Journal: bioRxiv

Article Title: Determinants of metal import and specificity in a bacterial transporter

doi: 10.64898/2026.03.30.714904

Figure Lengend Snippet: (a) Schematic of our fluorescent reporter system that leverages both transcriptional and translational control to couple intracellular Mn 2+ concentration to fluorescence. (b) Example green fluorescence distributions (kernel density estimates) measured by flow cytometry for E. coli cells expressing WT DraNramp (yellow), M230A (orange), and two variants with low (N59D; teal) or no (D56A; dark gray) transport activity, alongside the pET28a empty vector (light gray). The x-axis is plotted on a logicle scale and events were gated to have similar cell sizes as measured by side scatter. (c) Dose-response curves normalized to the D56A data relating MnCl 2 concentration in the growth medium to fluorescence for the same variants as in (b), with overlaid fits to a sigmoid curve (resulting kinetic parameters listed in Supplementary Table 1). Error bars represent standard error of the mean from three replicates; sample raw distributions and an overview of the analysis are in . (d) At the bottom is a kernel density plot showing the distribution of cells in the first replicate of the evolution-guided library screen on the correlated green fluorescence (FITC-A) and side scatter area axes; approximate locations of the four sorted bins are overlaid. Above is the log-transformed enrichment of WT and two variants with intermediate (M230A) or no (D56G) Mn 2+ transport activity, highlighting how enrichment scores vary across bins for different variants. (e) Distribution of Mn 2+ activity scores for substitutions in the evolution-guided library across mutational depth. Scores range between ∼0 to ∼1 (representing no activity to WT-like levels of activity), with scores above 1 representing improved activity and scores below zero likely representing experimental noise. (f) Heatmap of Mn 2+ import scores for all variants with single mutation at positions with at least 5 measured variants, primarily from the binding-site library. White circles mark wildtype amino acids. Gray positions lack data.

Article Snippet: We transformed these ligation products into electrocompetent DH10B E. coli (GoldBio) and the requisite number of colonies were harvested from agar plates to bottleneck each subpool to ∼40X the subpool diversity to balance obtaining nearly every variant with attaining a reasonable barcode diversity.

Techniques: Control, Concentration Assay, Fluorescence, Flow Cytometry, Expressing, Activity Assay, Plasmid Preparation, Transformation Assay, Mutagenesis, Binding Assay

Journal: bioRxiv

Article Title: Determinants of metal import and specificity in a bacterial transporter

doi: 10.64898/2026.03.30.714904

Figure Lengend Snippet: (a) MgKO, a Mg 2+ -auxotrophic strain of E. coli lacking any genetically encoded Mg 2+ transporters, can only survive in low Mg 2+ when rescued with a functional Mg 2+ transporter. (b) Example growth curves in LB supplemented with 1 mM MgSO 4 with 20 µM IPTG, showing robust growth for the Mg 2+ -transporting M230A variant (orange), no growth for the non-transporting WT DraNramp (gold), and intermediate growth for two replicates of the evolution-guided library (purple and teal). (c) Growth rates, normalized to WT in 1 mM supplemental MgSO 4 , measured across concentrations of Mg 2+ supplemented into LB medium. Rates are higher for M230A (right) compared to WT (left). Error bars represent standard error of the mean across three replicates. (d) Distribution of Mg 2+ import scores from the combined libraries, colored by whether the M230 position is mutated, with the region with scores above 2 (representing clear Mg 2+ import) in an inset. (e) Growth rates of isolated clones for a selected set of variants with no M230 mutation that have Mg 2+ import scores significantly higher than WT. M230A is included as a positive control. Stars represent significance thresholds from a series of Welch’s t-tests against the WT with a Benjamini-Hochberg correction (*: p<0.05). (f) Heatmaps of Mg 2+ import scores for all mutations to TM6 on the WT (top) and M230A (bottom) backgrounds. Black circles mark wildtype amino acids. Gray positions lack data. The 230 column is identical between heatmaps. Above the heatmaps is a snapshot of TM6 (PDB ID: 8E6N). The black asterisk approximates the bound Mn 2+ ; Mg 2+ may or may not bind the same site in the M230A variant. (g) Twenty-eight variants from the evolution-guided library with scores significantly outside the distribution defined by WT barcodes (FDR < 0.05 via Student’s t-test with a Benjamini-Hochberg correction). Variants were clustered based on which residues (color-coded by chemical property) were mutated using clustermap in seaborn. Arrows above and corresponding boxed columns highlight the positions of observed sequence couplings (purple: positions 54 and 275; green: positions 232 and 381/382). The asterisk represents a deletion of residues 125-127. (h) Scatterplot of Mg 2+ import scores for all single mutations present on both the WT (horizontal axis) and M230A (vertical axis) background. The gray diagonal line represents mutations with the same overall activity level on both backgrounds, while the horizontal gray lines represent the WT score (0.00 ± 0.04) or M230A score (7.29 ± 0.62). The black curve represents a fit to a site-independent sigmoid model, demonstrating how the mutations deviate considerably from an additive model even accounting for sigmoidal global epistasis. The shaded region around the sigmoidal curve represents a 99.7% confidence interval. A selection of mutations that improve Mg 2+ on their own are highlighted in teal, and several M230 mutations (which by definition have the same effect on each background) in orange.

Article Snippet: We transformed these ligation products into electrocompetent DH10B E. coli (GoldBio) and the requisite number of colonies were harvested from agar plates to bottleneck each subpool to ∼40X the subpool diversity to balance obtaining nearly every variant with attaining a reasonable barcode diversity.

Techniques: Functional Assay, Variant Assay, Isolation, Clone Assay, Mutagenesis, Positive Control, Sequencing, Activity Assay, Selection

Journal: Biology Direct

Article Title: FAM83D facilitates EMT and metastasis of cervical cancer via interaction with GSK3β and inactivation of GSK3β/stabilization of Snail signaling

doi: 10.1186/s13062-026-00761-z

Figure Lengend Snippet: FAM83D was associated with metastasis of CC. ( A ) Western blotting was employed to examine the expression of FAM83D in HeLa and SiHa cells with stable overexpression or knockdown of FAM83D. ( B-C ) Colony formation assay was conducted to evaluate the clonogenic ability of HeLa and SiHa cells following the overexpression or knockdown of FAM83D. ( D ) CCK-8 assay was used to detect cell viability in FAM83D-silenced HeLa and SiHa cells treated with different concentrations of 5-fluorouracil, cisplatin, oxaliplatin, and paclitaxel for 48 h. The inhibition rate was subsequently calculated. ( E-I ) Subcutaneous injection of FAM83D-silenced and control HeLa cells into BALB/c Nude mice ( n = 6) was used to determine the tumorigenicity. The tumor growth curves, tumor volumes, and tumor weights were measured. ( J ) And then, the nude mice that developed liver metastases were counted. ( K ) Representative images of liver metastases developed from FAM83D-silenced and control HeLa cells were shown, while ( L ) H&E staining of liver metastases in nude mice was presented. * p < 0.05, ** p < 0.01, *** p < 0.001

Article Snippet: Human CC cell lines SiHa and HeLa were purchased from the American Type Culture Collection (ATCC).

Techniques: Western Blot, Expressing, Over Expression, Knockdown, Colony Assay, CCK-8 Assay, Inhibition, Injection, Control, Staining

Journal: Biology Direct

Article Title: FAM83D facilitates EMT and metastasis of cervical cancer via interaction with GSK3β and inactivation of GSK3β/stabilization of Snail signaling

doi: 10.1186/s13062-026-00761-z

Figure Lengend Snippet: FAM83D promoted the metastasis of CC cells in vitro and vivo. ( A-D ) Wound healing assay was conducted to determine the migratory ability, and ( E-H ) transwell assay was utilized to determine the migratory and invasive ability, respectively, in HeLa and SiHa cells exhibiting stable overexpression or silencing of FAM83D. ( I ) Lung metastasis in nude mice ( n = 6) by tail vein injection of HeLa cells with or without silencing of FAM83D. ( J-K ) The statistical data of lung metastasis occurring in the mouse model. ( L ) Representative images and H&E staining of lung metastasis in nude mice. * p < 0.05, ** p < 0.01, *** p < 0.001

Article Snippet: Human CC cell lines SiHa and HeLa were purchased from the American Type Culture Collection (ATCC).

Techniques: In Vitro, Wound Healing Assay, Transwell Assay, Over Expression, Injection, Staining

Journal: Biology Direct

Article Title: FAM83D facilitates EMT and metastasis of cervical cancer via interaction with GSK3β and inactivation of GSK3β/stabilization of Snail signaling

doi: 10.1186/s13062-026-00761-z

Figure Lengend Snippet: FAM83D promoted GSK3β/Snail signaling and EMT. ( A-D ) Western blotting for E-cadherin, N-cadherin, Vimentin, and FAM83D in HeLa and SiHa cells following stable overexpression or silencing of FAM83D. ( E-H ) Western blotting for Snail, β-catenin, GSK3β, and phosphorylated GSK3β (Ser9), and FAM83D protein changes in CC cells with stable overexpression or silencing of FAM83D. ( I-J ) Cycloheximide (CHX) chase assays for Snail stability in CC cells with stable overexpression of FAM83D. ( K-L ) Ubiquitination assay for Snail protein ubiquitination level in CC cells with stable overexpression or silencing of FAM83D in the presence or absence of LiCl. * p < 0.05, ** p < 0.01, *** p < 0.001

Article Snippet: Human CC cell lines SiHa and HeLa were purchased from the American Type Culture Collection (ATCC).

Techniques: Western Blot, Over Expression, Ubiquitin Proteomics

Journal: Biology Direct

Article Title: FAM83D facilitates EMT and metastasis of cervical cancer via interaction with GSK3β and inactivation of GSK3β/stabilization of Snail signaling

doi: 10.1186/s13062-026-00761-z

Figure Lengend Snippet: FAM83D interacted with GSK3β. ( A ) The possible interaction between FAM83D and GSK3β was screened from the IntACT database. ( B-D ) Co-IP assay for interaction between FAM83D and GSK3β in HeLa and SiHa cells with or without silencing of FAM83D. ( E-F ) Co-IP assay for interaction between FAM83D and phosphorylated GSK3β (Ser9) in HeLa and SiHa cells. ( G ) In vitro cell-free proteins binding assay for validation of interaction specificity between FAM83D and GSK3β. * p < 0.05, ** p < 0.01, *** p < 0.001

Article Snippet: Human CC cell lines SiHa and HeLa were purchased from the American Type Culture Collection (ATCC).

Techniques: Co-Immunoprecipitation Assay, In Vitro, Binding Assay, Biomarker Discovery

Journal: Biology Direct

Article Title: FAM83D facilitates EMT and metastasis of cervical cancer via interaction with GSK3β and inactivation of GSK3β/stabilization of Snail signaling

doi: 10.1186/s13062-026-00761-z

Figure Lengend Snippet: The role of GSK3β/Snail axis in FAM83D-regulated EMT and metastasis of CC. ( A-D ) Western blotting for expressions of E-cadherin, N-cadherin, Vimentin, Snail, GSK3β, p-GSK3β (Ser9), and FAM83D in CC cells following stable FAM83D knockdown, as well as treated with LiCl (10 mM and 15 mM in HeLa and SiHa cells, respectively) for 24 h. ( E-H ) Following the treatment of HeLa cells with LiCl (10 mM) for 24 h and SiHa cells with LiCl (15 mM) for 24 h, a transwell assay was conducted to assess the migratory and invasive capabilities of HeLa and SiHa cells with stable FAM83D knockdown. The counts of migratory and invasive cells were recorded and subjected to statistical analysis. * p < 0.05, ** p < 0.01, *** p < 0.001

Article Snippet: Human CC cell lines SiHa and HeLa were purchased from the American Type Culture Collection (ATCC).

Techniques: Western Blot, Knockdown, Transwell Assay