Journal: PLOS Pathogens

Article Title: CD56-mediated activation of human natural killer cells is triggered by Aspergillus fumigatus galactosaminogalactan

doi: 10.1371/journal.ppat.1012315

Figure Lengend Snippet: CD56 expression on naïve NK cells (Control) and NK cells stimulated for 3 h with A . fumigatus germ tubes, depending on pre-treatment of the germ tubes with proteinase K (A) or periodate (B). Data for 3 independent donors are shown. (C) Significantly differentially expressed genes after 3-h stimulation of NK cells with A . fumigatus germ tubes pre-treated (60 min) with periodate or untreated (0 min). Abbreviations: adj. p = adjusted p-value, D = donor, fc = fold change. (D) Volcano plot summarizing transcriptional changes in NK cells stimulated with periodate-treated A . fumigatus germ tubes compared to cells stimulated with untreated germ tubes. Genes with a log2 fold change > 0.5 and an adjusted p-value < 0.05 are highlighted. Selected immune-related genes have been labelled. (E) CD56 expression on naïve NK cells (Control) and NK cells stimulated for 3 h with A . fumigatus germ tubes, depending on the duration of periodate pre-treatment of the germ tubes. Representative histograms and data for cells from 5 independent donors are shown. (F) Chemokine and perforin release by naïve NK cells (Control) and NK cells stimulated for 3 h with A . fumigatus germ tubes, depending on the duration of periodate pre-treatment of the germ tubes. (A-B; E-F) Columns and error bars indicate means and standard deviations, respectively. Repeated measures one-way analysis of variance with Tukey’s post-hoc test. * p < 0.05, ** p < 0.01, *** p < 0.001.

Article Snippet: Hyphae were incubated with soluble CD56 (5 μg/mL, R&D Systems, Minneapolis, MN, USA, Cat#2408-NC) or BSA (control, 5 μg/mL, Roth, Karlsruhe, Germany, #0163.2) in colorless RPMI for 2 h at 37°C, 5% CO 2 .

Techniques: Expressing, Control

Journal: PLOS Pathogens

Article Title: CD56-mediated activation of human natural killer cells is triggered by Aspergillus fumigatus galactosaminogalactan

doi: 10.1371/journal.ppat.1012315

Figure Lengend Snippet: (A) CD56 binding to fungal carbohydrates and proteins, as determined by enzyme-linked immunosorbent assay. GAG = galactosaminogalactan, RodAp = surface rodlet protein/hydrophobin. N = 3 technical replicates. One-way analysis of variance (ANOVA) with Dunnett’s post-hoc test versus Control, i.e., no coating. (B) Pull-down assay of CD56 with urea-insoluble galactosaminogalactan (PGG) or β-1,3-glucan. Lane M: Protein marker; Lane A: loading buffer extract of GAG + CD56 pellet; Lane B: supernatant of GAG + CD56; Lane C: CD56 alone; Lane D: supernatant of β-1,3-glucan + CD56; Lane E: loading buffer extract of β-1,3-glucan + CD56 pellet. (C) CD56 and CD69 expression on naïve NK cells (Control) and NK cells stimulated for 24 h with different concentrations of PGG. Representative histograms and data for cells isolated from 3 independent donors are shown. (D) Chemokine release by naïve NK cells (Control) and NK cells stimulated for 24 h with different concentrations of PGG. N = 3 independent donors. (C-D) Repeated measures (RM) one-way ANOVA with Tukey’s post-hoc test. (E) CD56, CD69, and CD107a expression on naïve NK cells (Control) and NK cells stimulated for 6 h with A . fumigatus (ATCC46645, AF) or A . nidulans (ATCC11267, AN) at different multiplicities of infection (MOIs). Representative histograms for one donor at MOI 4 are shown. (F) MOI-dependent stimulation of NK-cellular chemokine secretion after 6-h stimulation with AF or AN. (E-F) N = 3 independent donors. RM one-way ANOVA with Dunnett’s post-hoc test versus Control, i.e., unstimulated NK cells (asterisks). In addition, AF and AN stimulation at each MOI was compared using paired t-Test (hash signs). (A-F) Columns and error bars indicate means and standard deviations, respectively. */# p < 0.05, **/## p < 0.01, *** p < 0.001.

Article Snippet: Hyphae were incubated with soluble CD56 (5 μg/mL, R&D Systems, Minneapolis, MN, USA, Cat#2408-NC) or BSA (control, 5 μg/mL, Roth, Karlsruhe, Germany, #0163.2) in colorless RPMI for 2 h at 37°C, 5% CO 2 .

Techniques: Binding Assay, Enzyme-linked Immunosorbent Assay, Control, Pull Down Assay, Marker, Expressing, Isolation, Infection

Journal: PLOS Pathogens

Article Title: CD56-mediated activation of human natural killer cells is triggered by Aspergillus fumigatus galactosaminogalactan

doi: 10.1371/journal.ppat.1012315

Figure Lengend Snippet: (A) Representative fluorescent micrographs (z-projection of 3–4 slices with 1 μm distance, representative dataset from ≥5 independent experiments) of hyphae of wild-type (WT) A . fumigatus Af293 and two galactosaminogalactan (GAG)-deficient A . fumigatus mutants (Δ uge3 and Δ agd3 ) co-cultured with soluble CD56, followed by staining with fluorescent anti-CD56 antibody. The shape of the hyphae is indicated by dotted lines The insets represent a control image (bottom; BSA stained with anti-CD56 antibody) of the respective strain. Scale: 10 μm. (B) CD56, CD69, and CD107a expression on naïve NK cells (Control) and NK cells stimulated for 6 h with WT Af293 or the GAG-deficient A . fumigatus mutants (Δ uge3 and Δ agd3 ) at different multiplicities of infection (MOIs). Representative histograms for one donor at MOI 4 are shown. (C) MOI-dependent induction of NK-cellular secretion of granzyme B, perforin, and chemokines after 6-h stimulation with WT Af293, Δ uge3 , and Δ agd3 . (B-C) N = 3 independent donors. Repeated measures (RM) one-way analysis of variance (ANOVA) with Dunnett’s post-hoc test versus Control, i.e., unstimulated NK cells (asterisks). In addition, results for stimulation with the 3 strains at each MOI was compared using RM one-way ANOVA with Dunnett’s post-hoc test versus WT (hash signs). Columns and error bars indicate means and standard deviations, respectively. */# p < 0.05, **/## p < 0.01, ***/### p < 0.001. (D) CLSM micrographs of NK cells co-cultured with WT Af293 and Δ agd3 A . fumigatus hyphae. CD56 was stained with anti-CD56 Alexa Fluor 647 (red) to assess the CD56 localization. Germ tubes could be detected via their auto-fluorescence (cyan). Scale: 10 μm.

Article Snippet: Hyphae were incubated with soluble CD56 (5 μg/mL, R&D Systems, Minneapolis, MN, USA, Cat#2408-NC) or BSA (control, 5 μg/mL, Roth, Karlsruhe, Germany, #0163.2) in colorless RPMI for 2 h at 37°C, 5% CO 2 .

Techniques: Cell Culture, Staining, Control, Expressing, Infection, Fluorescence

Journal: PLOS Pathogens

Article Title: CD56-mediated activation of human natural killer cells is triggered by Aspergillus fumigatus galactosaminogalactan

doi: 10.1371/journal.ppat.1012315

Figure Lengend Snippet: (A) CD56 binding to fully acetylated (aPGG), fully deacetylated (dePGG), and native galactosaminogalactan (PGG), was determined by enzyme-linked immunosorbent assay (ELISA). Additional conditions with incomplete ELISA setup were included to preclude unspecific binding of the secondary antibody or anti-CD56 to the carbohydrates. N = 3 technical replicates. One-way analysis of variance (ANOVA) with Tukey’s post-hoc test was performed for each assay setup. (B) CD56, CD69, and CD107a expression on naïve NK cells (Control) and NK cells stimulated for 24 h with PGG, aPGG, or dePGG. Representative histograms for cells isolated from one donor are shown. (C) NK-cellular chemokine secretion after 24-h stimulation with PGG, aPGG, or dePGG. (B-C) N = 6 independent donors. Repeated measures (RM) one-way ANOVA with Tukey’s post-hoc test. (D) CD56, CD69, and CD107a expression on naïve NK cells (Control) and NK cells stimulated for 24 h with GalN oligomers, GalNAc oligomers, or chitosan. NK-cellular chemokine secretion after 24-h stimulation with GalN oligomers, GalNAc oligomers, or chitosan. N = 4 independent donors. RM one-way ANOVA with Tukey’s post-hoc test. (A-D) * p < 0.05, ** p < 0.01, *** p < 0.001.

Article Snippet: Hyphae were incubated with soluble CD56 (5 μg/mL, R&D Systems, Minneapolis, MN, USA, Cat#2408-NC) or BSA (control, 5 μg/mL, Roth, Karlsruhe, Germany, #0163.2) in colorless RPMI for 2 h at 37°C, 5% CO 2 .

Techniques: Binding Assay, Enzyme-linked Immunosorbent Assay, Expressing, Control, Isolation

Journal: PLOS Pathogens

Article Title: CD56-mediated activation of human natural killer cells is triggered by Aspergillus fumigatus galactosaminogalactan

doi: 10.1371/journal.ppat.1012315

Figure Lengend Snippet: (A) CD56 and CD69 expression of unstimulated NK cells (Control) and A . fumigatus -stimulated NK cells (6 h) depending on the fungal strain (wild type [WT] Af293 or isogenic mutants with defective GAG biosynthesis) and its enzymatic pre-treatment. (B) Secretion of granzyme B and perforin by unstimulated NK cells (Control) and A . fumigatus -stimulated NK cells depending (6 h) on the fungal strain and its enzymatic pre-treatment. (A-B) N = 6 independent donors. Repeated measures (RM) one-way analysis of variance (ANOVA) with Dunnett’s post-hoc test versus Control, i.e., unstimulated NK cells (asterisks). Additionally, enzymatic pre-treatments of A . fumigatus WT and the Δ agd3 mutant, respectively, were compared using RM one-way ANOVA with Dunnett’s post-hoc test versus no enzymatic pre-treatment (hash signs). Columns and error bars indicate means and standard deviations, respectively. */# p < 0.05, **/## p < 0.01, ***/### p < 0.001.

Article Snippet: Hyphae were incubated with soluble CD56 (5 μg/mL, R&D Systems, Minneapolis, MN, USA, Cat#2408-NC) or BSA (control, 5 μg/mL, Roth, Karlsruhe, Germany, #0163.2) in colorless RPMI for 2 h at 37°C, 5% CO 2 .

Techniques: Expressing, Control, Mutagenesis

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 , Schematic of metastatic LUAD and patient-derived xenograft (PDX) engraftment. b , Box and whisker plots of L1CAM IHC H-score in patient-derived primary tumor and metastasis samples. Primary tumor, n = 15; metastases, n = 54. *** P = 0.0002. c , Box and whisker plots of L1CAM IHC H-score in the primary tumors in ( b ) and in PDXs derived from primary tumor or metastases. Primary tumors, n = 15; primary tumor-derived PDXs, n = 36; metastasis-derived PDXs, n = 70. ns, P = 0.8811; * P = 0.0294 (left), 0.0420 (right). d , Schematic of the oncogenic transformation of AT2 cells into lung adenocarcinoma in the KP GEMM ( left panel ), and box and whisker plots of the percentage of L1CAM + cells in the KP mouse lungs and tumor areas at different time points after lentivirus instillation ( right panel ). Week 0, n = 6; week 14-19, n = 6; week 20-32, n = 15. ns, P = 0.5710; ** P = 0.0032. e , Schematic of the tumor tissue section ( left panel ), and box and whisker plots of the percentage of L1CAM + cells in the tumor center and invasive front ( right panel ). Tumor center, n = 300 cells; invasive front, n = 258 cells. N = 7 image frames. * P = 0.0152. f , L1CAM immunofluorescence (IF) staining of LUAD patient tissue samples at the tumor center (P, papillary) or the invasive front (M, micropapillary). Magnified regions are indicated in red boxes. Scale bar, 20 μm. g , Confocal microscopy image of L1CAM IF staining and Hoechst counterstaining of nuclei in KP tumoroids grown for 5 days. Schematic of tumoroids generated from KP-derived cancer cells ( lower right panel ). Scale bar, 10 μm. h , Percentage of L1CAM + cells in KP primary tumors versus tumoroids. Primary tumor, n = 4 experiments; tumoroids, n = 6 experiments. Mean ± s.e.m. ** P = 0.0095. i , Widefield fluorescence microscopy image of KP tumoroids stained with calcein AM in KP tumor cells sorted by L1CAM expression and grown as tumoroids for 7 days. The magnified region showing a single cell is indicated by a dotted box and that of a tumoroid by a solid box. Scale bar, 400 μm. j , Box and whisker plots of tumoroids formed per 1,000 cells after 7 days in culture. L1CAM − , n = 16; L1CAM + , n = 16. **** P < 0.0001. k , Box and whisker plots of cross-section area per tumoroid in the experiment of panel ( I ). L1CAM - , n = 807; L1CAM + , n = 477. **** P < 0.0001. l , Schematic representation of the generation of L1cam knockout KP LUAD GEMMs. m , H&E staining of KP and KPL1 primary tumors (26 week post-Cre) followed by their histopathological grading using an automated deep neural network. Magnified regions are shown in a red square. Scale bar, 1 mm. n , Fraction of KP and KPL1 tumors (22-26 week post-Cre) based on histopathological grading. Grade 1, green ; Grade 2, orange ; Grade 3, blue ; Grade 4, red . KP, n = 4; KPL1, n = 4. Mean ± S.D. o , H&E staining of KP metastasis in the subcapsular sinus of the lymph node (top panel) or ribcage bone (bottom panel). Invasive front is shown with a dotted yellow line. T, tumor; LN, lymph node. Scale bar, 100 μm. p , Incidence of primary tumor and spontaneous metastases upon viral transduction in KP and KPL1 mice. The organ specificity of spontaneous metastasis is shown as a percentage of the total metastatic tumors. LN, lymph node. KP primary, n = 46; KPL1 primary, n = 30; KP met, n = 42; KPL1 met, n = 16. ns, P = 1; * P = 0.0122. q , Representative images of subcutaneous tumors formed 6 weeks after inoculating 500 KP or KPL1 cells in athymic mice. Scale bar, 2 mm. r , Limiting dilution assay of subcutaneous tumor formation at various doses of KP or KPL1 cells in athymic mice. n = 10 for each condition. s , Fluorescent images of GFP + KP or KPL1 cells seeded in lungs at week 1 after tail vein injection (10 5 cells each) into athymic mice. Scale bar, 10 μm. t , Quantification of KP and KPL1 cells seeded in lungs from the experiment in ( S ). KP, n = 9; KPL1, n = 10. ns, P = 0.3154. u , Representative image of ex vivo lung BLI at week 5 after tail vein injection of single-cell suspension of KP and KPL1 tumoroids (2 x 10 4 cells) into athymic mice. v , Quantification of ex vivo lung BLI signal in the experiment in ( u ). KP, n = 28; KPL1, n = 14. *** P = 0.0006. w , The KM plot showing the overall survival after performing tail vein injections with KP or KPL1 cells. KP, n = 10; KPL1, n = 10. **** P < 0.0001. Statistical significance was assessed using the two-tailed Mann-Whitney test ( b , e , h , j , k , n , t , v ), one-way analysis of variance followed by the Tukey test ( c , d ), two-tailed Fisher’s exact test ( p ) or log-rank (Mantel-Cox) test ( o , w ). Data are shown as a box (median ± 25-75%) and whisker (maximum to minimum values) plot ( b - e , j , k , t , v ).

Article Snippet: For immunofluorescence staining, samples were fixed in 4% PFA for 10 min and permeabilized with 0.5% of Triton X-100 in PBS for another 10 min. After incubating with 10% normal goat serum (Life Technologies Cat# 50062Z) for 1 h at room temperature, the samples were incubated with primary antibodies overnight at 4°C in blocking solution with antibodies against mouse L1CAM (Miltenyi Biotec Cat# 130-115-812, AB_2727206), human L1CAM (Santa Cruz Biotechnology Cat# sc-53386, RRID: AB_628937), Sox2 (Invitrogen Cat# 14-9811-82, RRID: AB_891383), CELSR1 (Millipore Sigma Cat# ABT119, RRID: AB_11215810), c-Jun(pS73) (Cell Signaling Technology Cat# 9164, RRID: AB_330892), GFP (Aves Labs Cat# GFP-1010, RRID: AB_2307313), mouse FZD6 (R&D Systems Cat# AF1526, RRID: AB_354842), human FZD6 (Abcam Cat# AB150545, RRID: AB_3697520), Sox9 (Invitrogen Cat# 14-9765-82, RRID:AB_2573006), Cleaved Caspase-3 (Cell Signaling Technology Cat# 9661, RRID: AB_2341188), anti-TTF1/NKX2-1 (abcam Cat# ab76013, RRID: AB_1310784), or human cytokeratin (Dako Cat# M3515, RRID: AB_2132885).

Techniques: Derivative Assay, Whisker Assay, Transformation Assay, Immunofluorescence, Staining, Confocal Microscopy, Generated, Fluorescence, Microscopy, Expressing, Knock-Out, Transduction, Limiting Dilution Assay, Injection, Ex Vivo, Suspension, 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 , Representative L1CAM IHC staining in primary tumors, pleural fluids, and metastatic samples from LUAD patients and three tissue microarrays (Array 1, green; Array 2, blue; Array 3, red) containing normal human lung tissue and LUAD PDX (Passage 0 or 1). Scale bar, 100 μm. b , Heatmap of L1CAM IHC staining quantification (H-score) in primary tumor, pleural fluid, metastatic samples, and PDXs from LUAD patients. Each column represents an individual sample. Samples are color-coded according to their site of tissue. c , Maximum projection of a PDX tumoroid fluorescently stained with L1CAM. Scale bar, 10 μm. d , Cross-section of a PDX tumoroid stained with L1CAM IF and DAPI (nuclei). Scale bar, 10 μm. e , Close up of a PDX tumoroid stained with L1CAM IF and DAPI. Scale bar, 5 μm.

Article Snippet: For immunofluorescence staining, samples were fixed in 4% PFA for 10 min and permeabilized with 0.5% of Triton X-100 in PBS for another 10 min. After incubating with 10% normal goat serum (Life Technologies Cat# 50062Z) for 1 h at room temperature, the samples were incubated with primary antibodies overnight at 4°C in blocking solution with antibodies against mouse L1CAM (Miltenyi Biotec Cat# 130-115-812, AB_2727206), human L1CAM (Santa Cruz Biotechnology Cat# sc-53386, RRID: AB_628937), Sox2 (Invitrogen Cat# 14-9811-82, RRID: AB_891383), CELSR1 (Millipore Sigma Cat# ABT119, RRID: AB_11215810), c-Jun(pS73) (Cell Signaling Technology Cat# 9164, RRID: AB_330892), GFP (Aves Labs Cat# GFP-1010, RRID: AB_2307313), mouse FZD6 (R&D Systems Cat# AF1526, RRID: AB_354842), human FZD6 (Abcam Cat# AB150545, RRID: AB_3697520), Sox9 (Invitrogen Cat# 14-9765-82, RRID:AB_2573006), Cleaved Caspase-3 (Cell Signaling Technology Cat# 9661, RRID: AB_2341188), anti-TTF1/NKX2-1 (abcam Cat# ab76013, RRID: AB_1310784), or human cytokeratin (Dako Cat# M3515, RRID: AB_2132885).

Techniques: Immunohistochemistry, Staining

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 , L1CAM IHC staining in lung and tumor sections in KP mice at baseline and at different time points after lentivirus instillation to induce adenocarcinoma formation. Yellow, week 0; orange, week 14; red, week 24. Scale bar, 50 μm. b , L1CAM IHC staining of the patient tissue sections including normal tissue and tumor. The invasive tumor front is indicated with a yellow line. Scale bar, 200 μm. c , H&E and L1CAM IHC staining of KP primary tumor sections (26 week post-Cre) showing the indicated histological subtypes. Scale bar, 50 μm. d , Fraction of histological subtypes observed in KP primary tumors. n = 203,870 cells. e , Percentage of L1CAM + cells in KP primary tumors with respect to histological subtypes. Lepidic ( n = 26,871); Papillary ( n = 88,381); Micropapillary (red colored, n = 4,116); Acinar ( n = 3,430); Solid ( n = 81,072). **** P < 0.0001. f , Representative GFP (cancer cells) IF and L1CAM IF images of primary tumor and an autochthonous lymph node metastasis from a KP mouse (35 week post-Cre). Scale bar, 20 μm. g , Quantification of L1CAM IF intensity in the experiment ( f ). LN, lymph node; AU, arbitrary unit. n = 24 image frames; N = 3 mice in each condition. **** P < 0.0001. h , H&E staining of KP tumoroids grown in Matrigel over the course of 7 days. Scale bar, 100 μm. I , Brightfield and GFP fluorescence microscopy images of KP tumoroids at day 7. Scale bar, 10 μm. j , Close up of L1CAM IHC staining of patient samples and KP tumors. Scale bar, 20 μm. Statistical significance was assessed using the two-tailed Mann-Whitney test ( g ) or one-way analysis of variance followed by the Tukey test ( e ). Data are shown as mean ± S.D. ( d , e ).

Article Snippet: For immunofluorescence staining, samples were fixed in 4% PFA for 10 min and permeabilized with 0.5% of Triton X-100 in PBS for another 10 min. After incubating with 10% normal goat serum (Life Technologies Cat# 50062Z) for 1 h at room temperature, the samples were incubated with primary antibodies overnight at 4°C in blocking solution with antibodies against mouse L1CAM (Miltenyi Biotec Cat# 130-115-812, AB_2727206), human L1CAM (Santa Cruz Biotechnology Cat# sc-53386, RRID: AB_628937), Sox2 (Invitrogen Cat# 14-9811-82, RRID: AB_891383), CELSR1 (Millipore Sigma Cat# ABT119, RRID: AB_11215810), c-Jun(pS73) (Cell Signaling Technology Cat# 9164, RRID: AB_330892), GFP (Aves Labs Cat# GFP-1010, RRID: AB_2307313), mouse FZD6 (R&D Systems Cat# AF1526, RRID: AB_354842), human FZD6 (Abcam Cat# AB150545, RRID: AB_3697520), Sox9 (Invitrogen Cat# 14-9765-82, RRID:AB_2573006), Cleaved Caspase-3 (Cell Signaling Technology Cat# 9661, RRID: AB_2341188), anti-TTF1/NKX2-1 (abcam Cat# ab76013, RRID: AB_1310784), or human cytokeratin (Dako Cat# M3515, RRID: AB_2132885).

Techniques: Immunohistochemistry, Staining, Fluorescence, Microscopy, 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 , Representative images of tumor-bearing KP and KPL1 mice analyzed by BLI at week 16 after viral transduction. b , Growth curve of KP and KPL1 primary tumor burden as determined by BLI. KP, n = 37; KPL1, n = 14. ns, P > 0.9999. c , Micro-CT scan images of normal lung and tumor-bearing lungs from KP and KPL1 mice at week 22 after viral transduction. Arrows , tumors; red h , heart. d , Tumor burden comparison between KP and KPL1 mice based on micro-CT scans at week 22. n = 38 KP mice, n = 10 KPL1 mice. ns, P = 0.3192. e , Tumor areas of KP and KPL1 mouse lungs at week 22-30 after viral transduction. n = 4 mice. ns, P = 0.6857. f , Graphical representation of mouse survival prior to reaching the target tumor burden, set at a luciferase bioluminescence flux of 10 7 photons/sec per mouse chest. KP, n = 35; KPL1, n = 14. ns, P = 0.7143. g , Quantification of tumoroids formed per 10,000 cells over 7 days. KP, n = 6; KPL1, n = 6. ns, P = 0.6991. h , Schematic of three different metastasis inoculation routes. i , Representative image of ex vivo lung BLI signals at week 5 after intratracheal delivery of single-cell suspension of KP and KPL1 tumoroids (2 x 10 4 cells) into athymic mice. j , Quantification of ex vivo lung BLI signal in the experiment in panel ( i ). KP, n = 8; KPL1, n = 6. ** P = 0.0027. k , Representative image of ex vivo lung BLI signals at week 3 after intracardiac injection of single-cell suspension of KP and KPL1 tumoroids (5 x 10 4 cells) into athymic mice. l , Quantification of ex vivo lung BLI signals in the experiment in panel ( k ). KP, n = 9; KPL1, n = 5. ** P = 0.0070. m , Representative image of ex vivo kidney BLI signals at week 3 after intracardiac injection of single-cell suspension of KP tumoroids or KPL1 tumoroids (5 x 10 4 cells). n , Quantification of ex vivo kidney BLI signals at week 3 after intracardiac injection of single-cell suspension of KP tumoroids or KPL1 tumoroids. KP, n = 9; KPL1, n = 5. * P = 0.0120. o , Representative image of ex vivo brain BLI signals at week 3 after intracardiac injection of single-cell suspension of KP tumoroids or KPL1 tumoroids (5 x 10 4 cells). p , Quantification of ex vivo brain BLI signals at week 3 after intracardiac injection of single-cell suspension of KP tumoroids or KPL1 tumoroids. KP, n = 9; KPL1, n = 5. * P = 0.0120. q , Representative image of ex vivo liver BLI signals at week 3 after intracardiac injection of single-cell suspension of KP tumoroids or KPL1 tumoroids (5 x 10 4 cells). r , Quantification of ex vivo liver BLI signals at week 3 after intracardiac injection of single-cell suspension of KP tumoroids or KPL1 tumoroids. KP, n = 9; KPL1, n = 5. * P = 0.0120. s , Western immunoblotting analysis of Ru631 PDX cells transduced with two different L1CAM shRNAs. t , H&E staining of lung sections four weeks after injecting Ru631 L1CAM knockdown via tail vein. Scale bar, 50 μm. u , Quantification of Ru631 tumors after tail vein injection. n = 4 mice per condition. * P = 0.0337; ** P = 0.0013. v , Western immunoblotting of L1CAM knockdowns with two different short hairpins in Ru323 PDXs. w , H&E staining of lung sections four weeks after injecting Ru323 with L1CAM knockdown via tail vein. Scale bar, 50 μm. x , Quantification of Ru323 tumors after tail vein injection. n = 5 mice per condition. *P = 0.0123; ** P = 0.0058. Data are shown as a box (median ± 25-75%) and whisker (maximum to minimum values) plot ( d , e , j , l , n , p , r ). Error bar indicates mean ± S.D. ( b ). Statistical significance was assessed using Kolmogorov-Smirnov test ( b ), two-tailed Mann-Whitney test ( d , e , g , j , l , n , p , r ) or one-way analysis of variance followed by the Tukey test ( u , x ).

Article Snippet: For immunofluorescence staining, samples were fixed in 4% PFA for 10 min and permeabilized with 0.5% of Triton X-100 in PBS for another 10 min. After incubating with 10% normal goat serum (Life Technologies Cat# 50062Z) for 1 h at room temperature, the samples were incubated with primary antibodies overnight at 4°C in blocking solution with antibodies against mouse L1CAM (Miltenyi Biotec Cat# 130-115-812, AB_2727206), human L1CAM (Santa Cruz Biotechnology Cat# sc-53386, RRID: AB_628937), Sox2 (Invitrogen Cat# 14-9811-82, RRID: AB_891383), CELSR1 (Millipore Sigma Cat# ABT119, RRID: AB_11215810), c-Jun(pS73) (Cell Signaling Technology Cat# 9164, RRID: AB_330892), GFP (Aves Labs Cat# GFP-1010, RRID: AB_2307313), mouse FZD6 (R&D Systems Cat# AF1526, RRID: AB_354842), human FZD6 (Abcam Cat# AB150545, RRID: AB_3697520), Sox9 (Invitrogen Cat# 14-9765-82, RRID:AB_2573006), Cleaved Caspase-3 (Cell Signaling Technology Cat# 9661, RRID: AB_2341188), anti-TTF1/NKX2-1 (abcam Cat# ab76013, RRID: AB_1310784), or human cytokeratin (Dako Cat# M3515, RRID: AB_2132885).

Techniques: Transduction, Micro-CT, Comparison, Luciferase, Ex Vivo, Suspension, Injection, Western Blot, Staining, Knockdown, 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 , Schematic summary of lung developmental continuum from early to late progenitor stages defined by specific transcription factors. b , Representative 3D maximum intensity projections (MIP) images of SOX2, NKX2-1 and SOX9 IF in the anterior foregut domain in embryonic day E9.5 mouse embryos and the distal lung bud tip in E10.5 mouse embryos. The dashed box demarcates the SOX2/NKX2-1 boundary in E9.5 and NKX2-1/SOX9 boundary in E10.5 as illustrated in the zoomed 2D (Z-Slice) panels on the right. All data were independently validated from replicate samples of at least n = 4 embryos with similar results obtained. c , SOX2, NKX2-1, and SOX9 IF staining in patient-derived primary tumor and metastasis tissue sections. Magnified regions are highlighted in yellow boxes. Scale bar, 50 μm. d , Fraction of cancer cells expressing SOX2, NKX2-1, or SOX9 by IF analysis in patient-derived primary tumor and metastasis tissue sections. Mean ± S.D. n = 6 patient samples each. ns, P = 0.4848 (NKX2-1), 0.3095 (SOX9); ** P = 0.0043. e , SOX2 and SOX9 IHC staining in LUAD patient-derived primary tumor and pleural metastasis samples. Magnified regions are highlighted in red boxes. Scale bar, 100 μm. f , Box and whisker plots of SOX2 and SOX9 IHC H-score in the patient-derived primary tumor and metastasis samples. Primary, n = 15; Metastasis, n = 7. ns, P = 0.1229; ** P = 0.0011. g , Relative median survival of LUAD patients based on the expression of SOX2 , NKX2-1 , and SOX9 in the primary tumor. Gene expression and survival data compiled from GEO, EGA and TCGA. SOX2 (low, n = 706; high, n = 705); NKX2-1 (low, n = 1083; high, n = 1083); SOX9 (low, n = 1083; high, n = 1083). * P = 0.0219; *** P = 0.0003; **** P < 0.0001. h , H&E staining of lung tissue sections harboring metastatic colonies upon tail vein injection of KP tumoroid cells expressing control or Sox2 short hairpins (sh). Magnified regions are indicated in red boxes. Scale bar, 5 mm. i,j , Number per lung ( I ) and percent area ( j ) of metastases in the experiment of panel ( h ). n = 10. **** P < 0.0001. k , UMAP of scRNA-seq data from autochthonous KP tumors collected 32 weeks after viral instillation. n = 4,489 cells. Transcriptionally distinct clusters were computed by Leiden algorithm and numbered. l , Heatmap showing imputed average LUAD developmental marker expression in the clusters from primary tumors ranked left to right according to the average L1cam expression level. m , UMAP of scRNA-seq data from KP tumoroids collected after 7 days. n = 12,962 cells. Transcriptionally distinct clusters were computed by Leiden algorithm and are represented by a different color and a number. n , Heatmap showing imputed average LUAD developmental marker expression in the clusters from 7-day tumoroids ranked left to right according to the average L1cam expression level. Lung developmental progenitor stages represented by the predominant expression of Sox2 ( early developmental stage), Nkx2-1 and Foxa2 ( middle progenitor stage), Hmga2 and Sox9 ( late progenitor stage) are indicated. o , Representative L1CAM and SOX2 IF staining of the invasive front ( dotted yellow line ) within an autochthonous KP tumor (32 week post-Cre) and of KP-derived tumoroids grown for 4 days or 7 days. T , tumor; N , normal tissue. Magnified regions are indicated in red boxes. Scale bar, 50 μm for primary tumor; 20 μm for tumoroids. p , Relative Sox2 mRNA expression of KP tumoroid-derived cells sorted by L1CAM expression. Mean ± S.E.M. n = 3. * P = 0.0258. q , L1CAM and SOX2 IHC staining of representative LUAD PDXs with high or low expression of L1CAM. Scale bar, 50 μm. r , Scatter plot and linear regression ( red line ) of L1CAM versus SOX2 IHC H-score data from LUAD PDXs. Data are log-transformed for visualization. n = 36. s , L1CAM and SOX2 IHC staining in serial sections of patient-derived bone metastasis and liver metastasis tissues. Scale bar, 100 μm. t , Scatter plot and linear regression ( red line ) of L1CAM versus SOX2 IHC H-score data from LUAD patient primary and metastasis samples. Data are log-transformed for visualization. n = 61 samples (primary, 15; metastasis, 46). Spearman correlation was used to calculate the relationship between L1CAM expression and SOX2 expression ( r,t ). Data are shown as a box (median ± 25-75%) and whisker (maximum to minimum values) plot ( f , i,j ). Statistical significance was assessed using the two-tailed Mann-Whitney test ( d , f , g , i , j ) or two-tailed t test after passing the Shapiro-Wilk normality test ( p ).

Article Snippet: For immunofluorescence staining, samples were fixed in 4% PFA for 10 min and permeabilized with 0.5% of Triton X-100 in PBS for another 10 min. After incubating with 10% normal goat serum (Life Technologies Cat# 50062Z) for 1 h at room temperature, the samples were incubated with primary antibodies overnight at 4°C in blocking solution with antibodies against mouse L1CAM (Miltenyi Biotec Cat# 130-115-812, AB_2727206), human L1CAM (Santa Cruz Biotechnology Cat# sc-53386, RRID: AB_628937), Sox2 (Invitrogen Cat# 14-9811-82, RRID: AB_891383), CELSR1 (Millipore Sigma Cat# ABT119, RRID: AB_11215810), c-Jun(pS73) (Cell Signaling Technology Cat# 9164, RRID: AB_330892), GFP (Aves Labs Cat# GFP-1010, RRID: AB_2307313), mouse FZD6 (R&D Systems Cat# AF1526, RRID: AB_354842), human FZD6 (Abcam Cat# AB150545, RRID: AB_3697520), Sox9 (Invitrogen Cat# 14-9765-82, RRID:AB_2573006), Cleaved Caspase-3 (Cell Signaling Technology Cat# 9661, RRID: AB_2341188), anti-TTF1/NKX2-1 (abcam Cat# ab76013, RRID: AB_1310784), or human cytokeratin (Dako Cat# M3515, RRID: AB_2132885).

Techniques: Staining, Derivative Assay, Expressing, Immunohistochemistry, Whisker Assay, Gene Expression, Injection, Control, Marker, Transformation 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 , SOX2 and NKX2-1 IF staining of patient-derived LUAD primary tumor tissue section. The invasive front is marked with a dotted yellow line, and the magnified low-grade and high-grade tumor areas are shown in red boxes. Scale bar, 1 mm. b , SOX2 and NKX2-1 IF staining of patient-derived primary tumor tissue section with different tumor grade areas. Invasive front is shown with a dotted yellow line. Scale bar, 20 μm. c , SOX2 and SOX9 IF staining in KP primary tumor and metastasis tissue sections collected simultaneously from the same mouse (35 week post-Cre). Scale bar, 100 μm. d , Western immunoblotting analysis of SOX2 expression after dox-induced Sox2 knockdown in KP tumoroids. e , f , Box and whisker plots of the imputed transcript levels of Sox2 ( e ) and L1cam ( f ) in the scRNA-seq transcriptional clusters from KP primary tumors. g , Scatter plot of L1cam versus Sox2 expression in the scRNA-seq dataset from KP primary tumors. Data from transcriptional cluster 9 are shown in black. H , I , Box and whisker plots of the imputed transcript levels of Sox2 ( h ) and L1cam ( i ) from 7-day tumoroids. Data from the cluster 12 are shown in red. j , Scatter plot of L1cam versus Sox2 expression in the scRNA-seq dataset from 7-day tumoroids. Data from transcriptional cluster 12 are shown in black. k , Proportion of SOX9 + cells detected by IF in lung colonies after injecting a high number (1 x 10 5 ) of KP or KPL1 cells via the tail vein into athymic mice. KP, n = 10; KPL1, n = 5. ** P = 0.0013. l , Relative expression of SOX9 in two independent KP tumoroid lines upon conditional knockdown of L1CAM. Bar graphs, mean ± S.D. n = 3 in each condition. ns, P = 0.8301; * P = 0.0202, 0.0305; ** P = 0.0094. m , Western immunoblotting analysis of L1CAM levels after L1CAM overexpression in KPL1 tumoroids. Data are shown as a box (median ± 25-75%) and whisker (maximum to minimum values) plot ( e , f , h , i , k ). Spearman correlation was used to calculate the relationship between L1CAM expression and SOX2 expression ( g , j ). Statistical significance was assessed using the two-tailed Mann-Whitney test ( k ) or one-way analysis of variance followed by the Tukey test ( l ).

Article Snippet: For immunofluorescence staining, samples were fixed in 4% PFA for 10 min and permeabilized with 0.5% of Triton X-100 in PBS for another 10 min. After incubating with 10% normal goat serum (Life Technologies Cat# 50062Z) for 1 h at room temperature, the samples were incubated with primary antibodies overnight at 4°C in blocking solution with antibodies against mouse L1CAM (Miltenyi Biotec Cat# 130-115-812, AB_2727206), human L1CAM (Santa Cruz Biotechnology Cat# sc-53386, RRID: AB_628937), Sox2 (Invitrogen Cat# 14-9811-82, RRID: AB_891383), CELSR1 (Millipore Sigma Cat# ABT119, RRID: AB_11215810), c-Jun(pS73) (Cell Signaling Technology Cat# 9164, RRID: AB_330892), GFP (Aves Labs Cat# GFP-1010, RRID: AB_2307313), mouse FZD6 (R&D Systems Cat# AF1526, RRID: AB_354842), human FZD6 (Abcam Cat# AB150545, RRID: AB_3697520), Sox9 (Invitrogen Cat# 14-9765-82, RRID:AB_2573006), Cleaved Caspase-3 (Cell Signaling Technology Cat# 9661, RRID: AB_2341188), anti-TTF1/NKX2-1 (abcam Cat# ab76013, RRID: AB_1310784), or human cytokeratin (Dako Cat# M3515, RRID: AB_2132885).

Techniques: Staining, Derivative Assay, Western Blot, Expressing, Knockdown, Whisker Assay, Over Expression, 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 , SOX2 IF staining of KP and KPL1 primary tumors (32 week post-Cre). Scale bar, 10 μm. b , Percentage of SOX2 + cells in the experiment from panel ( a ). n = 3 mice for each condition. * P = 0.0411. c , d , Relative L1cam mRNA expression and Sox2 mRNA expression in KP and KPL1 tumoroids. KP, n = 4; KPL1, n = 4. * P = 0.0286 ( c , d ). e , IF images of KP and KPL1 tumors in the lung after injecting a high number (1 x 10 5 ) of single cells from KP and KPL1 tumoroids into athymic mice via the tail vein. Magnified regions are highlighted in yellow boxes. Scale bar, 100 μm. f , Proportion of SOX2 + cells detected by IF in lung colonies after injecting a high number (1 x 10 5 ) of KP or KPL1 cells via the tail vein in athymic mice. KP, n = 10; KPL1, n = 5. * P = 0.0400. g , h , Relative expression of L1cam ( g ) and Sox2 ( h ) in two independent KP tumoroid lines upon Dox-dependent conditional knock down of L1cam . n = 4. **** P < 0.0001. i , j , Western immunoblot analysis ( i ) and quantification ( j ) of SOX2 and SOX9 levels in control and L1CAM-overexpressing (OE) KP tumoroids. n = 4. * P = 0.0286. k , l , H&E staining ( k ) and quantification ( l ) of subcutaneous tumors or lung metastases after injection of KPL1 cells with or without L1CAM overexpression into athymic mice, analyzed at 4 weeks after subcutaneous (subQ) injection or 5 weeks after tail vein (TV) injection. Scale bar, 1 mm (SubQ) and 100 μm (TV). n = 5. ns, P > 0.9999; * P = 0.0238. m , GFP (cancer cells) and SOX2 IF staining of control vs L1CAM overexpressing KPL1 cells in lung metastasis after 5 week-post tail vein injection. Magnified regions are highlighted in yellow boxes. Scale bar, 10 μm. n , Fraction of SOX2 + cells in the experiment from panel ( m ). Control, n = 5; L1CAM overexpression, n = 5. ** P = 0.0079. Data are shown as a box (median ± 25-75%) and whisker (maximum to minimum values) plot ( f ). Bar graphs, mean ± S.D. ( c , d , g , h , j ) or ± S.E.M. ( b , l , n ). Statistical significance was assessed using the two-tailed t test after passing the Shapiro-Wilk normality test ( b ), two-tailed Mann-Whitney test ( c , d , f , j , l , n ) or one-way analysis of variance followed by the Tukey test ( g , h ).

Article Snippet: For immunofluorescence staining, samples were fixed in 4% PFA for 10 min and permeabilized with 0.5% of Triton X-100 in PBS for another 10 min. After incubating with 10% normal goat serum (Life Technologies Cat# 50062Z) for 1 h at room temperature, the samples were incubated with primary antibodies overnight at 4°C in blocking solution with antibodies against mouse L1CAM (Miltenyi Biotec Cat# 130-115-812, AB_2727206), human L1CAM (Santa Cruz Biotechnology Cat# sc-53386, RRID: AB_628937), Sox2 (Invitrogen Cat# 14-9811-82, RRID: AB_891383), CELSR1 (Millipore Sigma Cat# ABT119, RRID: AB_11215810), c-Jun(pS73) (Cell Signaling Technology Cat# 9164, RRID: AB_330892), GFP (Aves Labs Cat# GFP-1010, RRID: AB_2307313), mouse FZD6 (R&D Systems Cat# AF1526, RRID: AB_354842), human FZD6 (Abcam Cat# AB150545, RRID: AB_3697520), Sox9 (Invitrogen Cat# 14-9765-82, RRID:AB_2573006), Cleaved Caspase-3 (Cell Signaling Technology Cat# 9661, RRID: AB_2341188), anti-TTF1/NKX2-1 (abcam Cat# ab76013, RRID: AB_1310784), or human cytokeratin (Dako Cat# M3515, RRID: AB_2132885).

Techniques: Staining, Expressing, Knockdown, Western Blot, Control, Injection, 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 , 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: For immunofluorescence staining, samples were fixed in 4% PFA for 10 min and permeabilized with 0.5% of Triton X-100 in PBS for another 10 min. After incubating with 10% normal goat serum (Life Technologies Cat# 50062Z) for 1 h at room temperature, the samples were incubated with primary antibodies overnight at 4°C in blocking solution with antibodies against mouse L1CAM (Miltenyi Biotec Cat# 130-115-812, AB_2727206), human L1CAM (Santa Cruz Biotechnology Cat# sc-53386, RRID: AB_628937), Sox2 (Invitrogen Cat# 14-9811-82, RRID: AB_891383), CELSR1 (Millipore Sigma Cat# ABT119, RRID: AB_11215810), c-Jun(pS73) (Cell Signaling Technology Cat# 9164, RRID: AB_330892), GFP (Aves Labs Cat# GFP-1010, RRID: AB_2307313), mouse FZD6 (R&D Systems Cat# AF1526, RRID: AB_354842), human FZD6 (Abcam Cat# AB150545, RRID: AB_3697520), Sox9 (Invitrogen Cat# 14-9765-82, RRID:AB_2573006), Cleaved Caspase-3 (Cell Signaling Technology Cat# 9661, RRID: AB_2341188), anti-TTF1/NKX2-1 (abcam Cat# ab76013, RRID: AB_1310784), or human cytokeratin (Dako Cat# M3515, RRID: AB_2132885).

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: For immunofluorescence staining, samples were fixed in 4% PFA for 10 min and permeabilized with 0.5% of Triton X-100 in PBS for another 10 min. After incubating with 10% normal goat serum (Life Technologies Cat# 50062Z) for 1 h at room temperature, the samples were incubated with primary antibodies overnight at 4°C in blocking solution with antibodies against mouse L1CAM (Miltenyi Biotec Cat# 130-115-812, AB_2727206), human L1CAM (Santa Cruz Biotechnology Cat# sc-53386, RRID: AB_628937), Sox2 (Invitrogen Cat# 14-9811-82, RRID: AB_891383), CELSR1 (Millipore Sigma Cat# ABT119, RRID: AB_11215810), c-Jun(pS73) (Cell Signaling Technology Cat# 9164, RRID: AB_330892), GFP (Aves Labs Cat# GFP-1010, RRID: AB_2307313), mouse FZD6 (R&D Systems Cat# AF1526, RRID: AB_354842), human FZD6 (Abcam Cat# AB150545, RRID: AB_3697520), Sox9 (Invitrogen Cat# 14-9765-82, RRID:AB_2573006), Cleaved Caspase-3 (Cell Signaling Technology Cat# 9661, RRID: AB_2341188), anti-TTF1/NKX2-1 (abcam Cat# ab76013, RRID: AB_1310784), or human cytokeratin (Dako Cat# M3515, RRID: AB_2132885).

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

doi: 10.1101/2025.08.22.671773

Figure Lengend Snippet: a , FZD6 IF staining with counterstained nuclei in KP tumoroids upon conditional knockdown of L1cam . Scale bar, 10 μm. b , Relative intensity of FZD6 IF quantified in KP tumoroids upon conditional knockdown of L1cam . Left to right, n = 25, 23, 35, 29, 39, 41, 45, 53 tumoroids. * P = 0.0440, *** P = 0.0010. c , Relative Fzd6 mRNA level in upon conditional knockdown of Fzd6 in KP tumoroids. n = 4 experiments. **** P < 0.0001. d , Relative Sox2 mRNA level upon conditional knockdown of FZD6 in KP tumoroids. n = 4 experiments. ** P = 0.0040. e , IF staining for L1CAM, c-Jun(pS73), and SOX2 in KP tumoroids ( upper panels ) and image segmentation to quantify the signal ( bottom panels ). Scale bar, 10 μm. f , Pie chart showing the percent of cells staining positive for c-Jun(pS73) and SOX2 in KP tumoroids. n = 649 cells. g , IF staining for cytokeratin, L1CAM, and c-Jun(pS73) in a patient-derived LUAD lymph node metastasis. Scale bar, 50 μm. h , Pie chart showing the percent of cells staining positive for c-Jun(pS73) and L1CAM in patient-derived LUAD lymph node metastases. n = 2,166 cells from 12 different lymph nodes. i , c-Jun(pS73) IF staining and counterstained nuclei in KP tumoroids upon conditional knockdown of L1CAM. Scale bar, 20 μm. j , Percentage of cells staining positive for c-Jun(pS73) after conditional knockdown of L1cam in KP tumoroids. Left to right, n = 94, 67, 87, 101, 38, 41, 36, 36 tumoroids. **** P < 0.0001. k , Western immunoblotting analysis of L1CAM, S73 phosphorylated and total c-Jun levels in H23 LUAD cells treated with 10 μM (JNK-IN-8) JNK inhibitor (JNKi) for 2 h. l , Sox2 mRNA relative expression level upon incubation of KP tumoroids with 20 μM JNK inhibitor for 24 h. n = 7 for KP tumoroid #1; 4 for KP tumoroid #2. *** P < 0.0006; * P = 0.0286. m , Western immunoblotting analysis of S73 phosphorylated and total c-Jun levels upon the treatments with anisomycin for 6 h. n , Sox2 mRNA relative expression level upon incubation of KP cells with anisomycin for 6 h. n = 3. ** P = 0.0064 (left), 0.0021 (right). The bar graph indicates mean ± S.E.M. ( b - d , j , l , n ). Statistical significance was assessed using the one-way analysis of variance followed by the Tukey test ( b - d , j , n ) or two-tailed Mann-Whitney test ( l ).

Article Snippet: For immunofluorescence staining, samples were fixed in 4% PFA for 10 min and permeabilized with 0.5% of Triton X-100 in PBS for another 10 min. After incubating with 10% normal goat serum (Life Technologies Cat# 50062Z) for 1 h at room temperature, the samples were incubated with primary antibodies overnight at 4°C in blocking solution with antibodies against mouse L1CAM (Miltenyi Biotec Cat# 130-115-812, AB_2727206), human L1CAM (Santa Cruz Biotechnology Cat# sc-53386, RRID: AB_628937), Sox2 (Invitrogen Cat# 14-9811-82, RRID: AB_891383), CELSR1 (Millipore Sigma Cat# ABT119, RRID: AB_11215810), c-Jun(pS73) (Cell Signaling Technology Cat# 9164, RRID: AB_330892), GFP (Aves Labs Cat# GFP-1010, RRID: AB_2307313), mouse FZD6 (R&D Systems Cat# AF1526, RRID: AB_354842), human FZD6 (Abcam Cat# AB150545, RRID: AB_3697520), Sox9 (Invitrogen Cat# 14-9765-82, RRID:AB_2573006), Cleaved Caspase-3 (Cell Signaling Technology Cat# 9661, RRID: AB_2341188), anti-TTF1/NKX2-1 (abcam Cat# ab76013, RRID: AB_1310784), or human cytokeratin (Dako Cat# M3515, RRID: AB_2132885).

Techniques: Staining, Knockdown, Derivative Assay, Western Blot, Expressing, Incubation, 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 , Venn diagram of transcription factors and chromatin modifiers differentially active in L1CAM + /SOX2 + KP tumoroid cells (scRNA-seq cluster #12, ) based on CHEA and ENCODE databases. b , PLA image of CHD1 and c-Jun in H23 LUAD cells. The PLA signals are shown as red dots overlayed with nuclei staining. Scale bar, 10 μm. c , Quantification of PLA signals per nucleus. Control, n = 63 cells; CHD1/c-Jun, n = 39 cells. **** P < 0.0001. d , Co-immunoprecipitation of CHD1 and c-Jun in KP tumoroids. e , Metaplots of CHD1 only (green), c-Jun only (blue), and CHD1/c-Jun overlap (red) ChIP-seq peak summits relative to peak center of CHD1 (left) or c-Jun (right) in H23 cells. f , Venn diagram showing the overlap between CHD1 and c-Jun genome-wide peaks. g , ChIP-seq analysis of CHD1 and c-Jun binding to the Sox2 locus in H23 cells. Sox2 enhancers ( red ), promoter and gene body ( green ) are indicated. h , ChIP-qPCR analysis of CHD1 binding to the SOX2 promoter in H23 cells with and without JNK inhibitor. n = 3 experiments. *** P = 0.0002. i , Co-immunoprecipitation of CHD1 and the transcriptional elongation factor RTF1 in H23 LUAD cells. j , Western immunoblotting analysis of SOX2 levels upon CHD1 knockout in H23 LUAD cells. k , Western immunoblotting analysis of SOX2 levels upon CHD1 knockdown in KP tumoroids using two different shRNAs. l , H&E staining of lung sections from NSG mice after tail-vein inoculation of H23 cells with CRISPR/Cas9-induced knockouts of CHD1 or L1CAM. Magnified regions are shown ( red boxes ). Scale bar, 100 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 ( j ). n = 6 per experimental condition. ** P = 0.0035, 0.0060, 0.0015 from left to right; * P = 0.0301 in ( m ). ** P = 0.0026, 0.0028, 0.0011 from left to right; *** P = 0.0006 in ( n ). o , Model of L1CAM-dependent PCP activation of c-Jun/CHD1 driven SOX2 expression in LUAD progenitors to generate a metastasis-initiating state. Data are shown as a box (median ± 25-75%) and whisker (maximum to minimum values) plot ( c , m , n ). The bar graph indicates mean ± S.E.M. ( h ). Statistical significance was assessed using a one-way analysis of variance followed by the Tukey test ( h , m , n ) or two-tailed Mann-Whitney test ( c ).

Article Snippet: For immunofluorescence staining, samples were fixed in 4% PFA for 10 min and permeabilized with 0.5% of Triton X-100 in PBS for another 10 min. After incubating with 10% normal goat serum (Life Technologies Cat# 50062Z) for 1 h at room temperature, the samples were incubated with primary antibodies overnight at 4°C in blocking solution with antibodies against mouse L1CAM (Miltenyi Biotec Cat# 130-115-812, AB_2727206), human L1CAM (Santa Cruz Biotechnology Cat# sc-53386, RRID: AB_628937), Sox2 (Invitrogen Cat# 14-9811-82, RRID: AB_891383), CELSR1 (Millipore Sigma Cat# ABT119, RRID: AB_11215810), c-Jun(pS73) (Cell Signaling Technology Cat# 9164, RRID: AB_330892), GFP (Aves Labs Cat# GFP-1010, RRID: AB_2307313), mouse FZD6 (R&D Systems Cat# AF1526, RRID: AB_354842), human FZD6 (Abcam Cat# AB150545, RRID: AB_3697520), Sox9 (Invitrogen Cat# 14-9765-82, RRID:AB_2573006), Cleaved Caspase-3 (Cell Signaling Technology Cat# 9661, RRID: AB_2341188), anti-TTF1/NKX2-1 (abcam Cat# ab76013, RRID: AB_1310784), or human cytokeratin (Dako Cat# M3515, RRID: AB_2132885).

Techniques: Staining, Control, Immunoprecipitation, ChIP-sequencing, Genome Wide, Binding Assay, ChIP-qPCR, Western Blot, Knock-Out, Knockdown, CRISPR, Whisker Assay, Activation Assay, Expressing, Two Tailed Test, MANN-WHITNEY

Journal: Journal of neurochemistry

Article Title: A specific isoform of glial cell line-derived neurotrophic factor family receptor alpha 1 regulates RhoA expression and glioma cell migration.

doi: 10.1111/j.1471-4159.2010.06975.x

Figure Lengend Snippet: Fig. 8 NCAM but not Ret mediated cell migration and RhoA expression in C6 cells. C6 cells transfected with control, GFRa1, Ret or NCAM siRNA. (a) C6 cell migration after siRNA transfection was examined by wound healing assay. (b) Quantification of C6 cell migration by trans-well migration assay. (c) Western blot analysis of GFRa1, Ret, NCAM and RhoA expressions in siR- NA-transfected C6 cells. **p < 0.01.

Article Snippet: Small interfering RNA duplexes specific for GFRa1a and GFRa1b were designed against the unique exon junctions. siRNAs for GFRa1a and GFRa1b were from Invitrogen; siRNAs for total GFRa1 and RhoA were from Integrated DNA Technologies (IDT, Coralville, IA, USA), siRNAs for Ret (sc-156121) and NCAM (sc156119) were from Santa Cruz Biotechnologies; luciferase control siRNA was obtained from Sigma.

Techniques: Migration, Expressing, Transfection, Control, Wound Healing Assay, Western Blot