Journal: bioRxiv

Article Title: The Salmonella effector SspH2 disrupts F-actin cytoskeletal organization to inhibit macrophage migration

doi: 10.64898/2026.10.14.759247

Figure Lengend Snippet: Caspase 1/11 -/-RAW264.7 cells were infected with WT (SL1344) or the indicated mutant strain of S. Typhimurium at an MOI of 30:1 (mock-infected samples were used as a control). Immediately after infection, the monolayer was scratched with a pipette tip and micrographs were recorded under 100X magnification immediately and one day after infection. The width of every scratch at three specific points was calculated for each time point using Fiji software, and the migration distance was calculated as the difference between the scratch width at day zero and day one. Representative images are shown (A, scale bar = 100 µm). N ≥ 3, means and standard errors of the means (SEM) are presented in B, * p-value < 0.05 in a two-sample two-tailed Student’s t test.

Article Snippet: The RAW264.7 (ATCC TIB-71) cell line (and all cell lines derived from this line) was cultured in RPMI1640 supplemented with 10% fetal bovine serum (FBS) at 37°C with 5% CO 2 .

Techniques: Infection, Mutagenesis, Control, Transferring, Software, Migration, Two Tailed Test

Journal: bioRxiv

Article Title: The Salmonella effector SspH2 disrupts F-actin cytoskeletal organization to inhibit macrophage migration

doi: 10.64898/2026.10.14.759247

Figure Lengend Snippet: RAW264.7 cells were genetically modified to express the indicated SPI-2 effectors tagged with GFP under a doxycycline-inducible promoter. A-B) Scratches were introduced into the RAW264.7 monolayers with a 200µl pipette tip and treated with or without doxycycline (10 ng/mL for SspH2- and 100 ng/mL for SseI-expressing cells). Micrographs were recorded under 100X magnification immediately and 1 day later. Average migration (normalized to controls without doxycycline treatment) for at least 3 separate experiments is shown in A (error bars represent the standard deviation), and representative micrographs are shown in B (scale bar = 100 µm). C-D) SPI-2 effector-expressing RAW264.7 cells (as described in A and B) were seeded onto coverglass and treated with 0 or 10 ng/mL doxycycline for one day before observing by time-lapse microscopy every 3 minutes over 3.5 hours at 600X. Cells were tracked using Fiji to measure cell migration velocity (C) and the number of times a cell changes its direction of movement by more than 90 degrees (D). Between 113 and 163 cells were tracked for each group, and the mean and SEM are indicated by horizontal lines and error bars, respectively. E) A schematic of the under-agarose assay to measure chemotaxis: darker pink represents agarose while lighter pink represents wells (holes) cut out of the agarose where either cells or 500 ng/mL CCL5 was added. The indicated SPI-2 effector-expressing RAW264.7 cells were seeded into the well designated for cells in a 1:1 mixture with RAW264.7 cells genetically modified to express mCherry alone as an internal control. In the presence of 10 ng/mL doxycycline, chemotaxis was measured as the number of SPI-2 effector-expressing GFP+ cells that migrate under the agarose toward CCL5 minus the number of cells that migrate away. This raw measurement was then normalized to the raw chemotaxis measurements of the mCherry+ internal control. F) The indicated SPI-2 effector-expressing cell lines were treated with doxycycline and allowed to chemotax under the agarose for 24 hours before taking images at 600X on either side of the well containing cells. Chemotaxis measurements normalized to the internal control are presented. The means of between ≥ 5 replicates are shown. All * indicate p-values < 0.05 in two-sample two-tailed Student’s t tests.

Article Snippet: The RAW264.7 (ATCC TIB-71) cell line (and all cell lines derived from this line) was cultured in RPMI1640 supplemented with 10% fetal bovine serum (FBS) at 37°C with 5% CO 2 .

Techniques: Genetically Modified, Transferring, Expressing, Migration, Standard Deviation, Time-lapse Microscopy, Chemotaxis Assay, Control, Two Tailed Test

Journal: bioRxiv

Article Title: The Salmonella effector SspH2 disrupts F-actin cytoskeletal organization to inhibit macrophage migration

doi: 10.64898/2026.10.14.759247

Figure Lengend Snippet: RAW264.7 cells genetically modified to express SspH2-GFP or mCherry alone (as described in ) were mixed together along with the parental cell line and seeded onto coverglass, then treated with doxycycline to induce SspH2-GFP expression. The next day, cells were fixed using paraformaldehyde and stained for F-actin using phalloidin-660. Confocal images were taken at 630X magnification and a representative image (z-slice) is shown in (A) with the boxed regions enlarged for two SspH2-GFP expressing cells in (B) and two control cells in (C) (scale bar = 21 µm). The total F-actin-associated fluorescent signal for each entire cell with and without SspH2-GFP expression was measured using Volocity software and presented in (D). At least 55 cells were assessed for each group, means ± SEM are indicated by horizontal line and error bars and * indicates p-value < 0.05 in a two-sample two-tailed Student’s t test.

Article Snippet: The RAW264.7 (ATCC TIB-71) cell line (and all cell lines derived from this line) was cultured in RPMI1640 supplemented with 10% fetal bovine serum (FBS) at 37°C with 5% CO 2 .

Techniques: Genetically Modified, Expressing, Staining, Confocal, Control, Software, Two Tailed Test

Journal: bioRxiv

Article Title: The Salmonella effector SspH2 disrupts F-actin cytoskeletal organization to inhibit macrophage migration

doi: 10.64898/2026.10.14.759247

Figure Lengend Snippet: A) SspH2-GFP-expressing RAW264.7 cells were transduced with a constitutive expression construct for LifeAct-mCherry that stains F-actin in live cells. Cultures were treated with (circles) or without (control, triangles) doxycycline, and fluoresecent and DIC images were taken every 20 minutes over 13 hours at 600X magnification. CellTK software was used to track individual cells and to measure the fluorescence intensities of mCherry (filled symbols) and GFP (empty symbols) for each individual cell at each time point. The means ± SEM are shown, the number of cells for each time point ranged between 37 (at early time points) and 139 (at later time points). Control F-actin intensities were significantly different from that of cells treated with doxycycline for all times > 6 h (p < 0.05) in two-sample two-tailed Student’s t tests. Representative videos are shown in S2 Video (0 doxycycline) and S3 Video (+doxycycline). B-C) DIC and fluorescent images of representative cells are shown after addition of 0 (B, “Control”) or 50 ng/mL (C, “+SspH2-GFP”) doxycycline at 0 (Bi-ii, Ci-iii) or 10 h later (Biii-iv, Civ-vi). F-actin was detected using LifeAct-mCherry (red, Bii,iv and Cii,v) and SspH2 using GFP (green, Ciii,vi). Arrow points to an example of F-actin-rich punctate structures arranged into a rosette/ring formation.

Article Snippet: The RAW264.7 (ATCC TIB-71) cell line (and all cell lines derived from this line) was cultured in RPMI1640 supplemented with 10% fetal bovine serum (FBS) at 37°C with 5% CO 2 .

Techniques: Expressing, Transduction, Construct, Control, Software, Fluorescence, Two Tailed Test

Journal: bioRxiv

Article Title: The Salmonella effector SspH2 disrupts F-actin cytoskeletal organization to inhibit macrophage migration

doi: 10.64898/2026.10.14.759247

Figure Lengend Snippet: A) The sspH2 gene was mutated to introduce an alanine in place of the catalytic cysteine residue at amino acid position 580 creating the catalytic mutant SspH2C580A. As described for , RAW264.7 cells were genetically modified to conditionally express SspH2C580A-GFP upon addition of doxycycline and to constitutively express the F-actin staining protein LifeAct-mCherry. Cultures were treated with (empty squares) or without (control, filled triangles) doxycycline, and then DIC and fluorescent images were taken every 20 minutes at 600X magnification. CellTK software was used to track individual cells and to measure the mCherry fluorescence intensities for each individual cell at each time point. The means ± SEM for each time point are shown, the number of cells for each time point was between 45 (at early time points) and 78 (at later time points). Excluding time = 8.67 h, control F-actin intensities were not significantly different from that of cells treated with doxycycline for all times shown (p > 0.05) in two-sample two-tailed Student’s t tests. B) After 12 h treatment with or without doxycycline, cells expressing SspH2 or the catalytic mutant SspH2C580A were tracked and the cell velocities were measured using Fiji. Between 41 and 69 cells were tracked for each group, the means ± SEM are shown, and * p-value < 0.05 in a two-sample two-tailed Student’s t test. C) The total β-actin was harvested from the indicated SspH2-expressing RAW264.7 cell lines (C580A = SspH2C580A mutant) treated with and without doxycycline (for one day) and quantified by western blot (normalized to GAPDH). The amount of total β-actin in doxycycline-treated samples was reported as a percent of samples without doxycycline treatment (control), and the p-value was calculated using a paired two-tailed Student’s t test. D) The fraction of β-actin existing as F-actin was quantified for the indicated SspH2-expressing RAW264.7 cell lines with and without doxycycline treatment for one day. As a positive control, RAW264.7 cells were treated for one day with 10 µM cytochalasin D (CytD), an inhibitor of actin polymerization that diminishes the cell’s F-actin fraction. The data for doxycycline-(or cytochalasin D-) treated samples are presented as the percent of non-treated samples (controls). * indicates p-value < 0.05 in a paired two-tailed Student’s t test. E) The frequency of cells containing rosette/ring-like formations of F-actin puncta after one day with doxycycline treatment was quantified for ≥ 4 replicates for each group, and then normalized to that of cells not treated with doxycycline. * p-value < 0.05 in a two-sample two-tailed Student’s t test. Images of SspH2-GFP-(F) or SspH2C580A-GFP-(G) expressing cells were taken one day after addition of doxycycline using DIC (i) and fluorescence (ii - iii), scale bar = 10 µm. To discern higher-order F-actin structures, images with similar and easily visible F-actin signal are shown here. Arrows indicate punctate F-actin-rich (podosome-like) structures arranged into rosette/ring-like formations.

Article Snippet: The RAW264.7 (ATCC TIB-71) cell line (and all cell lines derived from this line) was cultured in RPMI1640 supplemented with 10% fetal bovine serum (FBS) at 37°C with 5% CO 2 .

Techniques: Introduce, Residue, Mutagenesis, Genetically Modified, Staining, Control, Software, Fluorescence, Two Tailed Test, Expressing, Western Blot, Positive Control

Journal: bioRxiv

Article Title: The Salmonella effector SspH2 disrupts F-actin cytoskeletal organization to inhibit macrophage migration

doi: 10.64898/2026.10.14.759247

Figure Lengend Snippet: SspH2-GFP-expressing RAW264.7 cells (“parental” cell line) were transfected with an expression construct that co-expressed Cas9 and a small scaffold RNA targeting vcl , after which stable transfectants were selected. A) Scratches were introduced into confluent monolayers of the parental cell line (expressing wild-type vinculin) or cells stably expressing the small scaffold RNA targeting vcl , and treated with doxycycline to induce SspH2 (“-“ = 0 doxycylcline). The width of every scratch at three specific points was measured immediately after making the scratch and then again 48 hours later. Means ± SEM are shown for ≥ 6 replicates for each group, and * p-value < 0.05 in a two-sample two-tailed Student’s t test. B) Vinculin protein expression was assessed by western blot for the parental and vcl -targeted cell line. Vinculin signal was calculated as the vinculin intensity divided by the GAPDH intensity (and normalized to the value of the parental cells). C) A vinculin expression construct (p Vcl ) was used to complement the vcl mutant phenotype, and the indicated cell lines were subjected to time-lapse microscopy, taking images every 20 minutes for 12 hours at 600X magnification. Cells in these time-lapse videos were then tracked using Fiji’s Trackmate plugin software (finding at least 695 cell tracks per group) and the straight line speed is presented (means ± SEM are shown, * p-value < 0.05 in a Tukey-Kramer multiple comparisons test, using JMP statistical software).

Article Snippet: The RAW264.7 (ATCC TIB-71) cell line (and all cell lines derived from this line) was cultured in RPMI1640 supplemented with 10% fetal bovine serum (FBS) at 37°C with 5% CO 2 .

Techniques: Expressing, Transfection, Construct, Stable Transfection, Two Tailed Test, Western Blot, Mutagenesis, Time-lapse Microscopy, Software