EMBO/soda-nel huggingface dataset:
MYBL2 and ATM suppress replication stress in pluripotent stem cells (2021)
Figure 5

Figure 5. Replication speed phenotype in Mybl2Δ/Δ ESCs is epistatic with loss of MRE-11 nuclease activity.

A) Scheme of the procedure before DNA spreading. Cells were treated for 1.5 hours with 20uM MRe11 inhibitor (Mirin), before sequential addition of IdU and CldU for 20 minutes each.

B and C) Distribution curve of replication fork rates for Mybl2+/+ and Mybl2Δ/Δ ESCs treated with mirin. Statistical analysis was performed using the Mann Whitney U test. n=3 experimental replicates. The value of the mean fork length is indicated above arrow ( **** p< 0.0001)

D) Representative images of new firing origins, and elongating forks showing stable replication and fork instability.

E) Frequency of new firing origins (relative to total structures counted) from Mybl2+/+ and Mybl2Δ/Δ ESCs treated or not with Mirin. At least 450 replication structures were counted per treatment from 3 independent repeats. Error bars represent SEM. Statistical analysis using two- tailed unpaired t-test.

F) Distribution and the average IdU/CldU ratio in Mybl2+/+ and Mybl2Δ/Δ ESCs with or without MRE11 inhibitor treatment. The length of both incorporated labels for each elongating fork was measured in µm and the positive ratio was calculated. Percentage of highly unstable forks (ratio above 2) are indicated. Dotted line at 1 indicates positive ratio. Y-axis was cut to change scale and display large values. Statistical analysis was carried out using the Mann Whitney U test. At least 260 ratios were calculated per treatment group from 3 separate experiments.( **** p< 0.0001)
MYBL2 and ATM suppress replication stress in pluripotent stem cells (2021)
Figure 6

Figure 6. The replication defect observed after ATM inhibition and in Mybl2Δ/Δ ESCs is due to deregulation of cell cycle associated replication initiation control.

A, B) Scheme of the aim and procedure before DNA spreading. Cells were treated for 1.5 hours with ATM inhibitor (KU60019) or CDC7 inhibitor (PHA-767491) alone or in combination, before sequential addition of IdU and CldU for 20 minutes each.

C ) Frequency of new firing origins (relative to total structures counted) from Mybl2+/+ and Mybl2Δ/Δ ESCs treated or not with the indicated inhibitors: CDC7 inhibitor (PHA-767491) and/or ATM inhibitor (Ku60019). At least 300 replication structures were counted per treatment. Error bars represent SEM. Statistical analysis using two- tailed unpaired t-test (*p < 0.05).

D ) Replication rate (kb/min) of Mybl2+/+ and Mybl2Δ/Δ ESCs treated with the indicated inhibitors. Statistical analysis was carried out using an unpaired Mann Whitney U test (**** p< 0.0001). Minimum three independent experiments. At least 270 forks were scored for non-ATM inhibitor treated groups, and a minimum of 130 for ATM inhibitor treated groups.

E ) Fork stability (average IdU/CldU ratio) of Mybl2+/+ ESC and Mybl2Δ/Δ ESCs treated with the indicated inhibitors alone or in combination. At least 240 ratios were calculated for non-ATM inhibitor treated groups, and a minimm of 130 for ATM inhibitor treated groups. Y-axis was cut to change scale and display large values. Statistical analysis was carried out using Mann Whitney U test (**** p< 0.0001).

Data for Mybl2+/+ ESCs treated with ATM inhibitor alone or in combination with CDC7 inhibitor, was collected from two independent experiments. For the rest of the conditions, a minimum of three independent repeats was performed.

F) Immunoblot showing the levels of CDC7 and actin in the Mybl2+/+ and Mybl2Δ/Δ ESCs. Bar graph (lower panel) represents average band density of CDC7 in Mybl2Δ/Δ ESCs relative to actin and relative to Mybl2+/+ ESCs from three independent repeats. Error bars represent SD. Statistical analysis was carried out using a two-tailed unpaired t-test (ns=no significant).
Concomitant deletion of Ptpn6 and Ptpn11 in T cells fails to improve anticancer responses (2022)
Figure 1

Figure 1. CD4cre Ptpn6fl/fl mice and CD4cre Ptpn6/11fl/fl mice do not exhibit superior antitumor immunity

A) The schematic illustrates CD4cre-mediated deletion of Ptpn6 or Ptpn6/11 during thymic T cell development; double-negative thymocytes (DN) and double-positive thymocytes (DP) are depicted. Created with Biorender (Biorender.com).

B) Total T cells were enriched from spleens of Ptpn6fl/fl/Ptpn11fl/flWbm, CD4cre Ptpn6fl/fl/Ptpn11fl/flWbm, CD4cre Ptpn6fl/fl mice. Expression of Ptpn6 and Ptpn11 was tested by immunoblot in cell lysates, Gapdh used as loading control.

C and D) CD4cre Ptpn6fl/fl and control mice were subcutaneously injected with MC38 cells. Tumour growth in individual mice is shown for the indicated genotypes and treatments; number of mice eradicating the tumour is shown within the graphs (C). Survival curves are shown (D).

E and F) A representative flow cytometry plot (E), spleen cellularity, CD4+ T cell (gated as TCRβ+ CD4+) numbers, CD8+ T cell (gated as TCRβ+ CD8+) numbers, and percentages of CD44+ T cells (F) from the spleens of CD4cre Ptpn6fl/fl/Ptpn11fl/flWbm and control mice are shown.

G and H) Tumour growth in individual mice challenged with MC38 and subsequently treated with anti-PD-1 antibody or isotype control is shown for each indicated genotype; number of mice eradicating the tumour is shown within the graphs (G). Survival curves of CD4cre Ptpn6fl/fl/Ptpn11fl/flWbm and control mice is shown (H).

I, J) 10-12 days following MC38 tumour inoculation and treatment with anti-PD-1 antibody or isotype control, CD4cre Ptpn6/11wt/wt and CD4cre Ptpn6fl/fl/Ptpn11fl/flWbm mice were sacrificed, and TILs analysed. Graphs depict frequencies of CD8+ T cells (gated as CD45+ TCRβ+ CD8+) expressing IFN-γ and TNFα upon re-stimulation (G) and percentages of CD8+ T cells (H).

Data information: Results depict n = 8-10 mice/group (C, D) and n = 8-11 mice/group (G, H); statistical significance was calculated by log-rank (Mantel-Cox) test (D, H). Results depict mean ± SEM of n = 10-12 mice/group (F) and of n = 9-14 mice/group (I, J). Student's t test (unpaired, two-tailed) was used to compare differences between experimental groups (F, I, J). *p ≤0.05, **p ≤ 0.01, ***p ≤ 0.001, ****p ≤ 0.0001.
Concomitant deletion of Ptpn6 and Ptpn11 in T cells fails to improve anticancer responses (2022)
Figure 2

Figure 2. Deletion of Ptpn6/11 in effector cells does not lead to enhanced antitumour response.

A) The schematic illustrates GzmBcre-mediated deletion of Ptpn6/11 following cancer challenge; CD8+ T cells and cytotoxic T lymphocytes (CTLs) are depicted. Created with Biorender (Biorender.com).

B) Ptpn6 and Ptpn11 copy number, as estimated through high resolution mass spectrometry in GzmBcre Ptpn6/11fl/fl and control CTLs.

C and D) GzmBcre R26R-YFP reporter mice were injected subcutaneously with MC38 cells and analysed after 11 days. Graphs show the percentage of YFP+ cells among CD45+ CD8+ TILs (C) and the percentage of PD-1+ cells among CD45+ CD8+ YFP+ TILs (D). Results depict n = 4 mice (C and D).

E and F) GzmBcre Ptpn6/11fl/fl and control Ptpn6/11fl/fl mice were subcutaneously injected with MC38 cells. Survival curves and statistical comparisons between different groups are shown (E). The tumour start depicts the time (days) following tumour engraftment after which the tumour is palpable, while tumour endpoint depicts the time (days) from tumour start until the reaching the maximal allowed tumour size (F).

Data information: Results depict mean ± SD of n = 3 biological replicates (B), mean ± SEM of n = 4 biological replicates (C, D), mean ± SEM (F) of n = 11-12 mice/group (E and F); statistical significance was calculated by log-rank (Mantel-Cox) test (E), and Student's t test (unpaired, two-tailed) was used to compare differences between experimental groups (B, F). **p ≤ 0.01, ***p ≤ 0.001.
Concomitant deletion of Ptpn6 and Ptpn11 in T cells fails to improve anticancer responses (2022)
Figure 3

Figure 3. Deletion of Ptpn6 and Ptpn11 impacts on CD8+ T cell proteome and cell survival.

CTLs were generated from splenocytes of GzmBcre Ptpn6/11fl/fl and Ptpn6/11fl/fl mice by anti-CD3, anti-CD28, and IL-12 stimulation and maintained in IL-2.

A, B) CD8+ T cells (gated DAPI- cells) were analysed by flow cytometry for size, granularity, and surface expression of CD25 and PD-1 after 2 days (A) and after 5 days (B); a quantification of these parameters is shown in the graphs (A and B) and a representative flow cytometry plot is shown for FSC and SSC (A).

C) Numbers of CTLs (gated on CD8+ T cells) from Ptpn6/11fl/fl or GzmBcre Ptpn6/11fl/fl mice were counted daily by flow cytometry with the addition of DAPI to monitor dead cells.

D) Quantitative high-resolution mass spectrometry was used to resolve the proteome of Ptpn6/11fl/fl and GzmBcre Ptpn6/11fl/fl CTLs (day 7). The plot depicts protein copy number in control and GzmBcre Ptpn6/11fl/fl cells. Significantly altered proteins are indicated in red; selected examples are annotated. Results are based on n = 3 biological replicates and two-tailed, unequal-variance t-test on log10 transformed copy number per cell values was used to compare differences between experimental groups.

E) GO Term analysis shows the enriched pathways with at least 23 proteins and a p value <0.05, % indicate the % altered genes on the total genes in a given pathway.

F) Graph shows the percentage of sub-G1 cells as measured by flow cytometry-based cell cycle analysis of Ptpn6/11fl/fl and GzmBcre Ptpn6/11fl/fl CTLs (gated on CD8+ T cells) at the indicated days.

G) Graph illustrates the percentage of DAPI+ dead cells of Ptpn6/11fl/fl and GzmBcre Ptpn6/11fl/fl CTLs (gated on CD8+ T cells) at the indicated days as measured by flow cytometry. H) Protein copy number of Tnfrsf1b and Mcl-1 in GzmB cre Ptpn6/11fl/fl and control CTLs, as estimated through high resolution mass spectrometry.

Data information: Results depict mean ± SD of n = 3 (A, B, F, H) or 4 (C, G) biological replicates and Student's t test (unpaired, two-tailed) was used to compare differences between experimental groups (A, B, C, F, G, H). *p ≤0.05, **p ≤ 0.01, ***p ≤ 0.001, ****p ≤ 0.0001.

Expanded View Figure legends
Subunit composition of VRAC channels determines substrate specificity and cellular resistance to Pt-based anti-cancer drugs. (2015)
Figure 1

Loss‐of‐function screen using haploid KBM7 cells identifies LRCC8A and LRRC8D as determinants of carboplatin resistance

Outline of the loss‐of‐function screen.

Genes enriched for gene‐trap insertions in a carboplatin‐selected cell population compared to unselected control cells. Circles represent genes and their size corresponds to the number of independent insertions identified in the carboplatin‐selected population. Genes are ranked on the x‐axis based on chromosomal position.

Location of gene‐trap insertion sites (red arrowheads). White boxes indicate the 5′‐ and 3′‐untranslated regions, and the black boxes show the coding sequence in exons 3 and 4 (LRRC8A) and exon 3 (LRRC8D).

Loss of LRRC8D causes resistance to carboplatin and cisplatin, but not to oxaliplatin. Survival of parental, vector‐transduced, or LRRC8D‐deficient GT1 and GT2 KBM7 cells exposed for 96 h to increasing concentrations of cisplatin, carboplatin, and oxaliplatin. The corresponding IC50 values and 95% confidence interval (CI) are given in Appendix Table S2. Data are presented as mean ± SEM.
Subunit composition of VRAC channels determines substrate specificity and cellular resistance to Pt-based anti-cancer drugs. (2015)
Figure 4

LRRC8 subunit‐ and osmolarity‐dependent caspase induction in HCT116 cells

A, B

Cisplatin‐induced caspase activity in the continuous presence of 200 μM cisplatin under isotonic conditions (A), or after 1 h exposure to 200 μM cisplatin under iso‐ and hypotonic conditions (B), was followed over time in WT, LRRC8A
−/−, LRRC8D
−/−, and LRRC8
−/− HCT116 cells. Results from LRRC8A
−/− and LRRC8D
−/− were obtained with two different clonal cell lines each and averaged.

C

Caspase activation after 1‐h exposure to 4 μM staurosporine under iso‐ or hypotonic conditions of WT, LRRC8A
−/−, LRRC8D
−/−, and LRRC8
−/− HCT116 cells.

Data information: Data are presented as mean ± SEM, n = 3–6. *P < 0.05; **P < 0.01; and ***P < 0.001. Similar results were obtained in three independent experiments. Fold change in (A) refers to t = 0. Control experiments indicated that hypotonicity per se had no effect (Appendix Fig S4).
Subunit composition of VRAC channels determines substrate specificity and cellular resistance to Pt-based anti-cancer drugs. (2015)
Figure 6

LRRC8 subunit‐ and osmolarity‐dependent carboplatin/cisplatin transport

A

Carboplatin uptake into control KBM7 cells and two LRRC8D‐deficient clones (n = 6).

B

Hypotonicity‐stimulated cisplatin uptake in HEK WT cells was selectively blocked by 100 μM carbenoxolone (CBX), a non‐specific blocker of VRAC (n = 3).

C, D

Cisplatin uptake into HEK cells of indicated genotypes using 40 μM cisplatin under long‐term isotonic (C) or 200 μM cisplatin in short‐term hypo‐ and isotonic (D) conditions as function of time (n = 3). Similar results were obtained in HCT116 cells (Fig EV4).

E, F

Cisplatin uptake (200 μM) into HEK cells of indicated genotypes. LRRC8(B,C,E)
−/− and LRRC8(B,D,E)
−/− cells express only LRRC8A and LRRC8D, and LRRC8A and LRRC8C, respectively (n = 3 for WT, 6 for LRRC8D
−/−, 9 for LRRC8A
−/− in E; n = 3 in F).

G

Mean current densities of maximally activated ICl,vol at −80 mV. The number of cells is indicated for each column.

H

Ratio of LRRC8‐dependent swelling‐activated cisplatin uptake (60 min) to mean ICl,vol (as in G) as function of genotype.

Data information: Data are presented as mean ± SEM. *P < 0.05; **P < 0.01; and ***P < 0.001 (for (C, E) compared to LRRC8A−/− cells). Dotted lines in (D) and (F) highlight that there is no significant difference in isotonic 60‐min uptake between the genotypes.
Subunit composition of VRAC channels determines substrate specificity and cellular resistance to Pt-based anti-cancer drugs. (2015)
Figure 7

Cisplatin–DMSO inhibition of I<sub>Cl,vol</sub> depends on the LRRC8D subunit

Upper panel, example current traces (as in Fig 3A) of fully activated ICl,vol in HEK cells exposed to hypotonic solution containing vehicle (0.3% DMSO) or 200 μM cisplatin in 0.3% DMSO. Dashed lines indicate zero current. Lower panel, ICl,vol current densities (at −100 mV and 100 mV) of WT HEK cells treated with different cisplatin concentrations.

No effect of 200 μM cisplatin/DMSO on ICl,vol in LRRC8D
−/− HEK cells.

Data information: Data are presented as mean ± SEM; the number of experiments is given for each bar; *P < 0.05; **P < 0.01.
Subunit composition of VRAC channels determines substrate specificity and cellular resistance to Pt-based anti-cancer drugs. (2015)
Figure 8

LRRC8A/LRRC8D‐containing channels transport the cellular osmolyte taurine

A, B

Swelling‐induced efflux of 3[H]‐taurine from WT and LRRC8D
−/− HEK cells (A, B) and partial rescue by transient transfection of LRRC8D (B). Rescue is incomplete due to low transfection/expression efficiency of LRRC8D (Voss et al, 2014).

C–E

Swelling‐induced efflux of 3[H]‐taurine from LRRC8(B,C,E)
−/− (C), LRRC8(B,D,E)
−/− (D), or LRRC8(B,C,D)
−/− (E) HEK cells compared to WT cells.

F

Cisplatin‐induced taurine efflux (over 30 min) from WT and LRRC8A
−/− HEK cells after preincubation with 200 μM cisplatin for 4 or 12 h, or without cisplatin (0 h). Carbenoxolone (CBX; 100 μM) blocks taurine efflux from WT cells treated for 12 h with cisplatin, excluding taurine flux through unspecific leaks.

Data information: Data are presented as mean ± SEM. Dashed lines in (A–E) represent isotonic efflux from the same individual experiments. Red arrows, change to hypotonic solution. Each panel shows the average of two independent experiments (n = 4 for each experiments, i.e., n = 8 in total).
Porphyromonas gingivalis evasion of autophagy and intracellular killing by human myeloid dendritic cells involves DC-SIGN-TLR2 crosstalk. (2015)
Fig 1

High intracellular content of Mfa1<sup>+</sup>Pg within human MoDCs.

A) Transmission electron microscopy (TEM) of MoDCs infected with P. gingivalis for 2, 12 and 24 hours (left, middle and right panels). The sections show the intra-and extra-cellular contents of Cont. (un-infected), Pg381, Mfa1+Pg and FimA+Pg infected MoDCs for the different time points. B) The figure shows the intracellular content of P. gingivalis strains after 24 hours of infection. MoDCs infected with Pg381 or isogenic mutants strains were lysed and the survived intracellular bacteria were re-suspended and maintained in anaerobic broth for 5 days. The data represents CFU within MoDCs harvested from three healthy individuals. The means ±standard deviation (in triplicates) were analyzed by One-way ANOVA of different groups and Tukey’s test for multiple group comparisons within 3 different experiments (* statistical significance at the p<0.001). C) Growth curves of Pg381 and mutant strains (Mfa1+Pg and Fima+Pg) in anaerobic condition in the absence of DCs. The means ±standard deviations were analyzed by One-way ANOVA of different groups and Tukey’s test for multiple group comparisons within 3 different experiments.
Porphyromonas gingivalis evasion of autophagy and intracellular killing by human myeloid dendritic cells involves DC-SIGN-TLR2 crosstalk. (2015)
Fig 2

Mfa1<sup>+</sup>Pg up-regulate the expression of DC-SIGN in human MoDCs.

A) DC-SIGN mRNA expression in P. gingivalis-infected MoDCs at 0.1, 1 and 10 MOIs. The figure shows the gene expression after 12 hours of Pg381 and mutant strains infections. The target gene (DC-SIGN) was normalized using the endogenous control GAPDH (ΔCt) and fold regulations were calculated using 2-(ΔΔCt) method. The statistical analysis was performed using the t-test, which accounts for the clustering of infected and un-infected controls within 3 different experiments (* p<0.001). B) Immuno-electron microscopy of un-infected MoDCs (Cont.) (upper panel), MoDCs infected with Pg381 (middle panel) and Mfa1+Pg mutants (lower panel). Gold particles (marked with red rings) for positive DC-SIGN were detected in the cell membrane and cytoplasm of cells infected with Mfa1+Pg strains. Minimal positive staining for DC-SIGN was detected in the membranes of MoDCs infected with Pg381, while no cytoplasmic gold labeling was detected in these cells. C) Flow cytometry analysis of surface DC-SIGN in human MoDCs after infection with Pg381, Mfa1+Pg and FimA+Pg. The analysis of the intensity used Kruskal-Wallis test analysis of different groups and Dunn’s test for multiple comparisons 3 different experiments (* p<0.01).
Porphyromonas gingivalis evasion of autophagy and intracellular killing by human myeloid dendritic cells involves DC-SIGN-TLR2 crosstalk. (2015)
Fig 3

Lower intracellular content of Mfa1<sup>+</sup>Pg in MoDCs treated with Rapamycin.

A) Pg381 and mutant strains count after 24 hr incubation with human MoDCs with/without Rapamycin treatment. The survived bacteria were measured after maintaining the lysed MoDCs suspension in anaerobic broth for 5 days. The plot represents the means ±standard deviation of CFU within MoDCs harvested from three healthy individuals (* P<0.001). The analysis of readings used One-way AVOVA analysis of different groups and Tukey’s test for multiple comparisons. B) Epifluorescence microscopy images of MoDCs treated with Rapamycin 1 hour after P. gingivalis infections. LC3-II (red-fluorescent dye) and the bacterial strains (green CFSE) were studied in MoDCs 11 hours after Rapamycin treatment (12 hours after infections). C) Quantifications of the fluorescent intensity of CFSE-labeled P. gingivalis and LC3-II signals within infected MoDCs using NIS-Elements BR software. One-way ANOVA analysis was used to compare the means of intensity of different groups and Tukey’s test for multiple comparisons (* P<0.001).
Porphyromonas gingivalis evasion of autophagy and intracellular killing by human myeloid dendritic cells involves DC-SIGN-TLR2 crosstalk. (2015)
Fig 4

Induction of autophagy impairs the survival of Mfa1<sup>+</sup>Pg strain within MoDCs.

A) Survival of P. gingivalis strains within MoDCs after 6, 12, 24 and 48 hours. Blue lines show P. gingivalis strains survival within MoDCs and their survival in anaerobic condition in the absence of DCs are showed in black lines. The effect of rapamycin on Mfa1+Pg, Pg381 and FimA+Pg survival within MoDCs are shown in figures B, C and D respectively. A three-factor repeated measures ANOVA using mixed models was used to test the effect of strain and rapamycin treatment over time on OD reading. The survival curves for the strains are showed in blue, while the effect of rapamycin treatments are in red. Bacterial survivals in the absence of MoDCs with and without rapamycin are plotted in grey and black, respectively. Statistical analysis showed that the strain by rapamycin treatment overtime interaction indicates the pattern of means in each strain (Mfa1+Pg, Pg381 and FimA+Pg) between treated (rapamycin) and untreated were significantly different overtime (p-value <0.=001).
Porphyromonas gingivalis evasion of autophagy and intracellular killing by human myeloid dendritic cells involves DC-SIGN-TLR2 crosstalk. (2015)
Fig 5

Low LC3-II signals in human MoDCs infected with Mfa1<sup>+</sup>Pg.

A) Epifluorescence microscopy images of MoDCs infected with Pg381, Mfa1+Pg, FimA+Pg and MFB strains after 12 hours. LC3-II was detected in red-fluorescent (Texas red) dye and the bacterial strains were pre-labeled with CFSE (green). Co-localization of P. gingivalis and LC3-II showed in the right panels. B) Quantifications of the fluorescent intensity of LC3-II within infected MoDCs using NIS-Elements BR software. One-way ANOVA analysis was used to compare the means of intensity of different groups and Tukey’s test for multiple comparisons of three different experiments (* p<0.001, # p<0.01). C) Bacterial uptake by MoDCs was determined by CFSE fluorescent intensity (* p<0.001, # p<0.01).
Porphyromonas gingivalis evasion of autophagy and intracellular killing by human myeloid dendritic cells involves DC-SIGN-TLR2 crosstalk. (2015)
Fig 6

Blocking of DC-SIGN inhibits Mfa1<sup>+</sup>Pg uptake and restores basal LC3-II signal in MoDCs.

A) Immuno-fluorescence images of LC3-II and P. gingivalis within MoDCs pre-treated with GP120 (DC-SIGN blocker) after 12 hours of infection. LC3-II was detected in red-fluorescent dye and the bacterial strains were pre-labeled with green CFSE. B) Fluorescent intensity of LC3-II and P. gingivalis strains signal (in triplicates) were statistically analyzed (* and # p<0.001). C) Flow cytometry of MoDCs treated with siRNA for DC-SIGN (si-DC-SIGN) after 12 hours. Left panels show the decrease of DC-SIGN in Cont. (un-infected), Pg381 and Mfa1+Pg-infected MoDCs. D) Epifluorescence microscopy images of MoDCs (si-DC-SIGN) infected with Pg381 and Mfa1+Pg. LC3-II was detected in red-fluorescent dye and the bacterial strains were pre-labeled with green CFSE. E) The figure shows the CFU counts of the P. gingivalis strains with MoDCs that lack DC-SIGN (si-DC-SIGN). The analysis of readings used One-way ANOVA analysis of different groups and Tukey’s test for multiple comparisons (* p<0.001).
Porphyromonas gingivalis evasion of autophagy and intracellular killing by human myeloid dendritic cells involves DC-SIGN-TLR2 crosstalk. (2015)
Fig 7

Low LC3-II expression in MoDCs infected with <italic>P. gingivalis</italic> expressing Mfa1.

A) Western blot detection of LC3 in MoDCs infected with Pg381 and mutant strains for 12 hours. B) The mean ±standard deviation of LC3-II intensity represents three different experiments and the values were analyzed using Kruskal-Wallis test analysis of different groups and Dunn’s test for multiple comparisons. C) Blot shows the autophagy flux test after blocking the lysosomal fusion with Bafilomycin in MoDCs. Cells were treated with Bafilomycin for 4 hours at 4nM. D) LC3-II intensity in MoDCs infected with Pg381 strains with and without Bafilomycin. The analysis of the intensity of three different experiments used Kruskal-Wallis test analysis of different groups and Dunn’s test for multiple comparisons.