Mutagenesis, Labeling, Construct, Transgenic Assay, Expressing, Marker

Topological mapping of selected A. thaliana OMM-TA proteins in differentially permeabilized BY-2 cells . Non-transformed (A) or transiently-transformed (B–D) BY-2 cells were formaldehyde fixed and permeabilized (as indicated above each set of images) with either Triton X-100, which perforates all cellular membranes, or, digitonin, which selectively permeabilizes the plasma membrane, then cells were processed for immuno-epifluorescence microscopy. Also indicated in each panel is the name of the immunostained transiently-expressed Myc-tagged protein or endogenous protein (i.e., E1β, CoxII or α-tubulin). Note that the presence or absence of immunofluorescence reflects whether the protein (epitope) was accessible to the applied antibodies. For instance, similar to α-tubulin in cytoplasmic microtubules (A) , N-terminal Myc-tagged TOM9-2 (B) and all other known or putative TA proteins examined (C) , but not endogenous E1β in the mitochondrial matrix or the control protein, Myc-TOM40 (B) , were immunodetected in digitonin-permeabilized cells. Note also in (D) that Myc-TraB-HA did not colocalize with endogenous mitochondrial CoxII, indicating that the expressed protein, unlike Myc-TraB (Figure 1 ) is not properly targeted to mitochondria. Bar in (A,C) = 10 μm.
Figure Legend Snippet: Topological mapping of selected A. thaliana OMM-TA proteins in differentially permeabilized BY-2 cells . Non-transformed (A) or transiently-transformed (B–D) BY-2 cells were formaldehyde fixed and permeabilized (as indicated above each set of images) with either Triton X-100, which perforates all cellular membranes, or, digitonin, which selectively permeabilizes the plasma membrane, then cells were processed for immuno-epifluorescence microscopy. Also indicated in each panel is the name of the immunostained transiently-expressed Myc-tagged protein or endogenous protein (i.e., E1β, CoxII or α-tubulin). Note that the presence or absence of immunofluorescence reflects whether the protein (epitope) was accessible to the applied antibodies. For instance, similar to α-tubulin in cytoplasmic microtubules (A) , N-terminal Myc-tagged TOM9-2 (B) and all other known or putative TA proteins examined (C) , but not endogenous E1β in the mitochondrial matrix or the control protein, Myc-TOM40 (B) , were immunodetected in digitonin-permeabilized cells. Note also in (D) that Myc-TraB-HA did not colocalize with endogenous mitochondrial CoxII, indicating that the expressed protein, unlike Myc-TraB (Figure 1 ) is not properly targeted to mitochondria. Bar in (A,C) = 10 μm.

Techniques Used: Transformation Assay, Epifluorescence Microscopy, Immunofluorescence

Localization of various CTS mutant versions of mitochondrial-TA proteins in BY-2 cells . Shown on the left are schematic illustrations of wild-type and/or various CTS mutant versions of TraB, Cb5-6, or PMD2 and their corresponding localization (or lack thereof) to mitochondria in transformed BY-2 cells. The names of the mutants represent the specific amino acids in their modified CTSs. All constructs also possess an N-terminal-appended Myc-epitope tag. Shown for each construct is the corresponding C-terminal amino acid sequence, including putative TMD (underlined) and modified (or wild-type) CTS from TraB, Cb5-6 and PMD2; modified amino acid residues in the protein's CTS are bolded. Mitochondrial localization (indicated as “Yes” or “No”) was assessed based on colocalization (or lack thereof) of the expressed protein and endogenous mitochondrial CoxII. Shown on the right in both are representative immuno-epifluorescence micrographs illustrating the localization of the various constructs shown on the left . Each micrograph is labeled with the name of the expressed Myc-tagged wild-type and/or CTS mutant version of TraB, Cb5-6, or PMD2 or endogenous CoxII. Bar = 10 μm.
Figure Legend Snippet: Localization of various CTS mutant versions of mitochondrial-TA proteins in BY-2 cells . Shown on the left are schematic illustrations of wild-type and/or various CTS mutant versions of TraB, Cb5-6, or PMD2 and their corresponding localization (or lack thereof) to mitochondria in transformed BY-2 cells. The names of the mutants represent the specific amino acids in their modified CTSs. All constructs also possess an N-terminal-appended Myc-epitope tag. Shown for each construct is the corresponding C-terminal amino acid sequence, including putative TMD (underlined) and modified (or wild-type) CTS from TraB, Cb5-6 and PMD2; modified amino acid residues in the protein's CTS are bolded. Mitochondrial localization (indicated as “Yes” or “No”) was assessed based on colocalization (or lack thereof) of the expressed protein and endogenous mitochondrial CoxII. Shown on the right in both are representative immuno-epifluorescence micrographs illustrating the localization of the various constructs shown on the left . Each micrograph is labeled with the name of the expressed Myc-tagged wild-type and/or CTS mutant version of TraB, Cb5-6, or PMD2 or endogenous CoxII. Bar = 10 μm.

Techniques Used: Mutagenesis, Transformation Assay, Modification, Construct, Sequencing, Labeling

Localization of various C-terminal mutant and GFP fusions of selected mitochondrial-TA proteins in BY-2 cells . Shown on the left in both (A) and (B) are schematic illustrations of various C-terminal-mutant (i.e., truncated) versions or GFP fusions of various dibasic-motif-containing TA proteins (A) or TOM-TA proteins (B) and their corresponding intracellular localization in transformed BY-2 cells. The numbers in the name of each construct denote the number of residues that were either deleted from the C terminus of the Myc-tagged wild-type TA protein or fused to the C terminus of GFP, and the numbers above each illustration correspond to the N- and C-terminal amino acid residues of the TA protein. Portions of the TA protein are represented in the illustrations by white and black boxes, the latter denoting the putative TMD; green boxes denote GFP. Cyt, cytoplasm; DNE, did not express; ER, endoplasmic reticulum; mito, mitochondria. Shown on the right in both (A) and (B) are representative immuno-epifluorescence micrographs illustrating the localization of the various constructs shown on the left. Each micrograph is labeled with the name of either the transiently-expressed Myc-tagged C-terminal mutant or GFP fusion protein, the endogenous mitochondrial marker protein, CoxII, or ConA. Boxes in the top row of (A) represent the portions of cells shown at higher magnification in the panels to the right. Arrowheads indicate examples of the torus-shaped fluorescent structures containing GFP-TraB+C24 delineating the spherical structures attributable to matrix-localized CoxII, indicating that GFP-TraB+C24 localizes to the OMM. For all other expressed proteins, only general (i.e., lower magnification) fluorescence patterns were compared with those of mitochondrial CoxII or, in the case of GFP-TOM20-4+C37, ConA-stained ER. Note also that cells transformed with Myc-TOM9-1-C61, which did not display a detectable immunofluorescence signal, were identified based on the fluorescence attributable to co-expressed β-ATPase-GFP, serving as cell transformation and mitochondrial matrix marker protein. Bar in (A) = 10 μm.
Figure Legend Snippet: Localization of various C-terminal mutant and GFP fusions of selected mitochondrial-TA proteins in BY-2 cells . Shown on the left in both (A) and (B) are schematic illustrations of various C-terminal-mutant (i.e., truncated) versions or GFP fusions of various dibasic-motif-containing TA proteins (A) or TOM-TA proteins (B) and their corresponding intracellular localization in transformed BY-2 cells. The numbers in the name of each construct denote the number of residues that were either deleted from the C terminus of the Myc-tagged wild-type TA protein or fused to the C terminus of GFP, and the numbers above each illustration correspond to the N- and C-terminal amino acid residues of the TA protein. Portions of the TA protein are represented in the illustrations by white and black boxes, the latter denoting the putative TMD; green boxes denote GFP. Cyt, cytoplasm; DNE, did not express; ER, endoplasmic reticulum; mito, mitochondria. Shown on the right in both (A) and (B) are representative immuno-epifluorescence micrographs illustrating the localization of the various constructs shown on the left. Each micrograph is labeled with the name of either the transiently-expressed Myc-tagged C-terminal mutant or GFP fusion protein, the endogenous mitochondrial marker protein, CoxII, or ConA. Boxes in the top row of (A) represent the portions of cells shown at higher magnification in the panels to the right. Arrowheads indicate examples of the torus-shaped fluorescent structures containing GFP-TraB+C24 delineating the spherical structures attributable to matrix-localized CoxII, indicating that GFP-TraB+C24 localizes to the OMM. For all other expressed proteins, only general (i.e., lower magnification) fluorescence patterns were compared with those of mitochondrial CoxII or, in the case of GFP-TOM20-4+C37, ConA-stained ER. Note also that cells transformed with Myc-TOM9-1-C61, which did not display a detectable immunofluorescence signal, were identified based on the fluorescence attributable to co-expressed β-ATPase-GFP, serving as cell transformation and mitochondrial matrix marker protein. Bar in (A) = 10 μm.

Techniques Used: Mutagenesis, Transformation Assay, Construct, Labeling, Marker, Fluorescence, Staining, Immunofluorescence

Localization of various CTS mutants and hybrid versions of selected mitochondrial-TA proteins in BY-2 cells . Shown on the left in both (A) and (B) are schematic illustrations of various CTS mutant (truncated) or hybrid versions of selected dibasic-motif-containing TA proteins (A) and/or TOM-TA proteins (B) and their corresponding localization (or lack thereof) to mitochondria in transformed BY-2 cells. The names of the mutant and hybrid constructs represent either the specific amino acids in the CTS that were deleted from the protein or replaced with the CTS from another protein. All constructs possess an N-terminal-appended Myc-epitope tag. Shown for each construct is the corresponding C-terminal amino acid sequence, including putative TMD (underlined) and modified CTS (bolded), or lack thereof. Mitochondrial localization (indicated as “Yes” or “No”) was assessed based on colocalization (or lack thereof) of the expressed protein and the endogenous mitochondrial CoxII. Shown on the right in both (A) and (B) are representative immuno-epifluorescence micrographs illustrating the localization of the various constructs shown on the left. Each micrograph is labeled with the name of either the expressed Myc-tagged CTS mutant or hybrid protein or endogenous CoxII. Bar in (A) = 10 μm.
Figure Legend Snippet: Localization of various CTS mutants and hybrid versions of selected mitochondrial-TA proteins in BY-2 cells . Shown on the left in both (A) and (B) are schematic illustrations of various CTS mutant (truncated) or hybrid versions of selected dibasic-motif-containing TA proteins (A) and/or TOM-TA proteins (B) and their corresponding localization (or lack thereof) to mitochondria in transformed BY-2 cells. The names of the mutant and hybrid constructs represent either the specific amino acids in the CTS that were deleted from the protein or replaced with the CTS from another protein. All constructs possess an N-terminal-appended Myc-epitope tag. Shown for each construct is the corresponding C-terminal amino acid sequence, including putative TMD (underlined) and modified CTS (bolded), or lack thereof. Mitochondrial localization (indicated as “Yes” or “No”) was assessed based on colocalization (or lack thereof) of the expressed protein and the endogenous mitochondrial CoxII. Shown on the right in both (A) and (B) are representative immuno-epifluorescence micrographs illustrating the localization of the various constructs shown on the left. Each micrograph is labeled with the name of either the expressed Myc-tagged CTS mutant or hybrid protein or endogenous CoxII. Bar in (A) = 10 μm.

Techniques Used: Mutagenesis, Transformation Assay, Construct, Sequencing, Modification, Labeling

Localization of various CTS mutant versions of the mitochondrial-TA hybrid protein TraBΔCb5-6 CTS in BY-2 cells . Shown on the left are schematic illustrations of wild-type and various CTS mutant versions of the hybrid protein TraBΔCb5-6 CTS and their corresponding localization (or lack thereof) to mitochondria in transformed BY-2 cells. The names of the mutant constructs represent the specific amino acids in their CTSs. All constructs possess an N-terminal-appended Myc-epitope tag. Shown for each construct is the corresponding C-terminal amino acid sequence, including putative TraB TMD (underlined) and modified (or wild-type) Cb5-6 CTS; modified or additional amino acid residues inserted into the Cb5-6 CTS are bolded. Mitochondrial localization (indicated as “Yes” or “No”) was assessed based on colocalization (or lack thereof) of the expressed protein and endogenous mitochondrial CoxII. Shown on the right in both are representative immuno-epifluorescence micrographs illustrating the localization of the various constructs shown on the left . Each micrograph is labeled with the name of the expressed Myc-tagged TraBΔCb5-6 CTS or CTS mutant version of TraBΔCb5-6 CTS , or endogenous CoxII. Bar = 10 μm.
Figure Legend Snippet: Localization of various CTS mutant versions of the mitochondrial-TA hybrid protein TraBΔCb5-6 CTS in BY-2 cells . Shown on the left are schematic illustrations of wild-type and various CTS mutant versions of the hybrid protein TraBΔCb5-6 CTS and their corresponding localization (or lack thereof) to mitochondria in transformed BY-2 cells. The names of the mutant constructs represent the specific amino acids in their CTSs. All constructs possess an N-terminal-appended Myc-epitope tag. Shown for each construct is the corresponding C-terminal amino acid sequence, including putative TraB TMD (underlined) and modified (or wild-type) Cb5-6 CTS; modified or additional amino acid residues inserted into the Cb5-6 CTS are bolded. Mitochondrial localization (indicated as “Yes” or “No”) was assessed based on colocalization (or lack thereof) of the expressed protein and endogenous mitochondrial CoxII. Shown on the right in both are representative immuno-epifluorescence micrographs illustrating the localization of the various constructs shown on the left . Each micrograph is labeled with the name of the expressed Myc-tagged TraBΔCb5-6 CTS or CTS mutant version of TraBΔCb5-6 CTS , or endogenous CoxII. Bar = 10 μm.

Techniques Used: Mutagenesis, Transformation Assay, Construct, Sequencing, Modification, Labeling

38) Product Images from "TMV-Gate vectors: Gateway compatible tobacco mosaic virus based expression vectors for functional analysis of proteins"

Article Title: TMV-Gate vectors: Gateway compatible tobacco mosaic virus based expression vectors for functional analysis of proteins

Journal: Scientific Reports

doi: 10.1038/srep00874

Nuclear localizsation of NLS-mCherry expressed using TMV-Gate vectors. (A) Schematic diagram showing constructs used for analysis of subcellular localisation of NLS-mCherry. mCherry carrying a nuclear localization signal (NLS) was introduced unmodified into pMW388. A NLS-mCherry variant without translational stop codon was linked in-frame with the coding sequence of YFP-3xFLAG or CFP-3xHA in pMW390 and pMW391, respectively. Annotated features as per Figure 1 . (B) Epifluorescent microscopy images showing nuclear localisation of NLS-mCherry (upper panel), NLS-mCherry linked to YFP-3xFLAG (middle panel) or NLS-mCherry linked to CFP-3xHA in cells of N. benthamiana leaves infiltrated with A. tumefaciens carrying the constructs described in (A) and imaged 3 DPI. The nucleus was detected by staining cells with DAPI. Images were taken using YFP, CFP, mCherry or DAPI filter sets as indicated. Scale bar, 20 µM. DIC, differential interference contrast.
Figure Legend Snippet: Nuclear localizsation of NLS-mCherry expressed using TMV-Gate vectors. (A) Schematic diagram showing constructs used for analysis of subcellular localisation of NLS-mCherry. mCherry carrying a nuclear localization signal (NLS) was introduced unmodified into pMW388. A NLS-mCherry variant without translational stop codon was linked in-frame with the coding sequence of YFP-3xFLAG or CFP-3xHA in pMW390 and pMW391, respectively. Annotated features as per Figure 1 . (B) Epifluorescent microscopy images showing nuclear localisation of NLS-mCherry (upper panel), NLS-mCherry linked to YFP-3xFLAG (middle panel) or NLS-mCherry linked to CFP-3xHA in cells of N. benthamiana leaves infiltrated with A. tumefaciens carrying the constructs described in (A) and imaged 3 DPI. The nucleus was detected by staining cells with DAPI. Images were taken using YFP, CFP, mCherry or DAPI filter sets as indicated. Scale bar, 20 µM. DIC, differential interference contrast.

Techniques Used: Construct, Variant Assay, Sequencing, Microscopy, Staining

39) Product Images from "Structural Organization of DNA in Chlorella Viruses"

Article Title: Structural Organization of DNA in Chlorella Viruses

Journal: PLoS ONE

doi: 10.1371/journal.pone.0030133

SDS-PAGE pattern of proteins associated with PBCV-1 DNA. DNA was released from capsids by osmotic shock and separated from soluble proteins by centrifugation. The DNA-containing pellet was treated with DNase to release DNA-bound proteins. The framed bands were excised and used for MALDI TOF analysis. Lane 1: weight marker, lane 2: proteins obtained after DNAse treatment.
Figure Legend Snippet: SDS-PAGE pattern of proteins associated with PBCV-1 DNA. DNA was released from capsids by osmotic shock and separated from soluble proteins by centrifugation. The DNA-containing pellet was treated with DNase to release DNA-bound proteins. The framed bands were excised and used for MALDI TOF analysis. Lane 1: weight marker, lane 2: proteins obtained after DNAse treatment.

Techniques Used: SDS Page, Centrifugation, Marker

Ejection of viral DNA. A: Fluorescence images of C. variabilis with ejected DNA molecules. The incubation medium contained C. variabilis cells and virus PBCV-1 at an m.o.i. of ∼100 plus the fluorescent DNA stain DAPI. The image shows a chlorella cell (cc) and the viral DNA molecule, which is propelled away from the alga cell. B: Magnification of the area indicated by the box in A. Inset: same area as in B with conventional light microscopy and phase contrast. C: same as in A but with two DNA bands projecting away from a chlorella cell (cc). D: Magnification of area indicated by box in C with loop like DNA structure. E: Electron micrograph of viral DNA projecting away from host cell wall. The cell wall of the alga exhibits the typical hole (*), which the viruses digest for infection. From this hole two linear structures project towards the left side. The part marked in E is magnified in F and presented in artificial colors in order to highlight the linear structures projecting away from the cell wall hole. G: fluorescence intensity profile along DNA molecule between arrows in B . H: Histogram of distances between individual fluorescence maxima as in E from 30 ejected DNA molecules.
Figure Legend Snippet: Ejection of viral DNA. A: Fluorescence images of C. variabilis with ejected DNA molecules. The incubation medium contained C. variabilis cells and virus PBCV-1 at an m.o.i. of ∼100 plus the fluorescent DNA stain DAPI. The image shows a chlorella cell (cc) and the viral DNA molecule, which is propelled away from the alga cell. B: Magnification of the area indicated by the box in A. Inset: same area as in B with conventional light microscopy and phase contrast. C: same as in A but with two DNA bands projecting away from a chlorella cell (cc). D: Magnification of area indicated by box in C with loop like DNA structure. E: Electron micrograph of viral DNA projecting away from host cell wall. The cell wall of the alga exhibits the typical hole (*), which the viruses digest for infection. From this hole two linear structures project towards the left side. The part marked in E is magnified in F and presented in artificial colors in order to highlight the linear structures projecting away from the cell wall hole. G: fluorescence intensity profile along DNA molecule between arrows in B . H: Histogram of distances between individual fluorescence maxima as in E from 30 ejected DNA molecules.

Techniques Used: Fluorescence, Incubation, Staining, Light Microscopy, Infection

AFM images of viral DNA and associated proteins, which were isolated by osmotic shock. Scan of single PBCV-1 particles after osmotic shock in a height image A and in amplitude image B . The images reveal emerging DNA and protein particles from the disrupted virus. Magnification of DNA from disrupted virus with protein particles C . Proteins are absent after the sample was treated with proteinase K D . 3 dimensional image of individual BSA protein E and of individual purified 70 kDa PBCV-1 protein A278L. The latter is a putative DNA-binding protein coded by virus PBCV-1. Scale bars 100 nm in A–D and 2 nm in E and F.
Figure Legend Snippet: AFM images of viral DNA and associated proteins, which were isolated by osmotic shock. Scan of single PBCV-1 particles after osmotic shock in a height image A and in amplitude image B . The images reveal emerging DNA and protein particles from the disrupted virus. Magnification of DNA from disrupted virus with protein particles C . Proteins are absent after the sample was treated with proteinase K D . 3 dimensional image of individual BSA protein E and of individual purified 70 kDa PBCV-1 protein A278L. The latter is a putative DNA-binding protein coded by virus PBCV-1. Scale bars 100 nm in A–D and 2 nm in E and F.

Techniques Used: Isolation, Purification, Binding Assay

40) Product Images from "The AKT inhibitor triciribine in combination with paclitaxel has order-specific efficacy against Zfp217-induced breast cancer chemoresistance"

Article Title: The AKT inhibitor triciribine in combination with paclitaxel has order-specific efficacy against Zfp217-induced breast cancer chemoresistance

Journal: Oncotarget

doi: 10.18632/oncotarget.19308

Triciribine-paclitaxel (TCN→PAC) combination therapy increases survival and reduces tumor volume in immunocompetent mice with tumors that overexpress Zfp217 (A) Experimental overview. Vo-PyMT-Luc cells constitutively overexpressing vector or Zfp217 were orthotopically injected into recipient FVB mammary glands. Mice received weekly treatments of single or dual agent combination therapy. The dual agent regimen was administered with a delay of approximately 24 hours between delivery of the first and second drug. Tumor tissue was collected at death or at the terminal endpoint, when the tumor diameter reached 2.5 cm. (B) Kaplan-Meier curves of recipient FVB mice bearing orthotopically grown Vo-PyMT-Luc cells expressing vector received single or dual agent combination therapy or vehicle. Kaplan-Meier survival curves show no change in median survival between cohorts. (C) Kaplan-Meier curves of FVB mice bearing orthotopically grown Vo-PyMT-Luc cells that are constitutively expressing Zfp217 received single or dual agent combination therapy or vehicle. Kaplan-Meier survival curves show a significant decrease in median survival between single agent paclitaxel and dual agent triciribine-paclitaxel compared to vehicle (p=0.0003 and p=0.02 by log-rank test, respectively). (D) Tumor burden in vector expressing glands after treatment. Tumor volume was compared using linear regression analysis and the slopes of the lines were not significantly different between vehicle and chemotherapy treated vector expressing tumors. (E) Tumor burden in Zfp217 overexpressing glands after treatment. Tumor volume was compared using linear regression analysis and the slopes of the lines were significantly different between vehicle and triciribine-paclitaxel treated Zfp217 expressing tumors (p=0.001). (F) Quantification of K8 + K14 + cells. Bar graph showing results of number of K8 + K14 + cells per field for dual agent treated cohorts expressing vector or Zfp217. There was a significant decrease in the K8 + K14 + double positive cells for Zfp217 tumors treated with TCN-Pac compared to vector expressing tumors treated with Pac-TCN (p=0.03, by one-way ANOVA with Tukey’s multiple comparisons test).
Figure Legend Snippet: Triciribine-paclitaxel (TCN→PAC) combination therapy increases survival and reduces tumor volume in immunocompetent mice with tumors that overexpress Zfp217 (A) Experimental overview. Vo-PyMT-Luc cells constitutively overexpressing vector or Zfp217 were orthotopically injected into recipient FVB mammary glands. Mice received weekly treatments of single or dual agent combination therapy. The dual agent regimen was administered with a delay of approximately 24 hours between delivery of the first and second drug. Tumor tissue was collected at death or at the terminal endpoint, when the tumor diameter reached 2.5 cm. (B) Kaplan-Meier curves of recipient FVB mice bearing orthotopically grown Vo-PyMT-Luc cells expressing vector received single or dual agent combination therapy or vehicle. Kaplan-Meier survival curves show no change in median survival between cohorts. (C) Kaplan-Meier curves of FVB mice bearing orthotopically grown Vo-PyMT-Luc cells that are constitutively expressing Zfp217 received single or dual agent combination therapy or vehicle. Kaplan-Meier survival curves show a significant decrease in median survival between single agent paclitaxel and dual agent triciribine-paclitaxel compared to vehicle (p=0.0003 and p=0.02 by log-rank test, respectively). (D) Tumor burden in vector expressing glands after treatment. Tumor volume was compared using linear regression analysis and the slopes of the lines were not significantly different between vehicle and chemotherapy treated vector expressing tumors. (E) Tumor burden in Zfp217 overexpressing glands after treatment. Tumor volume was compared using linear regression analysis and the slopes of the lines were significantly different between vehicle and triciribine-paclitaxel treated Zfp217 expressing tumors (p=0.001). (F) Quantification of K8 + K14 + cells. Bar graph showing results of number of K8 + K14 + cells per field for dual agent treated cohorts expressing vector or Zfp217. There was a significant decrease in the K8 + K14 + double positive cells for Zfp217 tumors treated with TCN-Pac compared to vector expressing tumors treated with Pac-TCN (p=0.03, by one-way ANOVA with Tukey’s multiple comparisons test).

Techniques Used: Mouse Assay, Plasmid Preparation, Injection, Expressing

Increased expression of Zfp217 contributes to chemoresistance and an increase in a progenitor cell population (A) Experimental overview of orthotopic transplants and combination therapy in orthotopic mammary transplants of Vo-PyMT-Luc cells that constitutively overexpress vector or Zfp217. Mice received a single treatment of epothilone B, doxorubicin (Adriamycin), and cyclophosphamide combination therapy. Tumor tissue was collected at death or at the terminal endpoint when the tumor diameter reached 2.5 cm. (B) Kaplan-Meier survival curves of mice bearing orthotopic transplants of Vo-PyMT-Luc cells with constitutive overexpression of vector or Zfp217 after treatment with either EAC or vehicle. Kaplan-Meier survival curves show a significant decrease in median survival between vector and Zfp217 vehicle treated mice compared to mice that received EAC therapy (p=0.0003 and p=0.02 by log-rank test, respectively). The median survival significantly decreases after EAC therapy in mice overexpressing Zfp217 compared to vector (p=0.03 by log-rank test). (C) Tumor burden. Tumor volume was compared using linear regression analysis and the slopes of the lines were significantly different between vehicle and EAC treated vector and Zfp217 expressing tumors (p=0.0004 and p=0.0006, respectively). (D) K8 + K14 + cells. Tumor sections from FVB mice ± Zfp217 ± EAC were stained for keratin-8 (K8 + ) and keratin-14 (K14 + ) by immunofluorescence staining of tumor sections. Arrowheads point to examples of K8 + K14 + double positive cells. (E) Quantification of K8 + K14 + cells. Bar graph showing results of number of K8 + K14 + cells per field. There was a significant increase in the K8 + K14 + double positive cells for Zfp217 tumors treated with EAC compared to tumors expressing vector (p=0.01 by one-way ANOVA with Tukey’s multiple comparisons test). (F) Kaplan-Meier survival curves of mice bearing orthotopic transplants of Vo-PyMT-Luc cells with constitutive overexpression of vector or Zfp217 after treatment with EAC. Kaplan-Meier survival curves show a significant decrease in median survival between in Zfp217 + EAC treated mice compared to vector + EAC therapy (p=0.04 by log-rank test). The median survival significantly decreases after EAC therapy in mice overexpressing Zfp217 (26 days, median survival) compared to vector (35 days, median survival).
Figure Legend Snippet: Increased expression of Zfp217 contributes to chemoresistance and an increase in a progenitor cell population (A) Experimental overview of orthotopic transplants and combination therapy in orthotopic mammary transplants of Vo-PyMT-Luc cells that constitutively overexpress vector or Zfp217. Mice received a single treatment of epothilone B, doxorubicin (Adriamycin), and cyclophosphamide combination therapy. Tumor tissue was collected at death or at the terminal endpoint when the tumor diameter reached 2.5 cm. (B) Kaplan-Meier survival curves of mice bearing orthotopic transplants of Vo-PyMT-Luc cells with constitutive overexpression of vector or Zfp217 after treatment with either EAC or vehicle. Kaplan-Meier survival curves show a significant decrease in median survival between vector and Zfp217 vehicle treated mice compared to mice that received EAC therapy (p=0.0003 and p=0.02 by log-rank test, respectively). The median survival significantly decreases after EAC therapy in mice overexpressing Zfp217 compared to vector (p=0.03 by log-rank test). (C) Tumor burden. Tumor volume was compared using linear regression analysis and the slopes of the lines were significantly different between vehicle and EAC treated vector and Zfp217 expressing tumors (p=0.0004 and p=0.0006, respectively). (D) K8 + K14 + cells. Tumor sections from FVB mice ± Zfp217 ± EAC were stained for keratin-8 (K8 + ) and keratin-14 (K14 + ) by immunofluorescence staining of tumor sections. Arrowheads point to examples of K8 + K14 + double positive cells. (E) Quantification of K8 + K14 + cells. Bar graph showing results of number of K8 + K14 + cells per field. There was a significant increase in the K8 + K14 + double positive cells for Zfp217 tumors treated with EAC compared to tumors expressing vector (p=0.01 by one-way ANOVA with Tukey’s multiple comparisons test). (F) Kaplan-Meier survival curves of mice bearing orthotopic transplants of Vo-PyMT-Luc cells with constitutive overexpression of vector or Zfp217 after treatment with EAC. Kaplan-Meier survival curves show a significant decrease in median survival between in Zfp217 + EAC treated mice compared to vector + EAC therapy (p=0.04 by log-rank test). The median survival significantly decreases after EAC therapy in mice overexpressing Zfp217 (26 days, median survival) compared to vector (35 days, median survival).

Techniques Used: Expressing, Plasmid Preparation, Mouse Assay, Over Expression, Staining, Immunofluorescence

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Article Title: A mechanosensitive ion channel regulating cell volume
Article Snippet: .. The microfluidic chamber was also used for fluorescence measurements using a Zeiss upright microscope (Axio Imager M1, Zeiss), and the data were recorded using a charge-coupled device camera (Axiocam MRm, Zeiss). .. We used the filter set 38 HE EGFP (excitation: 470 ± 40 nm; dichroic filter: 495 nm; emission: 525 ± 50 nm, Zeiss).

Article Title: Intrinsic muscle clock is necessary for musculoskeletal health
Article Snippet: .. Images were captured with a Zeiss upright microscope (Axio Imager.M1, Zeiss, Dublin, CA, USA). .. Cross sectional area and centrally nucleated fibres were determined using the H & E stained sections as previously described (McCarthy et al .).

Article Title: Impression Cytology Is a Non-invasive and Effective Method for Ocular Cell Retrieval of Zika Infected Babies: Perspectives in OMIC Studies
Article Snippet: .. Images were acquired on Axio microscope using 20x/0.35 and 40x/0.55 Zeiss A-Plan objectives (Carl Zeiss, Jena, Germany, see text footnote 1) and Q-Capture PRO 7 software (Surrey, BC, Canada, see text footnote 2). .. Another cytocentrifuged preparation was also stained for morphological analyses.

Article Title: Impression Cytology Is a Non-invasive and Effective Method for Ocular Cell Retrieval of Zika Infected Babies: Perspectives in OMIC Studies
Article Snippet: .. Images were acquired using on Axio microscope with 20x/0.35 and 40x/0.55 Zeiss A-Plan objectives (Carl Zeiss, Jena, Germany ) and Q-Capture PRO 7 software (Surrey, BC, Canada ). .. Additionally, the cytospin procedure was used to concentrate cells in suspension on a microscope slide.

Article Title: Composition of Bacterial Communities Associated with Aurelia aurita Changes with Compartment, Life Stage, and Population
Article Snippet: .. Light microscopy and fluorescence microscopy were performed with an Axio Scope microscope and Axio Vision software (Zeiss, Jena, Germany). .. The entire three-dimensional structure of the polyp and its associated microbiota was recorded by scanning along the depth by using a TCS SP confocal laser scanning microscope (Leica, Wetzlar, Germany) and recording the stacks of cross sections simultaneously at the corresponding excitation wavelengths.

Light Microscopy:

Article Title: Composition of Bacterial Communities Associated with Aurelia aurita Changes with Compartment, Life Stage, and Population
Article Snippet: .. Light microscopy and fluorescence microscopy were performed with an Axio Scope microscope and Axio Vision software (Zeiss, Jena, Germany). .. The entire three-dimensional structure of the polyp and its associated microbiota was recorded by scanning along the depth by using a TCS SP confocal laser scanning microscope (Leica, Wetzlar, Germany) and recording the stacks of cross sections simultaneously at the corresponding excitation wavelengths.

other:

Article Title: Effect of lithium on behavioral disinhibition induced by electrolytic lesion of the median raphe nucleus
Article Snippet: The slides were photographed with an optical microscope (Scope.A1 andAxioCam Mrc, Carl Zeiss, LLC) and images analyzed with a computer.

Confocal Microscopy:

Article Title: Copper Transport Protein Antioxidant-1 Promotes Inflammatory Neovascularization via Chaperone and Transcription Factor Function
Article Snippet: .. Images were captured by Axio scope microscope or confocal microscopy and processed by AxioVision 4.8 or Zeiss LSM510 software, respectively. .. To quantify proliferating cells, mice were injected intraperitoneally with the thymidine analog 5-bromo-2′-deoxyuridine (BrdU; 40 mg/kg body weight, 500 μl, SigmaAldrich) at 12 hours and 1 hour before sacrifice asdescribed previously .

Software:

Article Title: Copper Transport Protein Antioxidant-1 Promotes Inflammatory Neovascularization via Chaperone and Transcription Factor Function
Article Snippet: .. Images were captured by Axio scope microscope or confocal microscopy and processed by AxioVision 4.8 or Zeiss LSM510 software, respectively. .. To quantify proliferating cells, mice were injected intraperitoneally with the thymidine analog 5-bromo-2′-deoxyuridine (BrdU; 40 mg/kg body weight, 500 μl, SigmaAldrich) at 12 hours and 1 hour before sacrifice asdescribed previously .

Article Title: Impression Cytology Is a Non-invasive and Effective Method for Ocular Cell Retrieval of Zika Infected Babies: Perspectives in OMIC Studies
Article Snippet: .. Images were acquired on Axio microscope using 20x/0.35 and 40x/0.55 Zeiss A-Plan objectives (Carl Zeiss, Jena, Germany, see text footnote 1) and Q-Capture PRO 7 software (Surrey, BC, Canada, see text footnote 2). .. Another cytocentrifuged preparation was also stained for morphological analyses.

Article Title: Impression Cytology Is a Non-invasive and Effective Method for Ocular Cell Retrieval of Zika Infected Babies: Perspectives in OMIC Studies
Article Snippet: .. Images were acquired using on Axio microscope with 20x/0.35 and 40x/0.55 Zeiss A-Plan objectives (Carl Zeiss, Jena, Germany ) and Q-Capture PRO 7 software (Surrey, BC, Canada ). .. Additionally, the cytospin procedure was used to concentrate cells in suspension on a microscope slide.

Article Title: Composition of Bacterial Communities Associated with Aurelia aurita Changes with Compartment, Life Stage, and Population
Article Snippet: .. Light microscopy and fluorescence microscopy were performed with an Axio Scope microscope and Axio Vision software (Zeiss, Jena, Germany). .. The entire three-dimensional structure of the polyp and its associated microbiota was recorded by scanning along the depth by using a TCS SP confocal laser scanning microscope (Leica, Wetzlar, Germany) and recording the stacks of cross sections simultaneously at the corresponding excitation wavelengths.

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    Carl Zeiss epifluorescence microscope images
    Identification of darts structures by DIC microscopy. Live samples of Arabidopsis line CS84739 were imaged by <t>epifluorescence</t> and DIC microscopy. ( a ) In most cells, DIC imaging cannot distinguish darts (highlighted by green fluorescence) from other organelles,
    Epifluorescence Microscope Images, supplied by Carl Zeiss, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/epifluorescence microscope images/product/Carl Zeiss
    Average 86 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
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    88
    Carl Zeiss epifluorescence microscopy images
    Effect of meconium on the structure of surfactant films. ( A ) <t>Epifluorescence</t> microscopy images from interfacial films of native surfactant containing a trace of the fluorescent probe BODIPY-PC, in the absence ( left pictures ) or in the presence ( right
    Epifluorescence Microscopy Images, supplied by Carl Zeiss, used in various techniques. Bioz Stars score: 88/100, based on 20 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/epifluorescence microscopy images/product/Carl Zeiss
    Average 88 stars, based on 20 article reviews
    Price from $9.99 to $1999.99
    epifluorescence microscopy images - by Bioz Stars, 2021-01
    88/100 stars
      Buy from Supplier

    Image Search Results


    Identification of darts structures by DIC microscopy. Live samples of Arabidopsis line CS84739 were imaged by epifluorescence and DIC microscopy. ( a ) In most cells, DIC imaging cannot distinguish darts (highlighted by green fluorescence) from other organelles,

    Journal: Acta histochemica

    Article Title: Overexpressed Arabidopsis Annexin4 accumulates in inclusion bodylike structures

    doi: 10.1016/j.acthis.2015.03.005

    Figure Lengend Snippet: Identification of darts structures by DIC microscopy. Live samples of Arabidopsis line CS84739 were imaged by epifluorescence and DIC microscopy. ( a ) In most cells, DIC imaging cannot distinguish darts (highlighted by green fluorescence) from other organelles,

    Article Snippet: Differential interference contrast and epifluorescence microscope images were collected on an Axioimager.M2 microscope (Zeiss) using the following objective lenses: 10x Plan Neofluoar (N.A.=0.3), 40x Plan Apochromat (N.A.=0.95), and 100x Plan Apochromat (N.A.=1.4).

    Techniques: Microscopy, Imaging, Fluorescence

    Dart-shaped structures in Arabidopsis line CS84739. Epifluorescence and Differential Interference Contrast (DIC) microscope images were collected from live samples. ( a–c ) GFP fluorescence is pseudocolored green and overlain on black and white

    Journal: Acta histochemica

    Article Title: Overexpressed Arabidopsis Annexin4 accumulates in inclusion bodylike structures

    doi: 10.1016/j.acthis.2015.03.005

    Figure Lengend Snippet: Dart-shaped structures in Arabidopsis line CS84739. Epifluorescence and Differential Interference Contrast (DIC) microscope images were collected from live samples. ( a–c ) GFP fluorescence is pseudocolored green and overlain on black and white

    Article Snippet: Differential interference contrast and epifluorescence microscope images were collected on an Axioimager.M2 microscope (Zeiss) using the following objective lenses: 10x Plan Neofluoar (N.A.=0.3), 40x Plan Apochromat (N.A.=0.95), and 100x Plan Apochromat (N.A.=1.4).

    Techniques: Microscopy, Fluorescence

    AnnAt4-mCherry fusion protein accumulates in dart-shaped structures. Epifluorescence microscope images were collected from live samples of Arabidopsis and Tobacco after agroinfiltration with an AnnAt4-mCherry construct. ( a–c ) Two cells from transgenic

    Journal: Acta histochemica

    Article Title: Overexpressed Arabidopsis Annexin4 accumulates in inclusion bodylike structures

    doi: 10.1016/j.acthis.2015.03.005

    Figure Lengend Snippet: AnnAt4-mCherry fusion protein accumulates in dart-shaped structures. Epifluorescence microscope images were collected from live samples of Arabidopsis and Tobacco after agroinfiltration with an AnnAt4-mCherry construct. ( a–c ) Two cells from transgenic

    Article Snippet: Differential interference contrast and epifluorescence microscope images were collected on an Axioimager.M2 microscope (Zeiss) using the following objective lenses: 10x Plan Neofluoar (N.A.=0.3), 40x Plan Apochromat (N.A.=0.95), and 100x Plan Apochromat (N.A.=1.4).

    Techniques: Microscopy, Construct, Transgenic Assay

    Darts are not a part of the endomembrane system or peroxisomes. Epifluorescence images were collected from line CS84739 after agroinfiltration with constructs for ( a–c ) ER-targeted mCherry; ( d–f ) Golgi-targeted mCherry; and ( g–i

    Journal: Acta histochemica

    Article Title: Overexpressed Arabidopsis Annexin4 accumulates in inclusion bodylike structures

    doi: 10.1016/j.acthis.2015.03.005

    Figure Lengend Snippet: Darts are not a part of the endomembrane system or peroxisomes. Epifluorescence images were collected from line CS84739 after agroinfiltration with constructs for ( a–c ) ER-targeted mCherry; ( d–f ) Golgi-targeted mCherry; and ( g–i

    Article Snippet: Differential interference contrast and epifluorescence microscope images were collected on an Axioimager.M2 microscope (Zeiss) using the following objective lenses: 10x Plan Neofluoar (N.A.=0.3), 40x Plan Apochromat (N.A.=0.95), and 100x Plan Apochromat (N.A.=1.4).

    Techniques: Construct

    Effect of meconium on the structure of surfactant films. ( A ) Epifluorescence microscopy images from interfacial films of native surfactant containing a trace of the fluorescent probe BODIPY-PC, in the absence ( left pictures ) or in the presence ( right

    Journal: Biophysical Journal

    Article Title: Meconium Impairs Pulmonary Surfactant by a Combined Action of Cholesterol and Bile Acids

    doi: 10.1016/j.bpj.2010.12.3715

    Figure Lengend Snippet: Effect of meconium on the structure of surfactant films. ( A ) Epifluorescence microscopy images from interfacial films of native surfactant containing a trace of the fluorescent probe BODIPY-PC, in the absence ( left pictures ) or in the presence ( right

    Article Snippet: Epifluorescence microscopy images were obtained in an Axioplan II microscope (Zeiss, Dublin, CA) and analyzed with Image J software.

    Techniques: Epifluorescence Microscopy