Journal: Stem cell research

Article Title: Adaptation of the AID system for stem cell and transgenic mouse research.

doi: 10.1016/j.scr.2020.102078

Figure Lengend Snippet: Fig. 2. Evaluation of TIR1 transgene expression in mice. (a) An outline of B6V1-TIR1 and 129V2-TIR1 transgenic mice analysis. (b) Western blot analysis of TIR1 expression in mESCs derived from B6V1-TIR1 and 129V2-TIR1 transgenic mice with or without the floxed STOP cassette, which was excised in vivo. (c) RT-PCR analysis of TIR1 expression in different tissues from wild type (WT) and B6V1-TIR1 transgenic mice: primary cultures of lung cells, tail tip fibroblasts (TTF) and cortex tissue. Mice were analyzed at postnatal day 2 through day 10. Two pairs of primers (TIR1-1 and TIR1-2) were used to detect RNA expression of TIR1. Gapdh was used as an internal control. Reverse transcriptase-negative reactions are provided for each sample. B6V1-TIR1 mESCs were used as a positive control for TIR1 expression (d) PCR genotyping of 12.5 dpc wild type (WT) and 129V2-TIR1 embryonic cortices showing in vivo excision of the floxed STOP cassette, mediated by Emx1-Cre. Two WT and two 129V2-TIR1 mice from breeding with Emx1-Cre mice were used for analysis. Arrows show 420 bp internal PCR control (Cpxm) band (Control), 281 bp Cre transgene (Cre), 1652 bp fragment of TIR1 transgene with STOP cassette (No excision) and 269 bp fragment of TIR1 transgene with excised STOP cassette (Excision). (e) RT-PCR analysis of TIR1 expression in 12.5dpc wild type (WT) and 129V2-TIR1 embryonic cortices from (d). Full-length blot and gels are presented in Fig. S10.

Article Snippet: To make TIR1 vector for conditional expression, this vector was cut at SgrDI restriction site between EF1α promoter and TIR1 sequence, and PCR-amplified floxed STOP cassette (Addgene, #85006) was incorporated using In Fusion HD cloning kit (Clontech).

Techniques: Expressing, Transgenic Assay, Western Blot, Derivative Assay, In Vivo, Reverse Transcription Polymerase Chain Reaction, RNA Expression, Control, Reverse Transcription, Positive Control

Journal: Free radical biology & medicine

Article Title: Quantification of reactive oxygen species production by the red fluorescent proteins KillerRed, SuperNova and mCherry.

doi: 10.1016/j.freeradbiomed.2019.12.008

Figure Lengend Snippet: (a) Absorbance spectrum of the photosensitizers Rose Bengal dye (0.0026 mg/mL), mCherry (0.22 mg/mL), tdKillerRed (0.25 mg/mL), and SuperNova (0.76 mg/mL). See Table 1 for quantification.

Article Snippet: SuperNova/pRSETB was a gift from Dr. Takeharu Nagai (Addgene plasmid # 53234) [ 8 ]. mCherry (pmCherry-C1) and tdKillerRed (#FP963, Evrogen) were amplified and ligated into pRSETB using BamHI and EcoRI.

Techniques:

Journal: Free radical biology & medicine

Article Title: Quantification of reactive oxygen species production by the red fluorescent proteins KillerRed, SuperNova and mCherry.

doi: 10.1016/j.freeradbiomed.2019.12.008

Figure Lengend Snippet: Photosensitizer absorbance at 561 nm.

Article Snippet: SuperNova/pRSETB was a gift from Dr. Takeharu Nagai (Addgene plasmid # 53234) [ 8 ]. mCherry (pmCherry-C1) and tdKillerRed (#FP963, Evrogen) were amplified and ligated into pRSETB using BamHI and EcoRI.

Techniques:

Journal: Free radical biology & medicine

Article Title: Quantification of reactive oxygen species production by the red fluorescent proteins KillerRed, SuperNova and mCherry.

doi: 10.1016/j.freeradbiomed.2019.12.008

Figure Lengend Snippet: (a) Rose Bengal, (b) tdKillerRed, Supernova, mCherry and control (no photosensitizer) were irradiated with equal molar absorptivity at 561 nm in the presence of DHE (100 μM) for quantification of 2-OHE+. Values are mean ± SD for n = 3 independent experiments.

Article Snippet: SuperNova/pRSETB was a gift from Dr. Takeharu Nagai (Addgene plasmid # 53234) [ 8 ]. mCherry (pmCherry-C1) and tdKillerRed (#FP963, Evrogen) were amplified and ligated into pRSETB using BamHI and EcoRI.

Techniques: Irradiation

Journal: Free radical biology & medicine

Article Title: Quantification of reactive oxygen species production by the red fluorescent proteins KillerRed, SuperNova and mCherry.

doi: 10.1016/j.freeradbiomed.2019.12.008

Figure Lengend Snippet: Superoxide and singlet oxygen quantum yield of mCherry, tdKillerRed, and SuperNova.

Article Snippet: SuperNova/pRSETB was a gift from Dr. Takeharu Nagai (Addgene plasmid # 53234) [ 8 ]. mCherry (pmCherry-C1) and tdKillerRed (#FP963, Evrogen) were amplified and ligated into pRSETB using BamHI and EcoRI.

Techniques:

Journal: Free radical biology & medicine

Article Title: Quantification of reactive oxygen species production by the red fluorescent proteins KillerRed, SuperNova and mCherry.

doi: 10.1016/j.freeradbiomed.2019.12.008

Figure Lengend Snippet: (a) xanthine/xanthine oxidase (X/XO) O2•- production was assessed using cytochrome c reduction assay. Xanthine oxidase (XO, 4 mU/mL) and xanthine (X, 1 mM) were incubated with catalase (CAT) superoxide dismutase (SOD) where indicated. * p < 0.05 X/XO+SOD vs X/XO and vs X/XO+CAT, one-way ANOVA, Tukey post hoc. (b) Time course (0–60 min) of X/XO O2•- generation was measured using HPLC separation of 2-OHE+ and then plotted against the expected O2•- production. (c) Rose Bengal O2•- production rate per light dose. (d) Fluorescent protein (mCherry, tdKillerRed and SuperNova) O2•- production per light dose. Data from (c) and (d) are derived from data presented in Fig. 2. *p < 0.05 SuperNova vs mCherry, ** p < 0.05 SuperNova and tdKillerRed vs mCherry, two-way ANOVA, Bonferroni post hoc. Values are mean ± SD for n = 3 independent experiments.

Article Snippet: SuperNova/pRSETB was a gift from Dr. Takeharu Nagai (Addgene plasmid # 53234) [ 8 ]. mCherry (pmCherry-C1) and tdKillerRed (#FP963, Evrogen) were amplified and ligated into pRSETB using BamHI and EcoRI.

Techniques: Incubation, Derivative Assay

Journal: Free radical biology & medicine

Article Title: Quantification of reactive oxygen species production by the red fluorescent proteins KillerRed, SuperNova and mCherry.

doi: 10.1016/j.freeradbiomed.2019.12.008

Figure Lengend Snippet: (a) Rose Bengal, (b) tdKillerRed, Supernova, mCherry and control (no photosensitizer) were irradiated with equal molar absorptivity at 561 nm in the presence of 0.1 μM SOSG. The initial fluorescence reading (Ex 525 nm; Em 550 nm) was subtracted from the post-illumination reading and presented as the relative fluorescence change. Values are mean ± SD for n = 3 independent experiments.

Article Snippet: SuperNova/pRSETB was a gift from Dr. Takeharu Nagai (Addgene plasmid # 53234) [ 8 ]. mCherry (pmCherry-C1) and tdKillerRed (#FP963, Evrogen) were amplified and ligated into pRSETB using BamHI and EcoRI.

Techniques: Irradiation, Fluorescence

Journal: Free radical biology & medicine

Article Title: Quantification of reactive oxygen species production by the red fluorescent proteins KillerRed, SuperNova and mCherry.

doi: 10.1016/j.freeradbiomed.2019.12.008

Figure Lengend Snippet: Superoxide and singlet oxygen quantum yield of fluorescent proteins relative to tdKillerRed.

Article Snippet: SuperNova/pRSETB was a gift from Dr. Takeharu Nagai (Addgene plasmid # 53234) [ 8 ]. mCherry (pmCherry-C1) and tdKillerRed (#FP963, Evrogen) were amplified and ligated into pRSETB using BamHI and EcoRI.

Techniques:

Journal: bioRxiv

Article Title: mTOR signaling governs the formation of epithelial apical projection via S6K1-RhoA and aPKC-Lgl2 axes

doi: 10.1101/2024.12.08.627438

Figure Lengend Snippet: (A) qPCR analysis of relative fold change of prkci gene (normalized to eef1a1 ) upon rapamycin treatment for 24 hours starting from 24 hpf. (B) aPKC localisation in the peridermal cells of the embryos at 48 hpf treated with rapamycin or DMSO for 24 hours and quantification (C) of aPKC intensity under these two conditions. mRNA expression profiles of prkci (D) and prkcz (E) in zebrafish embryonic epidermis as per the previous report (Cokus et al., 2019). (F) Alphafold2 model of wildtype aPKC structure highlighting the juxtaposition of Thr549 of TOR Interacting Motif (TIM) and Thr556 of turn motif. Western blot analysis (G) and its quantification (H) for pan phospho Thr level of the mentioned aPKC variants immunoprecipitated using HA antibody. All the experiments were performed three times independently. For data presented in (B) 60-61 cells were analyzed from 18 embryos. Asterisks denote significant differences when analyzed using the student’s t-test in (A, H) or the Mann-Whitney U test for (C). ns (non significant) p>0.05, * p<0.05, ** p<0.01, *** p<0.001. The error bars in (H) denotes SEM. The scale bar corresponds to 10μm.

Article Snippet: N-terminal HA-tagged zebrafish aPKC wildtype CDS (NM 131855) synthesis in pCS2+8 vector (a gift from Amro Hamdoun; Addgene plasmid # 34931), and aPKC T556E and aPKC T556A site-directed mutagenesis (SDM) were done by GeneArt, Life Technologies. aPKC T549E, aPKC T549A, aPKC A122E, and the combined mutations were achieved by SDM with the primers mentioned in supplementary information 1, using Q5 Polymerase (Neb) according to the protocol published earlier ( ). pcDNA3-EGFP-RhoA-Q63L (Addgene plasmid # 12968) was a gift from Gary Bokoch.

Techniques: Expressing, Western Blot, Immunoprecipitation, MANN-WHITNEY

Journal: bioRxiv

Article Title: mTOR signaling governs the formation of epithelial apical projection via S6K1-RhoA and aPKC-Lgl2 axes

doi: 10.1101/2024.12.08.627438

Figure Lengend Snippet: (A) Sequence homology alignment of TIM and turn motif of Human, Zebrafish, Drosophila and C.elegans PKCs including both conventional and atypical PKCs. Highlighted Threonine residues are potential mTORC2 phosphorylation sites in zebrafish and are relevant for this study. Western blot analysis (B) and quantification (C), using anti phospho aPKC Thr 555 antibody (Thr 556 in zebrafish), of the phosphorylation level at Thr 556 of aPKC in zebrafish embryos treated with DMSO and rapamycin for 48 hrs. Note that the levels of Phospho-aPKC and aPKC were normalized to their corresponding loading control (β-Actin). (D) Representative images of peridermal cells of rhodamine phalloidin stained has/apkc ( prkci -/- ) embryos at 48 hpf (D, G, J, M, and P) and distribution of microridge lengths (E, H, K, N, and Q) and mean microridge lengths (F, I, L, O, and R) from these embryos injected with plasmids containing HA-tagged aPKC wild type (D-F), aPKC (T556E) (G-I), aPKC (T556A) (J-L), aPKC (T549E) (M-O) and aPKC (T549A) (P-R). Note that embryos were immunostained with aPKC antibody (Grey in D, G, J, M, and P) for identifying clones. Experiments represented in (D-F), (G-I), and (P-R) were performed four times and the rest were performed three times, independently. For data represented in (E, F) n = 32 cells from N = 10 embryos, (H, I) n = 39 from N =15, (K, L) n = 31 from N = 7, (N, O) n = 41 from N = 9, and (Q, R) n = 48 from N = 10, were analyzed. Asterisks represent the significant difference by Mann Whitney U test for (F, I, L, O, and R) and student’s t-test for (C). Error bars in (C) denote SEM. ns (non significant) p>0.05, * p<0.05, *** p<0.001. The scale bar corresponds to 10μm.

Article Snippet: N-terminal HA-tagged zebrafish aPKC wildtype CDS (NM 131855) synthesis in pCS2+8 vector (a gift from Amro Hamdoun; Addgene plasmid # 34931), and aPKC T556E and aPKC T556A site-directed mutagenesis (SDM) were done by GeneArt, Life Technologies. aPKC T549E, aPKC T549A, aPKC A122E, and the combined mutations were achieved by SDM with the primers mentioned in supplementary information 1, using Q5 Polymerase (Neb) according to the protocol published earlier ( ). pcDNA3-EGFP-RhoA-Q63L (Addgene plasmid # 12968) was a gift from Gary Bokoch.

Techniques: Sequencing, Western Blot, Control, Staining, Injection, Clone Assay, MANN-WHITNEY

Journal: bioRxiv

Article Title: mTOR signaling governs the formation of epithelial apical projection via S6K1-RhoA and aPKC-Lgl2 axes

doi: 10.1101/2024.12.08.627438

Figure Lengend Snippet: Confocal images of peridermal cells from aPKC and rhodamine-phalloidin stained has/apkc ( prkci -/- ) embryos at 48 hpf (A, D, and I), and graphical representation of the distribution of microridge lengths (B, E, and J), and mean microridge lengths (C, F, and K) from these embryos injected with HA tagged aPKC (T549E/556E) (A-C), aPKC (T549A/556A) (D-F) or aPKC (T549A/556E) (I-K) plasmids. (G) Western blot shows changes in the level of pLgl2 (Ser 645/649) upon co-expressing the Lgl2 plasmid with different variants (as denoted) of aPKC in HEK293T cells and pulling down using GFP antibody. The relative difference in the pLgl2 level is quantified and represented in (H). Experiments represented in (I, K) were performed four times; the rest were performed three times independently. For data presented in (B, C) n = 50 cells from N = 15 embryos, (E, F) n = 61 from N =13, (J, K) n = 54 from N = 16 were analyzed. Asterisks denote significant differences when analyzed using the Mann-Whitney U test in (C, F, and K) and using the student’s t-test in (H). The error bars in (H) denote SEM. ns (non significant) p>0.05, * p<0.05. The scale bar corresponds to 10μm.

Article Snippet: N-terminal HA-tagged zebrafish aPKC wildtype CDS (NM 131855) synthesis in pCS2+8 vector (a gift from Amro Hamdoun; Addgene plasmid # 34931), and aPKC T556E and aPKC T556A site-directed mutagenesis (SDM) were done by GeneArt, Life Technologies. aPKC T549E, aPKC T549A, aPKC A122E, and the combined mutations were achieved by SDM with the primers mentioned in supplementary information 1, using Q5 Polymerase (Neb) according to the protocol published earlier ( ). pcDNA3-EGFP-RhoA-Q63L (Addgene plasmid # 12968) was a gift from Gary Bokoch.

Techniques: Staining, Injection, Western Blot, Expressing, Plasmid Preparation, MANN-WHITNEY

Journal: bioRxiv

Article Title: mTOR signaling governs the formation of epithelial apical projection via S6K1-RhoA and aPKC-Lgl2 axes

doi: 10.1101/2024.12.08.627438

Figure Lengend Snippet: Representative images of peridermal cells from aPKC and rhodamine phalloidin stained has/apkc ( prkci -/- ) embryos at 48hpf (A, D, G, J, and M) and violin plots of microridge length distributions (B, E, H, K, and N) and box-jitter plots of mean microridge lengths (C, F, I, L, and O) for these embryos injected with HA-tagged constitutively active aPKC (A122E) (A-C), aPKC (A122E/ T556A) (D-F), aPKC (A122E / T549E) (G-I), aPKC (A122E/T549E/556E) (J-L) and aPKC (A122E/T549A/556A) (M-O) plasmids. All the experiments were performed three times independently. For data presented in (B, C) n = 38 cells from N = 10 embryos, (E, F) n = 59 from N = 16, (H, I) n = 59 from N = 16, (K, L) n = 31 from N = 10, and (N, O) n = 35 from N = 10 were analyzed. Asterisks denote the significant difference when the data were analyzed using the Mann-Whitney U test for all the box-jitter plots. ns (non significant) p>0.05, ** p<0.01, **** p<0.0001. The scale bar corresponds to 10μm.

Article Snippet: N-terminal HA-tagged zebrafish aPKC wildtype CDS (NM 131855) synthesis in pCS2+8 vector (a gift from Amro Hamdoun; Addgene plasmid # 34931), and aPKC T556E and aPKC T556A site-directed mutagenesis (SDM) were done by GeneArt, Life Technologies. aPKC T549E, aPKC T549A, aPKC A122E, and the combined mutations were achieved by SDM with the primers mentioned in supplementary information 1, using Q5 Polymerase (Neb) according to the protocol published earlier ( ). pcDNA3-EGFP-RhoA-Q63L (Addgene plasmid # 12968) was a gift from Gary Bokoch.

Techniques: Staining, Injection, MANN-WHITNEY

Journal: bioRxiv

Article Title: mTOR signaling governs the formation of epithelial apical projection via S6K1-RhoA and aPKC-Lgl2 axes

doi: 10.1101/2024.12.08.627438

Figure Lengend Snippet: Line plot for effective strain (Si) calculated per residue between (A) aPKC Wt vs aPKC T549E (Magenta) and aPKC Wt vs aPKC T549A (Green), (B) aPKC Wt vs aPKC T556E (Magenta) and aPKC Wt vs aPKC T556A (Green), and (C) aPKC Wt vs aPKC T549E/556E (Magenta) and aPKC Wt vs aPKC T549A/556A (Green). Note that Si was calculated from five top AF2 predictions of the 3D model compared against each other. The average trend line (darker) with min and max Si (lighter shade) is represented in the plot. For better visualization, only residues from 530-580 are shown. (D) Visual representation of Si per residue of aPKC T549E/556E. The heat map denotes an index of low to high effective strain. The Average RMSD (E) and CVCF (F) of all backbone atoms calculated after simulating phosphorylation at either T556 (purple) or both at T556 and T549 (magenta) of aPKC. The shaded region denotes standard deviation across all the trajactories generated from MD simulation. (G) Visual comparison of distances between pT556.Cɑ of turn motif and R254.NH1 of Glycine-rich motif of simulated aPKC pT556 (purple) and aPKC pT556/pT549 (magenta). (H) Box-jitter plots (i)-(v) comparing the average distances between the residues mentioned in among aPKC pT556 and aPKC pT556/pT549 obtained from 10 iterations of MD simulation. Asterisks denote the significant difference by the student’s t-test for all box-jitter plots. ns (non significant) p>0.05, * p<0.05, ** p<0.01, *** p<0.001.

Article Snippet: N-terminal HA-tagged zebrafish aPKC wildtype CDS (NM 131855) synthesis in pCS2+8 vector (a gift from Amro Hamdoun; Addgene plasmid # 34931), and aPKC T556E and aPKC T556A site-directed mutagenesis (SDM) were done by GeneArt, Life Technologies. aPKC T549E, aPKC T549A, aPKC A122E, and the combined mutations were achieved by SDM with the primers mentioned in supplementary information 1, using Q5 Polymerase (Neb) according to the protocol published earlier ( ). pcDNA3-EGFP-RhoA-Q63L (Addgene plasmid # 12968) was a gift from Gary Bokoch.

Techniques: Residue, Standard Deviation, Generated, Comparison

Journal: bioRxiv

Article Title: mTOR signaling governs the formation of epithelial apical projection via S6K1-RhoA and aPKC-Lgl2 axes

doi: 10.1101/2024.12.08.627438

Figure Lengend Snippet: (A) and pT556/pT549 (B). The colored lines are from 10 individual iterations and the black line is the average trend line. The line plot of CVCF vs Time (C) of all 10 trajectories from MD Simulation of aPKC pT556 (purple) and pT556/pT549 (magenta) shows plateaus which indicates the metastable stages of the protein. The comparison of these metastable states between aPKC pT556 and aPKC pT556+pT549 is shown in average residence time plot (D). The X-axis has been represented in -Log 10 for better visualization. The average CVCF trace of the residues surrounding the region of ATP binding cleft of aPKC calculated within the region of 3Å (E), 5Å (F), 7Å (G), and 10Å (H) of ATP.

Article Snippet: N-terminal HA-tagged zebrafish aPKC wildtype CDS (NM 131855) synthesis in pCS2+8 vector (a gift from Amro Hamdoun; Addgene plasmid # 34931), and aPKC T556E and aPKC T556A site-directed mutagenesis (SDM) were done by GeneArt, Life Technologies. aPKC T549E, aPKC T549A, aPKC A122E, and the combined mutations were achieved by SDM with the primers mentioned in supplementary information 1, using Q5 Polymerase (Neb) according to the protocol published earlier ( ). pcDNA3-EGFP-RhoA-Q63L (Addgene plasmid # 12968) was a gift from Gary Bokoch.

Techniques: Comparison, Binding Assay

Journal: bioRxiv

Article Title: mTOR signaling governs the formation of epithelial apical projection via S6K1-RhoA and aPKC-Lgl2 axes

doi: 10.1101/2024.12.08.627438

Figure Lengend Snippet: Proposed model representing the two mTOR signaling axes regulating the NMII activity. mTORC1 and its substrate S6K1 downregulates the RhoA/ROCK activity whereas, mTORC2 regulates aPKC activity via differential phosphorylation. Both of these axes converge on NMII which influences microridge pattern formation.

Article Snippet: N-terminal HA-tagged zebrafish aPKC wildtype CDS (NM 131855) synthesis in pCS2+8 vector (a gift from Amro Hamdoun; Addgene plasmid # 34931), and aPKC T556E and aPKC T556A site-directed mutagenesis (SDM) were done by GeneArt, Life Technologies. aPKC T549E, aPKC T549A, aPKC A122E, and the combined mutations were achieved by SDM with the primers mentioned in supplementary information 1, using Q5 Polymerase (Neb) according to the protocol published earlier ( ). pcDNA3-EGFP-RhoA-Q63L (Addgene plasmid # 12968) was a gift from Gary Bokoch.

Techniques: Activity Assay