Journal: Cell cycle (Georgetown, Tex.)

Article Title: Leucine zipper and ICAT domain containing (LZIC) protein regulates cell cycle transitions in response to ionizing radiation.

doi: 10.1080/15384101.2019.1601476

Figure Lengend Snippet: Figure 1. The loss of LZIC expression alters transcriptome under basal conditions and in response to ionizing radiation. (A) Venn diagram comparing the numbers of genes with significantly altered log-fold changes between LZIC KO clone vs CRISPR control cells in control and IR conditions. (B) Heatmaps representing Z-scores for genes comparing transcriptomic profiles in CRISPR control to LZIC KO Clone 1 cell lines in response to 5 Gy IR. (C) Heatmaps representing Z-scores for comparison of transcriptional profiles between CRISPR control and LZIC KO Clone 1 either under basal condition or following IR exposure. (D) Hallmark gene groups analyzed in GSEA and their associated FDR q-value (E) Barcode plots for significant GSEA hallmark gene groups. Microarray was repeated on two separate biological repeats with two technical repeats of each condition.

Article Snippet: LZIC knock-out line generation LZIC-targeting CRISPR-based knockout plasmid kit was purchased from Origene.

Techniques: Expressing, CRISPR, Control, Comparison, Microarray

Journal: Cell cycle (Georgetown, Tex.)

Article Title: Leucine zipper and ICAT domain containing (LZIC) protein regulates cell cycle transitions in response to ionizing radiation.

doi: 10.1080/15384101.2019.1601476

Figure Lengend Snippet: Figure 2. Late G2/M checkpoint arrest is perturbed following LZIC loss. (A) Cell cycle analysis of cell lines by propidium iodide staining. Cell lines were treated with 5 Gy IR and following a 24hr incubation harvested for analysis. Graphs are based on four separate biological repeats (B) Cell cycle analysis of LZIC KO Clone 2 and stable re-expression of LZIC-flag. Graphs based on three separate biological repeats. (C) p53 phosphorylation status in all cell lines, over a 4-h time course, following treatment with IR. A representative blot is shown from three separate biological repeats. (D) Activation of early G2/M checkpoint induction, following treatment with IR. The inclusion of Parental + ATMi provides a positive control for loss of early G2/M checkpoint activation. Graphs based on three separate biological repeats. (E) Quantification of phosphorylated serine 10 on Histone 3. Cells were treated with 2 Gy IR and harvested at 24 h. Images indicate staining profile with arrows to denote the quantified cells. Graphs based on three separate biological repeats. All statistical significance was determined with unpaired student T-Test, * = p-value < 0.05, n.s = non-significant. CRISPR control and LZIC KO Clones were compared to the parental line.

Article Snippet: LZIC knock-out line generation LZIC-targeting CRISPR-based knockout plasmid kit was purchased from Origene.

Techniques: Cell Cycle Assay, Staining, Incubation, Expressing, Phospho-proteomics, Activation Assay, Positive Control, CRISPR, Control, Clone Assay

Journal: Cell cycle (Georgetown, Tex.)

Article Title: Leucine zipper and ICAT domain containing (LZIC) protein regulates cell cycle transitions in response to ionizing radiation.

doi: 10.1080/15384101.2019.1601476

Figure Lengend Snippet: Figure 3. Cellular signaling for activation of G2/M checkpoint in response to IR is perturbed in LZIC KO cells. (A) Analysis of ATR and ATM phosphorylation status at 8 and 24-h post-treatment with 5 Gy IR. (*) indicates protein band corresponding to ATR protein. (B) Western blot analysis of checkpoint proteins at 8 h and 24 h in all cell types following treatment with 5 Gy IR. (C) Western blot analysis of Mitosis promoting factors at 8 and 24 h following treatment with 5 Gy IR. (D) Western blot analysis of major G2/M phosphatases, PP1 and PP2A, at 8 and 24 h following treatment with IR. (E) Schematic diagram of regulatory cascade showing key proteins involved in the G2/M cell cycle progression and their DNA damage-induced phosphorylation sites. All western blots shown are representative image of three separate biological repeats.

Article Snippet: LZIC knock-out line generation LZIC-targeting CRISPR-based knockout plasmid kit was purchased from Origene.

Techniques: Activation Assay, Phospho-proteomics, Western Blot

Journal: Cell cycle (Georgetown, Tex.)

Article Title: Leucine zipper and ICAT domain containing (LZIC) protein regulates cell cycle transitions in response to ionizing radiation.

doi: 10.1080/15384101.2019.1601476

Figure Lengend Snippet: Figure 4. Loss of LZIC leads to genome instability and poor prognosis in clear cell renal carcinoma. (A) Schematic outlining the development of aneuploidy following loss of G2/M checkpoint control. (B) Metaphase spread quantification of chromosome numbers from Parental, CRISPR control line, and LZIC KO Clone 1 and 2. Data from 3 biological repeats counting at least 17 spreads per replicate. Statistical significance was determined using unpaired student T-Test, *** = p-value < 0.001, n.s = non-significant. CRISPR control and LZIC KO clones were compared to the parental line in the untreated condition. (C) Kaplan Meier plot showing overall survival of patients stratified by LZIC expression. The calculated hazard ratios and significance is also included.

Article Snippet: LZIC knock-out line generation LZIC-targeting CRISPR-based knockout plasmid kit was purchased from Origene.

Techniques: Control, CRISPR, Clone Assay, Expressing

Journal: eBioMedicine

Article Title: SLK is mutated in individuals with a neurodevelopmental disorder

doi: 10.1016/j.ebiom.2025.105725

Figure Lengend Snippet: The effect of SLK deficiency on Paxillin localization and neuronal differentiation . (a) Immunofluorescence staining for Paxillin and Vinculin in fibroblasts derived from the index individual in Family 1 (IV:1) (SLK) and control (Control/WT) cell lines. Green and red represent Vinculin and Paxillin, respectively, while yellow indicates their colocalization. The scale bar in the bottom right corner denotes a length of 20 μm. (b) Anti-SLK immunostaining on control and SLK mutant patient fibroblasts, scale bar = 50 μM. ( c i ) SLK expression intensity in control vs SLK mutant fibroblasts (p < 0.0001 (unpaired t-test with Welch's correction)). (c ii ) Normalized SLK expression percentage in control vs SLK mutant fibroblasts (p < 0.0001 (unpaired t-test with Welch's correction)). (c iii ) Percentage change in cell density between control and SLK mutant fibroblasts (Kruskal–Wallis test, p = 0.1039). (d) Schematic representation of the transdifferentiation protocol (top panel). Representative images of control and SLK patient derived fibroblasts (PT D3 refers to 3 days post transduction with Brn2 and Ascl1 lentivirus, scale bar = 300 μM), transdifferentiated neurons (PT D12, 12 days after transduction; scale bar = 300 μM) and transdifferentiated fibroblasts were stained with Tubulin β-III and Map2. Scale bar = 50 μM. (e i ) Statistical analysis of the transdifferentiated SLK patient derived fibroblasts. (p = 0.0256 (Mann Whitney test of normalized cluster size)) and cell body diameter (e ii , p = 0.1229 (Unpaired t-test with Welch's correction of the number of neurites)). (e iii , p = 0.0471(Unpaired t-test with Welch's correction of the number of neurites)) and length of neurites (e iv , p = 0.7643 (Unpaired t-test with Welch's correction of the number of neurites)). (f) Schematic representation of lentiviral shRNA mediated knockdown (KD) of SLK followed by transdifferentiation to produce neurons (upper panel). shRNA mediated knockdown (KD) of SLK derived fibroblasts indicating the efficiency of the KD (bottom panel, Scale bar = 50 μM). (g i ) Analysis in fibroblasts reveals that the shRNA mediated KD of SLK resulted in a marked reduction in SLK expression intensity (p < 0.0001 (Mann–Whiteny test)). ( g ii ) Normalized SLK expression percentage decrease (Control vs shRNA mediated KD of SLK fibroblasts, (p < 0.0001 (Mann–Whiteny test)). (g iii ) Percentage change in cell density between control, scrambled shRNA and shRNA mediated KD of SLK fibroblasts (p = 0.0179 (Kruskal–Wallis test)). (h) Representative images of control, scrambled shRNA and shRNA mediated KD of SLK transdifferentiated neurons at 20× magnification. Scale bar = 300 μM. Please note the reduction in the SLK intensity in neurons derived from shRNA KD fibroblasts. (i i ) Normalized percentage change in SLK expression intensity (Control vs scrambled shRNA vs shRNA mediated KD of SLK fibroblasts, p = 0.0089 (Brown-Forsythe and Welch ANOVA tests) and ( i ii , p = 0.1773 (Kruskal–Wallis test, of the diameter of cell body)), number of neurites (I iii , p = 0.0178 (Kruskal–Wallis test, of the diameter of cell body)) and neurite length (I iv , p < 0.0001 (Kruskal–Wallis test, of the diameter of cell body)).

Article Snippet: SLK Human shRNA plasmid kit (Locus ID 9748, Cat.# TL320620, OriGene) was used for generating shRNA lentivirus to perform silencing of the SLK gene in fibroblasts.

Techniques: Immunofluorescence, Staining, Derivative Assay, Control, Immunostaining, Mutagenesis, Expressing, Transduction, MANN-WHITNEY, shRNA, Knockdown