Journal: bioRxiv

Article Title: L1CAM signaling through planar cell polarity generates SOX2 + metastatic progenitors in lung adenocarcinoma

doi: 10.1101/2025.08.22.671773

Figure Lengend Snippet: a , Scatter plot of accumulated enrichment score and negative log-transformed false discovery rate (FDR) of pathways associated with the L1CAM + /SOX2 + tumoroid cell cluster defined by scRNA-seq. The pathway with the highest total enrichment score and the lowest FDR is labeled in red. b , Scatter plot showing the expanded list of individual WNT/PCP-related terms. c , Schematic representations of L1CAM and PCP complex components at a cell-cell junction. d , Heatmap displaying the average expression of core PCP components in the KP tumoroid cell clusters defined in . The clusters are ranked left to right according to the average L1cam expression level. e , L1CAM and CELSR1 IF staining in KP tumoroid-derived cell monolayer. The merged image is magnified for visualization ( red box ). Scale bar, 10 μm. f , L1CAM and CELSR1 IF staining of lung metastasis one week after tail vein injection of H23 LUAD cells into athymic mice. Scale bar, 10 μm. g , Co-immunoprecipitation of L1CAM and the PCP component CELSR1 in H23 LUAD cell lysates. h , Quantification of L1CAM/CELSR1 PLA dots per cell in H23 LUAD cells. Negative PLA control, n = 25 cells; L1CAM/CELSR1 PLA, n = 31 cells. **** P < 0.0001. i , Control and L1CAM-knockout (KO) H23 cell monolayers were subjected to cytokeratin, CELSR1, and SOX2 IF staining. Scale bar, 20 μm. j , Fraction of SOX2 + cells quantified in control versus L1CAM-knockout H23 cell monolayers. Mean ± S.D. Control, n = 43 cells; L1CAM KO, n = 58 cells. * P = 0.0488. k , Western immunoblotting analysis of control and L1CAM-knockdown ( shL1CAM ) PDXs after incubating the cells with 1 μM bafilomycin A for 24 h. l , H&E staining of lung sections of athymic mice after tail-vein inoculation of L1CAM-knockout H23 cells with or without SOX2 overexpression. Scale bar, 20 μm. m , n , Box and whisker plots showing the number ( m ) and percent area ( n ) of metastatic lesions per lung in the experiments of panel ( l ). n = 5 for L1CAM KO-1; 10 for L1CAM KO-2. * P = 0.0317; ** P = 0.0012 in ( m ). ** P = 0.0079; *** P = 0.0002 ( n ). Data are shown as a box (median ± 25-75%) and whisker (maximum to minimum values) plot ( h , m , n ). Statistical significance was assessed using the two-tailed Mann-Whitney test ( h , j , m , n ).

Article Snippet: Once membranes were blocked with Intercept Tris-buffered saline (TBS) blocking buffer (LICOR Biosciences Cat# 927-60001) for 1 h at room temperature, they were probed with antibodies against c-Jun (Cell Signaling Technology Cat# 9165, RRID: AB_2130165), c-Jun(pS73) (Cell Signaling Technology Cat# 9164, RRID: AB_330892), CHD1 (Cell Signaling Technology Cat# 4351, RRID: AB_11179073), β-actin (Sigma-Aldrich Cat# A1978, RRID: AB_476692), L1CAM (BioLegend Cat# 826701, RRID: AB_2564904), Sox2 (Cell Signaling Technology Cat# 3579, RRID: AB_2195767), CELSR1 (Bethyl Cat# A304-432A, RRID: AB_2620626), and RTF1 (Cell Signaling Technology Cat# 14737, RRID: AB_2798594) in blocking buffer for overnight at 4°C.

Techniques: Transformation Assay, Labeling, Expressing, Staining, Derivative Assay, Injection, Immunoprecipitation, Control, Knock-Out, Western Blot, Knockdown, Over Expression, Whisker Assay, Two Tailed Test, MANN-WHITNEY

Journal: bioRxiv

Article Title: L1CAM signaling through planar cell polarity generates SOX2 + metastatic progenitors in lung adenocarcinoma

doi: 10.1101/2025.08.22.671773

Figure Lengend Snippet: a , WNT/PCP pathway scoring as the top hit among highly enriched genes in L1CAM + /SOX2 + KP tumoroid cells. Top 3000 ranked differentially expressed genes in cluster #12 versus the rest of cell population in day-7 KP tumoroids were analyzed to determine uniquely enriched biological pathways from the Gene Ontology (GO), Reactome, and PANTHER databases. 30 pathways shared among these three analyses were concatenated into three related processes. 13 of these pathways were from the WNT/PCP-related process and had the lowest accumulated false discovery rate (FDR). Within the WNT/PCP-related processes, the PCP and non-canonical WNT signaling pathways showed the highest enrichment score. b , Heatmap displaying the average expression of core PCP components in the primary tumor clusters defined in . The clusters are ranked left to right according to the average L1cam expression level. c , L1CAM and CELSR1 IF staining in KP tumoroids. The magnified region is indicated by red or white boxes . Scale bar, 10 μm. d , Image analysis workflow for detecting the colocalization of L1CAM and CELSR1 at cell-cell junctions using a steerable filter to extract curvilinear image features. e , Computer vision-driven detection and segmentation of L1CAM and CELSR1 at cell-cell junctions through steerable filtering. The thickness of segmented junctions was dilated for visualization purposes. Scale bar, 1 μm. f , L1CAM and CELSR1 PLA fluorescence in H23 LUAD cells. The PLA signals are shown as red dots with nuclei counterstaining. Scale bar, 10 μm. g , L1CAM western immunoblotting analysis in parental and L1CAM knockout H23 cells. h , Western immunoblotting analysis of SOX2 overexpression in H23 LUAD cells upon CRISPR/Cas9-mediated L1CAM knockout, clones 1 and 2. i. L1CAM and CELSR1 IF staining in HEK293T cells engineered to overexpress L1CAM versus control. Scale bar, 10 μm. j , L1CAM and CELSR1 segmentation analysis at cell-cell junctions in L1CAM overexpressing HEK293T cells. The region of higher magnification is indicated by a red box . k , Quantification of CELSR1 at cell-cell junctions in control or L1CAM overexpressing HEK293T cells. Control, n = 3406 junctions; L1CAM OE, n = 6508 junctions. **** P < 0.0001. l , L1CAM and FZD6 IF staining in control or L1CAM overexpressing HEK293T cells. Scale bar, 10 μm. m , Western immunoblotting analysis of SOX2 overexpression in CHD1 knockout H23 cells. n , Box and whisker plots showing the size of metastatic lesions per lung in SOX2 overexpressing, CHD1 knockout H23 cells. n = 9 for CHD1 KO-1; 10 for CHD1 KO-2. ns, P = 0.2805; * P = 0.0415. Data are shown as a box (median ± 25-75%) and whisker (maximum to minimum values) plot ( k , n ). Statistical significance was assessed using the one-way analysis of variance followed by the Tukey test ( k ) or two-tailed Mann-Whitney test ( n ).

Article Snippet: Once membranes were blocked with Intercept Tris-buffered saline (TBS) blocking buffer (LICOR Biosciences Cat# 927-60001) for 1 h at room temperature, they were probed with antibodies against c-Jun (Cell Signaling Technology Cat# 9165, RRID: AB_2130165), c-Jun(pS73) (Cell Signaling Technology Cat# 9164, RRID: AB_330892), CHD1 (Cell Signaling Technology Cat# 4351, RRID: AB_11179073), β-actin (Sigma-Aldrich Cat# A1978, RRID: AB_476692), L1CAM (BioLegend Cat# 826701, RRID: AB_2564904), Sox2 (Cell Signaling Technology Cat# 3579, RRID: AB_2195767), CELSR1 (Bethyl Cat# A304-432A, RRID: AB_2620626), and RTF1 (Cell Signaling Technology Cat# 14737, RRID: AB_2798594) in blocking buffer for overnight at 4°C.

Techniques: Protein-Protein interactions, Expressing, Staining, Fluorescence, Western Blot, Knock-Out, Over Expression, CRISPR, Clone Assay, Control, Whisker Assay, Two Tailed Test, MANN-WHITNEY

Journal: bioRxiv

Article Title: ZER1 contributes to the carcinogenic activity of high-risk HPV E7 proteins

doi: 10.1101/2022.07.18.500567

Figure Lengend Snippet: (A) Schematic of HPV16-HPV18 E7 chimeras. (B) N/Tert-1 keratinocytes stably expressing wild-type HPV16 E7- or HPV18 E7-FlagHA, the HPV16-HPV18 E7-FlagHA chimeras, or an empty vector were subjected to anti-HA immunoprecipitation. Whole cell lysates (left) and immunoprecipitates (right) were separated by SDS/PAGE and Western blotted with antibodies to HA, ZER1, and RB1.

Article Snippet: Membranes were blocked in 5% nonfat dried milk in TBST (Tris-buffered saline pH 7.4 with 0.05% Tween 20) and then incubated with primary antibodies to ZER1 (GeneTex), CUL2 (Bethyl), RB1 (Calbiochem/EMD), UBR4 (gift of Yoshihiro Nakatani), GAPDH (Invitrogen), Actin (Millipore), HA-HRP (Roche), or PTPN14 (Cell Signaling Technology).

Techniques: Stable Transfection, Expressing, Plasmid Preparation, Immunoprecipitation, SDS Page, Western Blot

Journal: bioRxiv

Article Title: ZER1 contributes to the carcinogenic activity of high-risk HPV E7 proteins

doi: 10.1101/2022.07.18.500567

Figure Lengend Snippet: (A) Protein sequence alignment of high-risk HPV E7 proteins tested for ZER1 interaction in . Sequences were aligned using ClustalWS and amino acids colored according to the ClustalX color scheme. Arrows indicate amino acid residues unique to HPV16 E7. The LxCxE motif responsible for RB1 binding is underlined with a black bar. (B) Schematic of HPV16 E7 mutants. Amino acids indicated by the arrows were substituted with alanine residues. (C) N/Tert-1 cells stably expressing Flag-HA-tagged wild-type HPV16 E7, HPV18 E7, the HPV16 E7 mutants, or an empty vector were subjected to anti-HA immunoprecipitation. Whole cell lysates (left) and immunoprecipitates (right) were separated by SDS/PAGE and Western blotted with antibodies to HA, ZER1, and RB1. (D) Schematic of additional HPV16 E7 mutants. Amino acids indicated by the arrows were substituted with alanine residues. (E) N/Tert-1 cells stably expressing Flag-HA-tagged wild-type HPV16 E7, HPV18 E7, the HPV16 E7 mutants, or an empty vector were subjecteded to anti-HA immunoprecipitation. Whole cell lysates and immunoprecipitates were separated by SDS/PAGE and Western blotted with antibodies to HA, ZER1, CUL2, and RB1.

Article Snippet: Membranes were blocked in 5% nonfat dried milk in TBST (Tris-buffered saline pH 7.4 with 0.05% Tween 20) and then incubated with primary antibodies to ZER1 (GeneTex), CUL2 (Bethyl), RB1 (Calbiochem/EMD), UBR4 (gift of Yoshihiro Nakatani), GAPDH (Invitrogen), Actin (Millipore), HA-HRP (Roche), or PTPN14 (Cell Signaling Technology).

Techniques: Sequencing, Binding Assay, Stable Transfection, Expressing, Plasmid Preparation, Immunoprecipitation, SDS Page, Western Blot

Journal: bioRxiv

Article Title: ZER1 contributes to the carcinogenic activity of high-risk HPV E7 proteins

doi: 10.1101/2022.07.18.500567

Figure Lengend Snippet: N/Tert-1 keratinocytes stably expressing wild-type HPV16 E7-Flag-HA were fractionated on a Superose 6 column. Five hundred-microliter fractions were collected and the even numbered fractions were subjected to anti-HA immunoprecipitation. The unfractionated whole cell lysate (WCL) and immunoprecipitates were separated by SDS/PAGE and Western blotted with antibodies to HA, ZER1, CUL2, RB1, and UBR4.

Article Snippet: Membranes were blocked in 5% nonfat dried milk in TBST (Tris-buffered saline pH 7.4 with 0.05% Tween 20) and then incubated with primary antibodies to ZER1 (GeneTex), CUL2 (Bethyl), RB1 (Calbiochem/EMD), UBR4 (gift of Yoshihiro Nakatani), GAPDH (Invitrogen), Actin (Millipore), HA-HRP (Roche), or PTPN14 (Cell Signaling Technology).

Techniques: Stable Transfection, Expressing, Immunoprecipitation, SDS Page, Western Blot

Journal: bioRxiv

Article Title: ZER1 contributes to the carcinogenic activity of high-risk HPV E7 proteins

doi: 10.1101/2022.07.18.500567

Figure Lengend Snippet: (A) Western blot of N/Tert-1 keratinocytes stably expressing empty vector, Flag-HA-tagged wild-type HPV16 E7, or the HPV16 E7 mutants DLYC and E80A/D81A. Protein lysates were separated by SDS/PAGE and Western blotted with antibodies to the hypo-phosphorylated form of RB1, HA, and actin. (B) N/Tert-1 cells stably expressing empty vector, Flag-HA-tagged wild-type HPV16 E7, or the HPV16 E7 mutants DLYC and E80A/D81A were treated with 40 ug/mL cycloheximide (CHX) and harvested at the indicated time points. Protein lysates were separated by SDS/PAGE and Western blotted with antibodies to RB1, HA, and actin. The experiment was repeated three times and a representative image is shown. (C) Primary HFK were transduced with retroviral vectors encoding HPV16 E7, HPV16 E7 E80A/D81A, or GFP as a control. Poly(A)-selected RNA was analyzed by RNA-seq. Heat map showing the expression patterns of selected DNA replication initiation genes.

Article Snippet: Membranes were blocked in 5% nonfat dried milk in TBST (Tris-buffered saline pH 7.4 with 0.05% Tween 20) and then incubated with primary antibodies to ZER1 (GeneTex), CUL2 (Bethyl), RB1 (Calbiochem/EMD), UBR4 (gift of Yoshihiro Nakatani), GAPDH (Invitrogen), Actin (Millipore), HA-HRP (Roche), or PTPN14 (Cell Signaling Technology).

Techniques: Western Blot, Stable Transfection, Expressing, Plasmid Preparation, SDS Page, Transduction, Retroviral, Control, RNA Sequencing