Journal: Nature Communications

Article Title: Synergistic activation of the human phosphate exporter XPR1 by KIDINS220 and inositol pyrophosphate

doi: 10.1038/s41467-025-58200-y

Figure Lengend Snippet: Cryo-EM maps ( a ) and structural models ( b ) of the outward-open state XPR1 (XPR1 OUT ) from the side view and extracellular view. The two protomers of XPR1, lipids, and InsP 6 molecules are colored light pink, light green, magenta, and red, respectively. The densities of the Pi are shown as blue meshes as an insert. Cryo-EM maps ( c ) and structural models ( d ) of the closed state XPR1 with SPX (XPR1 C_SPX ) from the side view and extracellular view. The color schemes are the same as in ( a , b ). e Structural comparison of XPR1 C (cyan) and XPR1 OUT (light pink and light green), superimposed based on the TMDs. f Schematic diagram of the secondary structure features of full-length XPR1 OUT . The TMD and the SPX domains are presented in gray and pink. The Glu622/Phe623 motif near the C-terminal of XPR1 is labeled in purple. InsP 6 is labeled in red. g Schematic diagram of the binding between KIDINS220 (1–432) and XPR1 in the outward-open state conformation. The XPR1 OUT atomic model was superimposed into the XPR1 OUT state density map after an additional two rounds of 3D classification at a low counter level. The two protomers of XPR1. and InsP 6 molecules are presented in light pink, light green, and red, respectively. The cryo-EM density maps of XPR1 and KIDINS220 at low counter level are shown in transparent mode and colored gray and orange, respectively. The binding pattern is based on the Alphafold2 prediction result presented in Supplementary Fig. . h Superimposition of our XPR1 OUT structure with the previously reported XPR1-SPX-1,5-InsP 8 crystal structure by aligning one of the SPX domains. The InsP 6 molecules and the protomer of XPR1 OUT_A were colored in light pink and the protomer of XPR1 OUT_B in light green. The InsP 8 molecule and the SPX A protomer of the XPR1-SPX-1,5-InsP 8 complex were colored in dark blue, and the SPX B protomer in light blue.

Article Snippet: The following primary antibodies were utilized in this study: Anti-mCherry Tag Mouse Monoclonal Antibody (9D3) (Abbkine, ABT2080, diluted 1:5000), KIDINS220 Monoclonal antibody (Proteintech, 66748-1-Ig, diluted 1:6000) and GAPDH antibody (Ray Antibody, RM2002, diluted 1:3000).

Techniques: Cryo-EM Sample Prep, Comparison, Labeling, Binding Assay

Journal: Nature Communications

Article Title: Synergistic activation of the human phosphate exporter XPR1 by KIDINS220 and inositol pyrophosphate

doi: 10.1038/s41467-025-58200-y

Figure Lengend Snippet: a The Glu622/Phe623 motif and the cavity-forming helices from the TMD, i.e., TMs 5, 6, 7, 8, and 10. The Glu622/Phe623 motif is colored in purple. Putative interactive residues are displayed as sticks. b 32 Pi efflux of EV, WT-XPR1 and XPR1 mutants in the C-terminal plug-in loop. The numbers of biologically independent experiments are identical to ( c ). c 32 P efflux percentages of EV and XPR1 mutants in ( b ) at 2 h normalized against WT-XPR1. From left to right: n = 4, 24, 7, 3, and 5 biologically independent experiments. P values are 4 × 10 −4 , 1, <1 × 10 −4 , 0.0191, and <1 × 10 −4 . Data are shown as mean ± s.d. in ( b , c ). P values were obtained by a two-tailed unpaired t- test with Welch’s correction in ( c ). d Western blot analysis of the binding capacity between endogenous KIDINS220 and over-expressed WT-XPR1 or XPR1 (1–630)-E622A/F623A in HEK293T cells using co-immunoprecipitation. n = 1 experiment representative of n = 2 independent transfections. Cryo-EM maps ( e ) and structural models ( f ) of the inward-facing XPR1-E622A/F623A mutant (XPR1 IN ) from side view and intracellular view. The color schemes are the same as in Figs. . g The densities of Pi and the surrounding residues at the intracellular cavity are shown as blue meshes. h Structural comparison of XPR1 OUT (green) and XPR1 IN (light pink and light green), superimposed based on the TMD. The directions of the α3 helix backbones are indicated by arrows. The amino acid residues at the N- and C- ends of the α3 helices are labeled. Taking the α3 helix as an example, the SPX domain rotates nearly 180°.

Article Snippet: The following primary antibodies were utilized in this study: Anti-mCherry Tag Mouse Monoclonal Antibody (9D3) (Abbkine, ABT2080, diluted 1:5000), KIDINS220 Monoclonal antibody (Proteintech, 66748-1-Ig, diluted 1:6000) and GAPDH antibody (Ray Antibody, RM2002, diluted 1:3000).

Techniques: Two Tailed Test, Western Blot, Binding Assay, Immunoprecipitation, Transfection, Cryo-EM Sample Prep, Mutagenesis, Comparison, Labeling

Journal: Nature Communications

Article Title: Synergistic activation of the human phosphate exporter XPR1 by KIDINS220 and inositol pyrophosphate

doi: 10.1038/s41467-025-58200-y

Figure Lengend Snippet: A schematic representation of the proposed XPR1 activation mechanism. Under non-activating conditions, the TMD is locked in a closed state (State 1). When KIDINS220 is absent, elevated InsPs in the cytoplasm bind between the two SPX domains of XPR1, locking XPR1 in a closed conformation (State 2). Mutation of the Glu622/Phe623 motif which blocks the intracellular cavity induces XPR1 to transition to an inward-facing conformation (State 3). In the first three states, the flexible C-terminal helix (631–655) resides near the SPX domain. In the presence of KIDINS220, the C-terminal helix binds with KIDINS220 (1–432) and induces a further conformational change of the SPX domain possibly due to steric hindrance. Although the dynamics of the SPX domain are too strong to observe its specific orientation, subsequent results indicate that its conformation undergoes a 180° flip compared to State 2, with XPR1 still remaining in a closed conformation (State 4). InsPs bind at the interface between the SPX domain and the TMD, maintaining XPR1 in a closed conformation. However, this binding enhances the dynamics of the Glu622/Phe623 motif, thereby eliminating one of the barriers to activation (State 5). Upon the introduction of substrate phosphate ions, they promptly bind to XPR1, thereby inducing the conformational alteration of XPR1 into an outward-open state. This conformational change, in turn, promotes the export of these phosphate ions (State 6). Although the two halves of the XPR1 dimer in different states were depicted with the same behavior in our figure drawing, we hypothesize that their actual functional states might not be in synchronization. States 3–6 were observed in this study. State 2 is derived from PDB ID: 8X5F and State 1 is hypothesized. Created in BioRender. Zuo, P. (2025) https://BioRender.com/v23k582 .

Article Snippet: The following primary antibodies were utilized in this study: Anti-mCherry Tag Mouse Monoclonal Antibody (9D3) (Abbkine, ABT2080, diluted 1:5000), KIDINS220 Monoclonal antibody (Proteintech, 66748-1-Ig, diluted 1:6000) and GAPDH antibody (Ray Antibody, RM2002, diluted 1:3000).

Techniques: Activation Assay, Mutagenesis, Binding Assay, Functional Assay, Derivative Assay

Journal: Developmental cell

Article Title: Square cell packing in the Drosophila embryo through spatiotemporally regulated EGF receptor signaling

doi: 10.1016/j.devcel.2015.09.015

Figure Lengend Snippet: (A) Cells form a square cell grid (bracket) at the midline of the presumptive pharynx (left, stage 13), which ultimately forms the midline of the pharyngeal tube after involution (right, stage 15). Phosphotyrosine (green), β-catenin (red), and Par-3 (blue). (B,C) The percentage of 4-sided cells increased from 10±2% (mean±sem) in early Phase I to 59±4% in Phase III. The average number of neighbors decreased from 5.6±0.09 in early Phase I to 4.5±0.09 in late Phase I and 4.5±0.04 in Phase III. The percentage of 4-cell vertices significantly increased and the variance of the topological distribution significantly decreased in Phase III compared to early Phase I (p < 0.001, 4-cell vertices, p = 0.01, variance) or lateral cells in Phase III (p < 0.001, 4-cell vertices, p = 0.0002, variance) (unpaired t test). (D) Schematic of the square grid (yellow). (E–H) Wild-type embryos at stages 10 (E), 11 (F), 12 (G), or 13 (H) (β-catenin, white). (I,J) Stills from time-lapse movies of wild-type cells expressing β-catenin:GFP. (I) In Phase I (cell alignment, stage 11), rectangular cells align into rows on either side of the midline. (J) In Phase II (oriented division, stage 12), cells divide largely synchronously perpendicular to the midline. White lines, cells in anaphase or telophase. In Phase III (apicobasal cell elongation, stage 13), cells elongate along the apical-basal cell axis and midline cells delaminate from the epithelium, producing a compact square grid. t = 0 is the onset of Phase I (I) or the onset of the first division in Phase II (J). (n = 299–751 vertices in 86–398 cells in 4–7 embryos/stage) (*, p = 0.005 – 0.03, ** p < 0.005). Ventral views, anterior left. Arrowheads, ventral midline. Bars, 10 μm. See also Supplemental Movies 1 and 2.

Article Snippet: Antibodies were mouse Arm/β-catenin (1:25, Developmental Studies Hybridoma Bank, DSHB), rat DE-cadherin (1:25, DSHB), mouse Neurotactin (1:100, DSHB), guinea pig Bazooka/Par-3 (1:1000) ( Blankenship et al., 2006 ), mouse phosphotyrosine (1:250, 4G10 Millipore), rabbit GFP (1:100, Torrey Pines), mouse GFP (1:25, Roche), mouse α-tubulin (1:400, Sigma), and rabbit phospho-ERK1/2 (Thr202/Tyr204) (dpERK) (1:100, D13.14.4E, Cell Signaling).

Techniques: Expressing

Journal: Developmental cell

Article Title: Square cell packing in the Drosophila embryo through spatiotemporally regulated EGF receptor signaling

doi: 10.1016/j.devcel.2015.09.015

Figure Lengend Snippet: (A–F) Localization of Par-3, β-catenin, and myosin II (Myosin:GFP) in wild-type embryos in Phase I (stage 11) (A, B), Phase II (stage 12) (C–E), or Phase III (stage 13) (F). Myosin II (red) is parallel to the midline in all three phases (B, E, and F). Par-3 (green) is perpendicular to the midline in Phases I (A) and III (F). Par-3 levels were transiently reduced at the cortex of dividing cells (asterisks) and transiently localized to new interfaces between daughter cells (arrowheads) in Phase II (C,D). (G–J) Cells undergo apicobasal elongation in Phase III (early Phase III, left; late Phase III, right). Brackets indicate the lateral cell membrane. (K and L) Microtubules align with the apical-basal axis in Phase III. (M) Schematic. Arrowheads, ventral midline. Bars, 5 μm. See also Figure S1.

Article Snippet: Antibodies were mouse Arm/β-catenin (1:25, Developmental Studies Hybridoma Bank, DSHB), rat DE-cadherin (1:25, DSHB), mouse Neurotactin (1:100, DSHB), guinea pig Bazooka/Par-3 (1:1000) ( Blankenship et al., 2006 ), mouse phosphotyrosine (1:250, 4G10 Millipore), rabbit GFP (1:100, Torrey Pines), mouse GFP (1:25, Roche), mouse α-tubulin (1:400, Sigma), and rabbit phospho-ERK1/2 (Thr202/Tyr204) (dpERK) (1:100, D13.14.4E, Cell Signaling).

Techniques: Membrane

Journal: Developmental cell

Article Title: Square cell packing in the Drosophila embryo through spatiotemporally regulated EGF receptor signaling

doi: 10.1016/j.devcel.2015.09.015

Figure Lengend Snippet: (A) Stills from time-lapse movies of wild-type and pins mutant embryos expressing β-catenin:GFP. White lines indicate cells in anaphase or telophase. (B) Quantification of the cell division axis (0° is parallel to the ventral midline). All wild-type cells divided at 75–90° relative to the midline. Cell divisions were frequently misoriented in pins mutants (p < 0.001) (Chi square test). (C) Stills from a time-lapse movie of wild-type cells expressing Jupiter:mCherry. One spindle rotates 90° in the epithelial plane. (D) Pins/LGN accumulates at cell interfaces contacting midline cells in Phase II (stage 12). (E) The gap between the cell division axis and the cell long axis was significantly larger in pins mutants (p < 0.001) (unpaired t test). Boxes, 25th to 75th percentile; whiskers, 2.5th to 97.5th percentile; horizontal line, median; +, mean. Plot shows the distribution of average values across embryos. (F) The cell division axis and the cell long axis were both close to perpendicular to the ventral midline in wild type. The orientation of the cell long axis in pins mutants (85°±0.5°) was similar to wild type (86°±0.4°), but cell division occurred at a much wider range of orientations. (G) Angular distributions of the aligned centrosomes at prophase and spindles at prometaphase or anaphase. n = 73–77 cells in 3 embryos/genotype in B, E, and F and 82 cells in 5 embryos in G. Arrowheads, ventral midline. Bars, 10 μm in A,D, 5 μm in C. See also Figure S2.

Article Snippet: Antibodies were mouse Arm/β-catenin (1:25, Developmental Studies Hybridoma Bank, DSHB), rat DE-cadherin (1:25, DSHB), mouse Neurotactin (1:100, DSHB), guinea pig Bazooka/Par-3 (1:1000) ( Blankenship et al., 2006 ), mouse phosphotyrosine (1:250, 4G10 Millipore), rabbit GFP (1:100, Torrey Pines), mouse GFP (1:25, Roche), mouse α-tubulin (1:400, Sigma), and rabbit phospho-ERK1/2 (Thr202/Tyr204) (dpERK) (1:100, D13.14.4E, Cell Signaling).

Techniques: Mutagenesis, Expressing

Journal: Developmental cell

Article Title: Square cell packing in the Drosophila embryo through spatiotemporally regulated EGF receptor signaling

doi: 10.1016/j.devcel.2015.09.015

Figure Lengend Snippet: (A) Pins asymmetry was absent in spitz mutants (0/19 cells in 6 embryos), but occurred normally in pointed mutants (25/27 cells in 7 embryos), similar to wild type (33/35 cells in 5 embryos). (B) Quantification of the cell division axis (0° is parallel to the ventral midline). All wild-type cells divided at 75–90° relative to the midline. Cell divisions were frequently misoriented in spitz (62%) and pointed (52%) mutants (55–84 cells in 3–4 embryos/genotype). (C) Stills from time-lapse movies of wild-type, spitz, and pointed mutant embryos expressing β-catenin:GFP. White lines indicate the division axis. (D) The cell division axis correlates with the long axis of the cell in wild type. (E,F) Cells divided at a wider range of orientations and the cell division axis was not well correlated with the cell long axis in spitz (linear correlation coefficient R2 = 0.14) and pointed (R2 = 0.18) mutants. The orientation of the cell long axis in spitz mutants (55°±4°) was significantly different from wild type (86°±0.4°), but was less strongly affected in pointed mutants (64°±3°) (p = 0.06, spitz vs. pointed) (unpaired t test). Bars, 10 μm in A, 5 μm in C.

Article Snippet: Antibodies were mouse Arm/β-catenin (1:25, Developmental Studies Hybridoma Bank, DSHB), rat DE-cadherin (1:25, DSHB), mouse Neurotactin (1:100, DSHB), guinea pig Bazooka/Par-3 (1:1000) ( Blankenship et al., 2006 ), mouse phosphotyrosine (1:250, 4G10 Millipore), rabbit GFP (1:100, Torrey Pines), mouse GFP (1:25, Roche), mouse α-tubulin (1:400, Sigma), and rabbit phospho-ERK1/2 (Thr202/Tyr204) (dpERK) (1:100, D13.14.4E, Cell Signaling).

Techniques: Mutagenesis, Expressing

Journal: Cell proliferation

Article Title: HN1 Functions in Protein Synthesis Regulation via mTOR-RPS6 Axis and Maintains Nucleolar Integrity.

doi: 10.1111/cpr.13805

Figure Lengend Snippet: FIGURE 1 | (A) Changes in the half-life of the proteins were investigated using a Cycloheximide (1 μM) treatment time course (0–24 h) in cells with stabilised HN1 expression (OE) and found that the HN1 OE increased only the native HN1 abundance but did not influence Cdk2 and B-actin proteins' half-life significantly. The increases in expressions were noted with a red arrow and the decreases with a green arrow respectively. (B) Native HN1 expression stabilisation was quantitated from Western data and graphed. The ectopic expression of HN1 stabilised native form more than 2-fold despite CHX treatment. (C) shRNA mediated knockdown (KD) of HN1 expression reduced the half-life of the proteins tested signifi- cantly, where the decreases in expressions were shown in green arrows. (D) Ectopic HN1 expression increased native HN1's stability in PC3 cells. β- catenin and p62 were used as controls for proteosomal and autophagy-mediated degradations respectively. (E) GSK3B inhibitor SB216763 was given to shRNA-depleted HN1 KD PC3 cells in comparison to controls and flow cytometry analysis was performed to evaluate the cell cycle phases. (F) HN1 depletion reduced the cell viability by almost 20% (p < 0.05) even in untreated cells. The shRNA-mediated cell growth inhibition is augmented by GSK3Bi and proteosome inhibitors, (G) MG132, (H) bortezomib treatments. The cell viability significantly decreased further down to 40% with inhibitors, concentration-dependently (p < 0.05).

Article Snippet: Antibody Technique Manufacturer and catalogue Dilution Anti- HN1 IB, IF, IP Invitrogen—PA5109824 1/750, 1/300, 2 μg Anti- Cdk2 IB Santa Cruz—sc- 6248 1/500 Anti- β- Actin IB Sigma—A3854 1/500.000 Anti- p62 IB Proteintech—18,420- 1- AP 1/10000 Anti- β- catenin IB Santa Cruz—sc59737 1/500 Anti- Cdk1 IB Abcam—ab131450 1/1000 Anti- p- 4EBP1(S65) IB, IF Santa Cruz—sc- 293,124 1/500, 1/200 Anti- 4EBP1/2/3 IB Santa Cruz—sc- 271,947 1/500 Anti- 4EBP1 IB Santa Cruz—sc- 9977 1/500 Anti- p- GSK3β(S9) IB Santa Cruz—sc- 11,757 1/500 Anti- p- GSK3β (Tyr 216) IB Santa Cruz—sc- 135,653 1/500 Anti- p- p70S6K1(S434) IB, IF Santa Cruz—sc- 8416 1/500, 1/200 Anti- p- RPS6(S235/236) IB, IF Santa Cruz—sc- 293,144 1/500, 1/200 Anti- RPS6 IB, IF Santa Cruz—sc- 74,459 1/500, 1/200 Anti- UBF IB, IF Santa Cruz—sc- 13,125 1/500, 1/200 Anti- mTOR IB Santa Cruz—sc- 517,464 1/500 Anti- nucleolin IB, IF Santa Cruz—sc- 8031 1/500, 1/200 Anti- BrdU IF, FC Santa Cruz—sc- 32,323 1/200, 1/200 Anti- GAPDH IB Ambion—AM4300 1/240.000 Anti- lamin A/C IB Santa Cruz—sc- 376,248 1/500 Anti- rabbit IB Jackson Immuno Research—111- 035- 003 1/25.000 Anti- mouse IB Jackson Immuno Research—515- 035- 003 1/25.000 Alexa488 (anti- mouse IgG) IF Thermo A- 21202 1/600 Alexa594 (anti- mouse IgG) IF Thermo A- 21203 1/600 Alexa488 (anti- rabbit IgG) IF Thermo A- 11008 1/600 Alexa594 (anti- rabbit IgG) IF Jackson Immuno Research 711- 585- 152 1/600 13652184, 0, D ow nloaded from https://onlinelibrary.w iley.com /doi/10.1111/cpr.13805 by IN A SP - N E PA L , W iley O nline L ibrary on [25/01/2025].

Techniques: Expressing, Western Blot, shRNA, Knockdown, Comparison, Flow Cytometry, Inhibition, Concentration Assay

Journal: Cell proliferation

Article Title: HN1 Functions in Protein Synthesis Regulation via mTOR-RPS6 Axis and Maintains Nucleolar Integrity.

doi: 10.1111/cpr.13805

Figure Lengend Snippet: FIGURE 2 | (A) Subcellular fractionation was performed in PC3 cells when HN1 KD or OE. Asynchronous or Thymidine synchronised cells were used to evaluate the timing of alteration when HN1 expression is changed. It is observed that histone protein expressions decreased in HN1 KD cells, but not in OE cells significantly in comparison to controls. (B) The observation was repeated with chromatin-enriched lysates. Intriguingly shHN1 cells exhibited lower-sized histones in Coomassie gels in both KD and OE phenotypes. (C) p-GSK3β(S9) and p-4EBP1(S65) phosphorylations were studied w/wo Akti (wortmannin) and PI3Ki (LY294002) treatments for 24 h in HN1 OEcells. Clearly HN1 OE stabilised both p-GSK3β(S9) and p- 4EBP1(S65) phosphorylations. (D) p-4EBP1/2/3 phosphorylations were also examined w/wo GSK3Bi (SB216763) treatment and observed that HN1 OE increased p-4EBP1/2/3 phosphorylations and proteosome inhibitors MG132 and bortezomib stabilised the protein levels as well as phosphorylations in HN1 OE PC3 cells in comparison to vector controls. (E) p-GSK3β(S9) and p-4EBP1(S65) phosphorylations were studied w/wo Akti (wortmannin) and PI3Ki (LY294002) treatments for 24 h in HN1 KD cells. HN1 depletion decreased both 4EBP1 and its S65 phosphorylations together with clear changes in p-GSK3β(S9). The cells that are treated with LY294002 but not wortmannin exhibited lower expression of 4EBP1 with clearly suppressed S65 phosphorylations. The use of both reagents together resulted in a significant (more than 5-fold) decrease in 4EBP1 expression consistent with reduced p-GSK3β(S9). (F) 4EBP1 and its S65 phosphorylation together with clear changes in p-GSK3β(S9) and p-GSK3β(T216) phosphorylations were studied w/wo GSK3Bi (SB216763) and proteosome inhibitor treatments for 24 h in HN1 KDcells. HN1 depletion decreased both 4EBP1 and its S65 phosphorylations together with p-GSK3β(S9) and p-GSK3β(T216) phosphorylations. The cells that are treated with SB216763 exhibited decreased S65 phosphorylation and expression of 4EBP1. The use of proteosome inhibitors resulted in marginal stabilisation in 4EBP1 level consistent with in- creased p-GSK3β(S9). (G) Using immunofluorescent microscopy HN1 colocalization with p-4EBP1(S65) was examined when HN1 was depleted and overexpressed in comparison to controls in PC3 cells. The speckled distribution of HN1 and p-4EBP1(S65) phosphorylations colocalized but the dis- tribution did not significantly change.

Article Snippet: Antibody Technique Manufacturer and catalogue Dilution Anti- HN1 IB, IF, IP Invitrogen—PA5109824 1/750, 1/300, 2 μg Anti- Cdk2 IB Santa Cruz—sc- 6248 1/500 Anti- β- Actin IB Sigma—A3854 1/500.000 Anti- p62 IB Proteintech—18,420- 1- AP 1/10000 Anti- β- catenin IB Santa Cruz—sc59737 1/500 Anti- Cdk1 IB Abcam—ab131450 1/1000 Anti- p- 4EBP1(S65) IB, IF Santa Cruz—sc- 293,124 1/500, 1/200 Anti- 4EBP1/2/3 IB Santa Cruz—sc- 271,947 1/500 Anti- 4EBP1 IB Santa Cruz—sc- 9977 1/500 Anti- p- GSK3β(S9) IB Santa Cruz—sc- 11,757 1/500 Anti- p- GSK3β (Tyr 216) IB Santa Cruz—sc- 135,653 1/500 Anti- p- p70S6K1(S434) IB, IF Santa Cruz—sc- 8416 1/500, 1/200 Anti- p- RPS6(S235/236) IB, IF Santa Cruz—sc- 293,144 1/500, 1/200 Anti- RPS6 IB, IF Santa Cruz—sc- 74,459 1/500, 1/200 Anti- UBF IB, IF Santa Cruz—sc- 13,125 1/500, 1/200 Anti- mTOR IB Santa Cruz—sc- 517,464 1/500 Anti- nucleolin IB, IF Santa Cruz—sc- 8031 1/500, 1/200 Anti- BrdU IF, FC Santa Cruz—sc- 32,323 1/200, 1/200 Anti- GAPDH IB Ambion—AM4300 1/240.000 Anti- lamin A/C IB Santa Cruz—sc- 376,248 1/500 Anti- rabbit IB Jackson Immuno Research—111- 035- 003 1/25.000 Anti- mouse IB Jackson Immuno Research—515- 035- 003 1/25.000 Alexa488 (anti- mouse IgG) IF Thermo A- 21202 1/600 Alexa594 (anti- mouse IgG) IF Thermo A- 21203 1/600 Alexa488 (anti- rabbit IgG) IF Thermo A- 11008 1/600 Alexa594 (anti- rabbit IgG) IF Jackson Immuno Research 711- 585- 152 1/600 13652184, 0, D ow nloaded from https://onlinelibrary.w iley.com /doi/10.1111/cpr.13805 by IN A SP - N E PA L , W iley O nline L ibrary on [25/01/2025].

Techniques: Fractionation, Expressing, Comparison, Plasmid Preparation, Phospho-proteomics, Microscopy

Journal: Cell proliferation

Article Title: HN1 Functions in Protein Synthesis Regulation via mTOR-RPS6 Axis and Maintains Nucleolar Integrity.

doi: 10.1111/cpr.13805

Figure Lengend Snippet: FIGURE 3 | (A) 4EBP1, RPS6, UBF, and mTOR expressions, as well as p-P70S6K1(S434) and p-RPS6(S235/236) phosphorylations, were examined in cytoplasmic/nuclear-fractionated cell lysates in asynchronized and thymidine synchronised HN1 depleted (KD) and HN1 OE PC3 cells. Here, 4EBP1, RPS6, UBF and mTOR expressions as well as p-RPS6(S235/236) and p-P70S6K1(S434) phosphorylations significantly decreased in HN1 depleted cells, consistent with increases in HN1 OE cells in comparison to controls. Here A; represents asynchronized cells. Zero represents thymidine syn- chronised but not released, and four represents 4 h released cells from synchronizations. (B) RPS6, and (C) p-RPS6(S235/236) were examined in HN1 depleted PC3 cells using immunofluorescent microscopy. (D) When UBF and HN1 colocalizations were examined in both HN1 depleted (KD) and shcontrol PC3 cells, it was found that the nucleolar (proximal to NOR region) localization of UBF expression changed in KD cells in comparison to controls, correlated with western blots. (E) Specked localizations of p-P70S6K1(S434) and p-RPS6(S235/236) (same as in C) phosphorylations increased in the S phase clearly indicating a cytoplasmic granulation and retention of p-RPS6(S235/236) in HN1 KD cells.

Article Snippet: Antibody Technique Manufacturer and catalogue Dilution Anti- HN1 IB, IF, IP Invitrogen—PA5109824 1/750, 1/300, 2 μg Anti- Cdk2 IB Santa Cruz—sc- 6248 1/500 Anti- β- Actin IB Sigma—A3854 1/500.000 Anti- p62 IB Proteintech—18,420- 1- AP 1/10000 Anti- β- catenin IB Santa Cruz—sc59737 1/500 Anti- Cdk1 IB Abcam—ab131450 1/1000 Anti- p- 4EBP1(S65) IB, IF Santa Cruz—sc- 293,124 1/500, 1/200 Anti- 4EBP1/2/3 IB Santa Cruz—sc- 271,947 1/500 Anti- 4EBP1 IB Santa Cruz—sc- 9977 1/500 Anti- p- GSK3β(S9) IB Santa Cruz—sc- 11,757 1/500 Anti- p- GSK3β (Tyr 216) IB Santa Cruz—sc- 135,653 1/500 Anti- p- p70S6K1(S434) IB, IF Santa Cruz—sc- 8416 1/500, 1/200 Anti- p- RPS6(S235/236) IB, IF Santa Cruz—sc- 293,144 1/500, 1/200 Anti- RPS6 IB, IF Santa Cruz—sc- 74,459 1/500, 1/200 Anti- UBF IB, IF Santa Cruz—sc- 13,125 1/500, 1/200 Anti- mTOR IB Santa Cruz—sc- 517,464 1/500 Anti- nucleolin IB, IF Santa Cruz—sc- 8031 1/500, 1/200 Anti- BrdU IF, FC Santa Cruz—sc- 32,323 1/200, 1/200 Anti- GAPDH IB Ambion—AM4300 1/240.000 Anti- lamin A/C IB Santa Cruz—sc- 376,248 1/500 Anti- rabbit IB Jackson Immuno Research—111- 035- 003 1/25.000 Anti- mouse IB Jackson Immuno Research—515- 035- 003 1/25.000 Alexa488 (anti- mouse IgG) IF Thermo A- 21202 1/600 Alexa594 (anti- mouse IgG) IF Thermo A- 21203 1/600 Alexa488 (anti- rabbit IgG) IF Thermo A- 11008 1/600 Alexa594 (anti- rabbit IgG) IF Jackson Immuno Research 711- 585- 152 1/600 13652184, 0, D ow nloaded from https://onlinelibrary.w iley.com /doi/10.1111/cpr.13805 by IN A SP - N E PA L , W iley O nline L ibrary on [25/01/2025].

Techniques: Comparison, Microscopy, Expressing, Western Blot

Journal: Cell proliferation

Article Title: HN1 Functions in Protein Synthesis Regulation via mTOR-RPS6 Axis and Maintains Nucleolar Integrity.

doi: 10.1111/cpr.13805

Figure Lengend Snippet: FIGURE 4 | (A) Immunoprecipitations were performed with anti-HN1 antibody using HN1 overexpressing PC3 cell lysates and, RPS6 interac- tion was identified by Western blots. (B) Additionally, mTOR and RPS6 expressions as well as p-RPS6(S235/236) and p-p70S6K(S434) phosphorylations were examined in cytoplasmic/nuclear-fractionated cell lysates in asynchronized HN1 depleted (KD) MDA-MB231 cells. Although there was not a significant variation in mTOR, P70S6K and nucleolin expressions, in control, but not in shHN1 depleted lysates two close bands for RPS6 were ob- served. Thus, the p-RPS6(S235/236) phosphorylation was examined and observed that the phosphorylated RPS6 accumulated in nuclear lysates with a clear decrease in HN1 depletion. This implies that nuclear retention occurs due to less phosphorylation in HN1 KD in comparison to controls. (C) Immunoprecipitations were also performed with anti-HN1 antibody using HN1 overexpressing MDA-MB231 cell lysates and, RPS6, nucleolin as well as mTOR interactions were confirmed by western blots. All IP experiments were duplicates and immunoblots were at least triplicates. (D) Also, nocodazole (16 h) synchronised PC3 cell lysates were used to enrich the G2 population and the HN1 interactions with mTOR and Nucleolin were demonstrated. (E) HN1 colocalizations with RPS6, and p-RPS6(S235/236) and UBF were examined in HN1-depleted MDA-MB231 cells in comparison to control cells. The cells that were negative for expression were marked with blue and the positives with red arrows. (F) An arbitrary expression cutoff was applied and the negative/positive cells that are expressing the protein of interest were counted from shHN1 cells versus controls. The num- ber of cells counted for p-RPS6(S235/236) were (n = 207) (p < 0.001) of MDA-MB231 and (n = 62) (p = 0.29) of MCF10A. (G) Again the number of cells counted for RPS6 was n = 683 (p < 0.05) and (H) it was n = 80 for UBF (p < 0.001) in MDA-MB231. The cells were counted using ImageJ software and statistically significant (p values/p values) differences were found. The scale bar was 4 μm. (I) HN1 depletion and overexpression were confirmed in MDA-MB231 cells in comparison to controls. B-actin was also shown as a loading control.

Article Snippet: Antibody Technique Manufacturer and catalogue Dilution Anti- HN1 IB, IF, IP Invitrogen—PA5109824 1/750, 1/300, 2 μg Anti- Cdk2 IB Santa Cruz—sc- 6248 1/500 Anti- β- Actin IB Sigma—A3854 1/500.000 Anti- p62 IB Proteintech—18,420- 1- AP 1/10000 Anti- β- catenin IB Santa Cruz—sc59737 1/500 Anti- Cdk1 IB Abcam—ab131450 1/1000 Anti- p- 4EBP1(S65) IB, IF Santa Cruz—sc- 293,124 1/500, 1/200 Anti- 4EBP1/2/3 IB Santa Cruz—sc- 271,947 1/500 Anti- 4EBP1 IB Santa Cruz—sc- 9977 1/500 Anti- p- GSK3β(S9) IB Santa Cruz—sc- 11,757 1/500 Anti- p- GSK3β (Tyr 216) IB Santa Cruz—sc- 135,653 1/500 Anti- p- p70S6K1(S434) IB, IF Santa Cruz—sc- 8416 1/500, 1/200 Anti- p- RPS6(S235/236) IB, IF Santa Cruz—sc- 293,144 1/500, 1/200 Anti- RPS6 IB, IF Santa Cruz—sc- 74,459 1/500, 1/200 Anti- UBF IB, IF Santa Cruz—sc- 13,125 1/500, 1/200 Anti- mTOR IB Santa Cruz—sc- 517,464 1/500 Anti- nucleolin IB, IF Santa Cruz—sc- 8031 1/500, 1/200 Anti- BrdU IF, FC Santa Cruz—sc- 32,323 1/200, 1/200 Anti- GAPDH IB Ambion—AM4300 1/240.000 Anti- lamin A/C IB Santa Cruz—sc- 376,248 1/500 Anti- rabbit IB Jackson Immuno Research—111- 035- 003 1/25.000 Anti- mouse IB Jackson Immuno Research—515- 035- 003 1/25.000 Alexa488 (anti- mouse IgG) IF Thermo A- 21202 1/600 Alexa594 (anti- mouse IgG) IF Thermo A- 21203 1/600 Alexa488 (anti- rabbit IgG) IF Thermo A- 11008 1/600 Alexa594 (anti- rabbit IgG) IF Jackson Immuno Research 711- 585- 152 1/600 13652184, 0, D ow nloaded from https://onlinelibrary.w iley.com /doi/10.1111/cpr.13805 by IN A SP - N E PA L , W iley O nline L ibrary on [25/01/2025].

Techniques: Western Blot, Control, Phospho-proteomics, Comparison, Expressing, Software, Over Expression

Journal: Cell proliferation

Article Title: HN1 Functions in Protein Synthesis Regulation via mTOR-RPS6 Axis and Maintains Nucleolar Integrity.

doi: 10.1111/cpr.13805

Figure Lengend Snippet: FIGURE 5 | (A–D) NOR staining was performed without KCl treatments in KD cells and with KCl in both conditions, which are the HN1 deple- tion (KD) as well as HN1 overexpression (OE) in PC3 cells by silver nitrate staining (AgNOR). The staining of NOR regions also demonstrates that the HN1 depletion increases the number of nucleolar structures with severe form changes as well as staining intensity. (B) Quantitation of the NOR count was performed and exhibited a significantly higher number of NOR per nucleolus (p < 0.001). (C) Areas of each NOR region were also quanti- tated and plotted for each nucleus in shHN1 cells versus controls and found that HN1 KD cells exhibited significantly lower mean area for NORs as ratios (p < 0.001). (D) HN1 OE cells exhibiting highly granular nucleus having larger nucleoli with denser NOR staining in comparison to controls. (E–H) NOR staining was also performed without KCl treatments in KD cells and with KCl in both conditions of HN1, which are the HN1 depleted (KD) as well as HN1 overexpressed (OE) in the MDA-MB231 cell line. (F, G) Mean area and the NOR counts were not significantly different (p > 0.05). (I) Mean nuclear size was calculated and plotted from PC3 cells, and (J) from MDA-MB231 cells. HN1 KD cells exhibited significantly larger nucle- ar sizes in both cell lines (p < 0.001). The statistical significance of the data was calculated and given in graphs as either *, ** and *** corresponding p > 0.05, 0.01 or 0.001 respectively.

Article Snippet: Antibody Technique Manufacturer and catalogue Dilution Anti- HN1 IB, IF, IP Invitrogen—PA5109824 1/750, 1/300, 2 μg Anti- Cdk2 IB Santa Cruz—sc- 6248 1/500 Anti- β- Actin IB Sigma—A3854 1/500.000 Anti- p62 IB Proteintech—18,420- 1- AP 1/10000 Anti- β- catenin IB Santa Cruz—sc59737 1/500 Anti- Cdk1 IB Abcam—ab131450 1/1000 Anti- p- 4EBP1(S65) IB, IF Santa Cruz—sc- 293,124 1/500, 1/200 Anti- 4EBP1/2/3 IB Santa Cruz—sc- 271,947 1/500 Anti- 4EBP1 IB Santa Cruz—sc- 9977 1/500 Anti- p- GSK3β(S9) IB Santa Cruz—sc- 11,757 1/500 Anti- p- GSK3β (Tyr 216) IB Santa Cruz—sc- 135,653 1/500 Anti- p- p70S6K1(S434) IB, IF Santa Cruz—sc- 8416 1/500, 1/200 Anti- p- RPS6(S235/236) IB, IF Santa Cruz—sc- 293,144 1/500, 1/200 Anti- RPS6 IB, IF Santa Cruz—sc- 74,459 1/500, 1/200 Anti- UBF IB, IF Santa Cruz—sc- 13,125 1/500, 1/200 Anti- mTOR IB Santa Cruz—sc- 517,464 1/500 Anti- nucleolin IB, IF Santa Cruz—sc- 8031 1/500, 1/200 Anti- BrdU IF, FC Santa Cruz—sc- 32,323 1/200, 1/200 Anti- GAPDH IB Ambion—AM4300 1/240.000 Anti- lamin A/C IB Santa Cruz—sc- 376,248 1/500 Anti- rabbit IB Jackson Immuno Research—111- 035- 003 1/25.000 Anti- mouse IB Jackson Immuno Research—515- 035- 003 1/25.000 Alexa488 (anti- mouse IgG) IF Thermo A- 21202 1/600 Alexa594 (anti- mouse IgG) IF Thermo A- 21203 1/600 Alexa488 (anti- rabbit IgG) IF Thermo A- 11008 1/600 Alexa594 (anti- rabbit IgG) IF Jackson Immuno Research 711- 585- 152 1/600 13652184, 0, D ow nloaded from https://onlinelibrary.w iley.com /doi/10.1111/cpr.13805 by IN A SP - N E PA L , W iley O nline L ibrary on [25/01/2025].

Techniques: Staining, Over Expression, Quantitation Assay, Comparison

Journal: Cell proliferation

Article Title: HN1 Functions in Protein Synthesis Regulation via mTOR-RPS6 Axis and Maintains Nucleolar Integrity.

doi: 10.1111/cpr.13805

Figure Lengend Snippet: FIGURE 6 | (A) 18S and 28S rRNA levels were relatively quantitated using qRT-PCR using a double set of specific primers. It was found that the 18S rRNA level slightly but significantly (p < 0.001) decreased in LNCaP cells, and increased in PC3 cells, but did not change in MDA-MB231 cells in HN1 KD samples in comparison to controls. The slight alterations in 28S rRNA level were also observed in both LNCaP and PC3 cell lines but they were not significant. (The data obtained from two different experiments and six to eight replicates for each exp.). (B, C) Same batch of whole cell (2 × 106) lysates were used in polysome analysis and observed that 80S and polysome abundance significantly (p < 0.001) decreased in HN1 KD PC3, and (D, E) MDA-MB231 cells in comparison to controls. (The data were obtained from two different sets of shHN1 experiments and four technical replicates for each cell line). Polysome profiles of control and silenced cells were not overlayed to make it easier to observe subtle differences. The area under each region (e.g., free RNA, 40S, 60S, 80S, and polysomes) was calculated and its percentage in the total area was demonstrated in the graph. The statistical significance of the data was calculated and given in graphs as either *, ** and *** corresponding p > 0.05, 0.01 or 0.001 respectively.

Article Snippet: Antibody Technique Manufacturer and catalogue Dilution Anti- HN1 IB, IF, IP Invitrogen—PA5109824 1/750, 1/300, 2 μg Anti- Cdk2 IB Santa Cruz—sc- 6248 1/500 Anti- β- Actin IB Sigma—A3854 1/500.000 Anti- p62 IB Proteintech—18,420- 1- AP 1/10000 Anti- β- catenin IB Santa Cruz—sc59737 1/500 Anti- Cdk1 IB Abcam—ab131450 1/1000 Anti- p- 4EBP1(S65) IB, IF Santa Cruz—sc- 293,124 1/500, 1/200 Anti- 4EBP1/2/3 IB Santa Cruz—sc- 271,947 1/500 Anti- 4EBP1 IB Santa Cruz—sc- 9977 1/500 Anti- p- GSK3β(S9) IB Santa Cruz—sc- 11,757 1/500 Anti- p- GSK3β (Tyr 216) IB Santa Cruz—sc- 135,653 1/500 Anti- p- p70S6K1(S434) IB, IF Santa Cruz—sc- 8416 1/500, 1/200 Anti- p- RPS6(S235/236) IB, IF Santa Cruz—sc- 293,144 1/500, 1/200 Anti- RPS6 IB, IF Santa Cruz—sc- 74,459 1/500, 1/200 Anti- UBF IB, IF Santa Cruz—sc- 13,125 1/500, 1/200 Anti- mTOR IB Santa Cruz—sc- 517,464 1/500 Anti- nucleolin IB, IF Santa Cruz—sc- 8031 1/500, 1/200 Anti- BrdU IF, FC Santa Cruz—sc- 32,323 1/200, 1/200 Anti- GAPDH IB Ambion—AM4300 1/240.000 Anti- lamin A/C IB Santa Cruz—sc- 376,248 1/500 Anti- rabbit IB Jackson Immuno Research—111- 035- 003 1/25.000 Anti- mouse IB Jackson Immuno Research—515- 035- 003 1/25.000 Alexa488 (anti- mouse IgG) IF Thermo A- 21202 1/600 Alexa594 (anti- mouse IgG) IF Thermo A- 21203 1/600 Alexa488 (anti- rabbit IgG) IF Thermo A- 11008 1/600 Alexa594 (anti- rabbit IgG) IF Jackson Immuno Research 711- 585- 152 1/600 13652184, 0, D ow nloaded from https://onlinelibrary.w iley.com /doi/10.1111/cpr.13805 by IN A SP - N E PA L , W iley O nline L ibrary on [25/01/2025].

Techniques: Quantitative RT-PCR, Comparison, Control